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  • Articles  (1,175)
  • Occupational Health and Environmental Toxicology  (1,044)
  • fertilization  (132)
  • Molecular Sequence Data
  • Wiley-Blackwell  (1,175)
  • Biology  (1,175)
  • 1
    Electronic Resource
    Electronic Resource
    Motility and centrosomal organization during sea urchin and mouse fertilization (1986)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 6 (1986), S. 163-175 
    Details
    ISSN: 0886-1544
    Keywords: centrosomes ; fertilization ; mice ; microfilaments ; microtubules ; mitosis ; pericentriolar material ; sea urchins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.
    Additional Material: 15 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970060215
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  • 2
    Electronic Resource
    Electronic Resource
    Actin-mediated surface motility during sea urchin fertilization (1983)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 3 (1983), S. 513-524 
    Details
    ISSN: 0886-1544
    Keywords: fertilization ; actin ; microfilaments ; sea urchin ; cell division ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The sea urchin egg at fertilization is an ideal model in which to study actin-mediated surface activity. Electron microscopy of unfertilized eggs demonstrates the presence of thousands of well-arrayed short microvilli, which appear supported by cytochalasin-sensitive actin oligomers as detected with rhodamine-labeled phalloidin staining of permeabilized eggs. At insemination, the previously short microvilli elongate and cluster around the successful sperm during incorporation. Phalloidin staining demonstrates a tremendous recruitement of polymerized actin into the site of sperm incorporation, resulting in the formation of the fertilization cone. Fertilization of cytochalasin-treated eggs results in the normal activation of the metabolic and bioeletric events, but sperm incorporation does not occur since the localized actin assembly required for fertilization cone formation is precluded. After sperm incorporation, the entire fertilized surface is restructured, as a result of a massive polymerization of actin to produce a burst in microvillar elongation. Addition of cytochalasin to eggs immediately following sperm incorporation demonstrates the recruitment of actin assembly for the proper progression through the first cell cycle. During normal cell divison, the egg surface retains the long microvilli. The furrow which forms at cytokinesis does not appear as a unique new structure, but rather as a reorganization of the cortical microfilaments. Quantitative fluorescence microscopy argues against an increase in microfilaments during early cytokinesis. At the latest stages of cytokinesis, a thickening of the cortical actin is noted, which could possibly be interpreted as a contractile ring. A minor basal level of actin assembly with numerous nucleation sites in unfertilized eggs and a tremendous but localized assembly of microfilaments surrounding the sperm during incorporation, followed by a massive global microfilament assembly event to elongate the fertilized egg microvilli resulting later in the reorganization of these microfilaments to produce the forces necessary for cytokinesis, highlight the utility of the study of sea urchin eggs at fertilization for understanding actin-membrane interactions.
    Additional Material: 13 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970030518
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  • 3
    Electronic Resource
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    Microtubules in ascidian eggs during meiosis, fertilization, and mitosis (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 219-230 
    Details
    ISSN: 0886-1544
    Keywords: fertilization ; ooplasmic segregation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic rescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090304
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  • 4
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    Activation of maternal centrosomes in unfertilized sea urchin eggs (1992)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 61-70 
    Details
    ISSN: 0886-1544
    Keywords: activation ; fertilization ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 μM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material. © 1992 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970230107
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  • 5
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    Redistribution of actin and fascin in sea urchin eggs after fertilization (1980)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 1 (1980), S. 31-40 
    Details
    ISSN: 0886-1544
    Keywords: actin ; fascin ; actin cross-linking proteins ; fertilization ; microvilli ; sea urchin eggs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Following fertilization, the sea urchin egg cortex undergoes a structural change involving the assembly and organization of actin filaments into microvilli. Antifascin localizes this actin cross-linking protein in the microvilli of the fertilized egg cortex but no organized staining is present in the unfertilized cortex. Determination of the actin content of eggs using the DNAase I inhibition assay indicates that actin is about 1.4% of the total protein. Approximately 90% of this actin is soluble in low calcium isotonic extracts of unfertilized eggs while only 60-65% can be recovered in identical extracts of fertilized eggs. Similar measurements for fascin using a radioimmunoassay indicate this molecule represents about 0.3% of the total egg protein, essentially all of which is recovered in low calcium isotonic extracts of unfertilized eggs. After fertilization only 65-70% of this actin cross-linking protein is in the soluble phase. These results demonstrate a markedly different solubility for actin and fascin after fertilization, when the indirect immunofluorescence staining localizes fascin in the microvilli, and are consistent with the idea that fascin organizes newly polymerized actin filaments into the microvillar cores. A consideration of the amounts of actin and fascin incorporated into the cortex after fertilization and the number of microvilli on the egg surface indicates that the measured values are sufficient to account for the observed microvillar elongation.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970010104
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  • 6
    Electronic Resource
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    Wave of cortical actin polymerization in the sea urchin egg (1987)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 7 (1987), S. 46-53 
    Details
    ISSN: 0886-1544
    Keywords: actin filament ; fertilization ; fluorescent labeled phallotoxins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970070107
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  • 7
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    Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 85-96 
    Details
    ISSN: 0886-1544
    Keywords: fertilization ; echinoderm eggs ; egg cortex ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold-labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1-μm-thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin-filament phalloidin-binding sites by some cellular actin-binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090109
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  • 8
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    Wave of free calcium at fertilization in the sea urchin egg visualized with fura-2 (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 271-277 
    Details
    ISSN: 0886-1544
    Keywords: fertilization ; Ca2+ wave ; fura-2 ; sea urchin egg ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A wave front of increased free calcium traversing the egg at fertilization is demonstrated in the sea urchin Lytechinus pictus. The use of the fluorescent calcium chelator fura-2 in combination with low-light-level TV microscopy and image processing allows the visualization of the Ca2+ wave front with high spatial and temporal resolution. Such a wave is demonstrated as increased fluorescence after an excitation of 340-nm wavelength and as the reciprocal image in form of a reduced fluorescence when excited at 380 nm. The band-like appearance of the wave resembles the Ca2+ wave described for larger eggs of other species. In a dispermic egg the high resolution of the system used allows us to recognize two waves of Ca2+ originating from the respective points of sperm entry.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090309
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  • 9
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    A 70 kD microtubule-binding protein from starfish eggs: Purification, characterization, and localization during meiosis and mitosis (1990)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 168-180 
    Details
    ISSN: 0886-1544
    Keywords: microtubule-associated proteins (MAPs) ; taxol ; oocyte maturation ; fertilization ; cell division ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A microtubule-binding protein was purified from eggs of the starfish, Asterias amurensis, through several steps of purification including the taxol-dependent procedure [Vallee, 1982, J. Cell Biol. 92:435-442]. This protein consists of a single polypeptide chain having an apparent molecular mass of 70 kD determined by SDS-PAGE. The 70 kD protein was identified as a unique microtubulebinding protein, judging from electrophoretic mobility, cleavage pattern by limited proteolysis, heat stability, and immunocrossreactivity. The 70 kD protein binds to brain and egg microtubules. It does not promote assembly of brain tubulin, but promotes that of egg tubulin in vitro in a concentration-dependent manner.Using indirect immunofluorescence and immunoelectron microscopy with the anti-70 kD protein antibody, we analyzed the cellular localization of the 70 kD protein in starfish oocytes and eggs during both meiotic maturation (meicsis) and first cleavage (mitosis). Immunofluorescence studies showed that the 70 kD protein localized on microtubule structures spread widely throughout the cytoplasm, the sperm aster, and the microtubules making up the mitotic apparatus through both meiosis and mitosis. The antibody, however, did not recognize sperm axonemes. These results were confirmed by immunoelectron microscopy. Using a colloidal gold technique, the 70 kD protein was localized along the microtubules in vivo.This 70 kD protein is the first microtubule-binding protein that has been shown to localize along the microtubules in oocytes and eggs throughout meiosis and mitosis and to promote microtubule assembly. The 70 kD protein may be involved in the dynamic changes of microtubule structures occurring within oocytes and eggs.
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970150306
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  • 10
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    Microtubule-containing detergent-extracted cytoskeletons in sea urchin eggs from fertilization through cell division: Antitubulin immunofluorescence microscopy (1983)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 3 (1983), S. 213-226 
    Details
    ISSN: 0886-1544
    Keywords: microtubules ; fertilization ; cell division ; sea urchin ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The microtubule-containing structures that appear in eggs during fertilization and cell division in the sea urchins Lytechinus variegatus and Arbacia punctulata were detected by antitubulin immunofluorescence microscopy of detergent extracted cytoskeletal preparations. The extraction buffer, which is composed of 0.55 mM MgCl2, 10 mM EGTA, 25 mM MES, 25% glycerol, 1% Nonidet P-40, and 25 μM PMSF, pH 6.7, allows for dramatically improved fluorescent images compared to those obtained using conventional staining procedures, with residual background staining being reduced to near zero.The immunofluorescent images obtained using this technique provide information on several motile events that occur during the first cell cycle. This technique demonstrates that all of the cytoplasmic microtubules are associated with the incorporated sperm's centrioles during female pronuclear migration. This changes during the centration of the male and female pronuclei at which time a monastral array of microtubules forms in the egg's cytoplasm. A large proportion of the monastral microtubules do not appear to be associated with the centrioles. At prophase and early metaphase, the centrioles are the dominant microtubule organizing centers (MTOCs) consistent with mitotic theories that the kinetochore catches, but does not initiate, microtubules. Observations of intercentriolar distances show that there are three stages of pole separation during the first cell cycle. The initial separation occurs during pronuclear centration, the second during the streak stage, and the final one during the late stages of mitosis. At telophase, polar microtubules appear to extend into the cortex supporting the cell surface at all regions except the presumptive cleavage site.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970030303
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  • 11
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    Dithiothreitol prevents membrane fusion but not centrosome or microtubule organization during the first cell cycles in sea urchins (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 59-68 
    Details
    ISSN: 0886-1544
    Keywords: fertilization ; nucleus ; chromosomes ; cytoskeleton ; mitosis ; sulfhydryl groups ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Dithiothreitol (DTT), a disulfide reducing agent, inhibits the fusion of male and female pronuclei within the activated cytoplasm of sea urchin eggs. The migrations of the pronuclei are not affected by DTT, indicating that microtubule function is not impaired. Centrosomal antigens are detected in the sperm aster and in all subsequent microtubule-based configurations. Nuclear membranes never fuse and the chromatin of male and female pronuclei never mix in the DTT-treated cells. During prophase, when nuclear envelopes break down to undergo mitosis, both sets of chromosomes undergo condensation cycles independent from each other. Both pronuclei initially stain for centrosomal material and surrounding microtubules. With time, the female's centrosomal material as well as the microtubules disappear while the male forms a bipolar spindle. Interestingly, one pole of the paternal mitotic apparatus communicates with the separate maternal chromatin, forming a half spindle which moves the egg-derived chromatin towards its pole. At the time for cell division, the individual karyomeres are not able to fuse their nuclear membranes to reconstitute the blastomere nuclei. When DTT is applied at prometaphase of the first cell cycle, the chromosome cycle continues until next metaphase. Centrosomes also continue their cycle and undergo somewhat atypical splitting during the time for second telophase. Division furrows are initiated but aborted. These results support the hypothesis that disulfide groups are required for membrane fusion of the pronuclei, for membrane fusion of the karyomeres, and for the completion of the division furrow to achieve successful cell division. © 1994 Wiley-Liss, Inc.
    Additional Material: 2 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270107
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  • 12
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    Radioiodination studies of the envelopes from Xenopus laevis eggs (1983)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 22 (1983), S. 235-244 
    Details
    ISSN: 0730-2312
    Keywords: fertilization ; egg envelopes ; glycoproteins ; molecular topography ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: To investigate the molecular basis of the observed morphological and biological characteristics of coelomic egg envelopes (CE), vitelline envelopes (VE), and fertilization envelopes (FE) of Xenopus laevis eggs, envelopes were radioiodinated under a variety of conditions: in situ, isolated and intact, or solubilized. The distribution of 125I in envelope components was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each envelope type displayed unique profiles when iodinated in the intact state. A major constituent of VE, the 41,500 molecular weight component, was not labeled in the intact state, although the corresponding component of CE was heavily labeled. After dissociation of the envelope by guanidine-HCl or sodium dodecyl sulfate, all of the components could be radioiodinated. However, when the envelopes (VE and FE) were dissolved by heating and subsequently radioiodinated by lactoperoxidase, the resulting radioactivity profile was similar to that of the intact envelopes, suggesting that in the heat-dissolved envelope, the individual components retain similar structural relations as in the intact envelope. Quantitative but not qualitative differences were found between the inner and outer aspects of VE and FE. The significance of these findings is discussed in relation to what is known about the morphological, biological, and molecular properties of the envelopes.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/jcb.240220405
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  • 13
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    Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins (1991)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 47 (1991), S. 1-10 
    Details
    ISSN: 0730-2312
    Keywords: chromatin ; chromatin remodeling ; fertilization ; embriogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western imunoblot analysis with policlonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/jcb.240470102
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  • 14
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    An immunocytochemical localization of the cortical granule lectin in fertilized and unfertilized eggs of xenopus laevis (1978)
    New York, NY : Wiley-Blackwell
    Gamete Research 1 (1978), S. 13-18 
    Details
    ISSN: 0148-7280
    Keywords: immunoperoxidase localization ; fertilization ; Xenopus laevis ; cortical reaction ; block to polyspermy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: At fertilization, the vitelline envelope surrounding the egg of Xenopus laevis is modified by the addition of an electron-dense component termed the “F layer.” The F layer functions as a block to polyspermy and as a block to the escape of macromolecules from the perivitelline space, thereby causing an osmotically driven envelope elevation. F-layer formation has been hypothesized to result from interaction between a cortical-granule lectin, released in the cortical reaction, and a jelly-coat ligand. Evidence for this hypothesis was sought by determining the location of the cortical-granule lectin both before and after fertilization, using a specific antibody conjugated to horseradish peroxidase. The cortical-granule lectin was localized only in the cortical granules of the unfertilized egg and was located predominantly in the perivitelline space and the F layer of a fertilized egg. These observations support the hypothesis that the F layer is formed by a cortical-granule-Iectin-jelly layer-ligand interaction.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/mrd.1120010104
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  • 15
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    A new procedure for rapidly scoring acrosome reactions of human sperm (1980)
    New York, NY : Wiley-Blackwell
    Gamete Research 3 (1980), S. 211-216 
    Details
    ISSN: 0148-7280
    Keywords: acrosome ; human sperm ; lectin ; capacitation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Because the acrosome of human sperm is too small to be directly visualized by phase-contrast microscopy, acrosome reactions (that is loss of the acrosome) are generally not evaluated in studies of human sperm capacitation and fertilization. Nevertheless, it would be useful in such studies to have a technique for easily identifying and quantitating acrosome-reacted sperm. In this paper, we describe a method for labeling the human sperm acrosome with fluorescein-conjugated Ricinus communis agglutinin-60 (FITC-RCA); we show that in sperm without acrosomal caps, FITC-RCA labeling occurs either not at all or only in the equatorial segment of the acrosome. To determine if the absence of FITC-RCA labeling in the acrosomal cap region gives a reliable estimate of acrosome reactions, washed sperm or sperm incubated in a capacitating medium (BWW) were divided into two groups, which were then fixed for FITC-RCA labeling or transmission electron microscopy. Counts of acrosome reactions made by each method were similar, and we observed an increase in the percentage of reactions following incubation in BWW. We conclude that the FITC-TCA labeling technique is a reliable method for accurately scoring the percentage of acrosome-reacted human sperm.
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    URL: http://dx.doi.org/10.1002/mrd.1120030303
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  • 16
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    Properties of the first and second factors released after hamster sperm make contact with the zona pellucida prior to fertilization in vitro (1980)
    New York, NY : Wiley-Blackwell
    Gamete Research 3 (1980), S. 395-403 
    Details
    ISSN: 0148-7280
    Keywords: sperm-zona contact ; fertilization ; peptides ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An investigation was made as to the nature of two of the factors, termed S1, released within the first 30 minutes after contact is made between capacitated hamster sperm and the zona pellucida in vitro. Previous studies showed that these S1 factors were detected two and 20 to 25 minutes after the gametes were combined and that, based on filtration studies, the former possessed a molecular weight of less than 5,000 daltons. The present results show that the quantity of the 20-25-minute S1 factor released into the supernatant increased linearly as a function of the sperm concentration. This factor passed unimpeded through a filter with a 5,000 molecular weight cutoff but only 42% of the activity traversed a filter with a cutoff of 2,000 daltons. The two-minute S1 factor, in the virtual total absence of cells, was stable for 10 to 15 minutes, but lost significant activity upon longer incubation. Under the same conditions, the 20-25-minute factor lost approximately 25% of its activity within 15 minutes, but remained stable at this level for at least 45 minutes of incubation. Both S1 factors were not affected by a mixture of glycosidases, but were inactivated by subtilisin, trypsin, and leucine aminopeptidase which was contaminated with endopeptidases. The activity of the two-minute S1 factor appeared more susceptible to the action of the proteases than that of the 20-25-minute S1 factor. In contrast to previous results obtained with the two-minute S1 factor, the release of the 20-25-minute S1 factor was not inhibited by the inclusion of soybean trypsin inhibitor a t concentrations which are known to inhibit penetration of the zona by the sperm. The results suggest that the two- and 20-25-minute S1 factors are peptides which are not identical.
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    URL: http://dx.doi.org/10.1002/mrd.1120030410
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    Artificial fertilization of gametes from the South African clawed frog, Xenopus laevis (1980)
    New York, NY : Wiley-Blackwell
    Gamete Research 3 (1980), S. 45-57 
    Details
    ISSN: 0148-7280
    Keywords: clawed frog ; egg ; fertilization ; jelly coat ; motility ; sperm ; Xenopus laevis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A reproducible and effective method for fertilization eggs of Xenopus laevis was developed based of systematic manipulation of environmental factors. The effects of varying concentrations of individual components of a fertilization medium were tested by measuring jelly swelling, sperm motility, and sperm longevity. Results were used to develop an improved medium for fertilization, consisting of 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl2, 0.0625 mM MgCl2, 0.5 mM Na2HPO4, 2.5 mM HEPES, 1.9 mM NaOH, final pH(2°) 7.8.
    Additional Material: 12 Ill.
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    URL: http://dx.doi.org/10.1002/mrd.1120030106
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    Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction (1981)
    New York, NY : Wiley-Blackwell
    Gamete Research 4 (1981), S. 253-273 
    Details
    ISSN: 0148-7280
    Keywords: capacitation ; acrosome reaction ; fertilization ; lysophospholipids ; spermatozoa ; membrane fusion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of lipids on the survival, acrosome reaction, and fertilizing capacity of guinea pig spermatozoa were studied by incubating the spermatozoa in media containing various concentrations of the lipids. Lipids tested were: phosphatidyl-choline (PC), -ethanolamine (PE), -inositol (PI), -serine (PS), sphingomyelin (S), cholesterol (C), lysophosphatidyl-choline (LC), -ethanolamine (LE), -inositol (LI), -serine (LS), and glyceryl monooleate (M).When spermatozoa were incubated in a regular medium (containing 2 mM Ca2+) with M, the majority underwent the acrosome reaction within 1 hour. None of the other lipids were as effective as M, and some were totally ineffective under the same conditions. However, when spermatozoa were preincubated in Ca2+-free medium containing LC, LE, or LI, they gained the ability to undergo the acrosome reaction. One hour of preincubation in Ca2+-free medium with LC, LE, or LI was enough to render the vast majority of spermatozoa capable of undergoing the acrosome reaction in response to Ca2+. The optimum concentrations for LC, LE, and LI were approximately 85 μg/ml, 210 μg/ml, and 140 μg/ml, respectively. Spermatozoa that had undergone the acrosome reaction by pretreatment with LC, LE, or LI remained actively motile and were capable of fertilizing eggs. LS was totally ineffective in rendering the spermatozoa capable of undergoing the acrosome reaction, and in fact it inhibited the acrosome reaction by itself and also inhibited the LC-, LE-, or LI-mediated acrosome reaction. LS did not prevent acrosome-reacted spermatozoa from penetrating the zona pellucida, but did prevent sperm-egg fusion.Based on these findings, it is suggested that lysophospholipids are intricately involved in the sperm acrosome reaction and perhaps in sperm-egg fusion.
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    URL: http://dx.doi.org/10.1002/mrd.1120040402
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    Stimulation of endogenous protein phosphorylation in oocytes of Sabellaria alveolata (polychaete annelid) at meiosis reinitiation induced by protease, fertilization, or ionophore A 23187 (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 115-123 
    Details
    ISSN: 0148-7280
    Keywords: meiosis ; protein kinase ; protease ; fertilization ; activation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Incorporation of [32P]-phosphate into proteins was enhanced when Sabellaria oocytes were stimulated with specific protease to continue from prophase I block to metaphase I block. The rate of incorporation was increased 50 fold between onset of treatment and germinal vesicle breakdown (GVB). The same result was obtained when release from prophase block involved fertilization, or activation with ionophore A 23187. In all cases, meiosis was associated with phosphorylation of an 18,000 dalton protein, which is perhaps not labeled in prophase-blocked oocytes. Phosphorylation of a 38,000-40,000 dalton doublet of membrane proteins, which are among the main phosphorylated proteins in intact oocytes, was also strongly enhanced in vitro in homogenates prepared from oocytes following release from prophase block.
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    Fertilization of cumulus-free, zona-intact mouse ova in vitro at high and low sperm concentrations (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 61-70 
    Details
    ISSN: 0148-7280
    Keywords: mouse ; spermatozoa ; concentration ; fertilization ; ova ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of varying the sperm concentration between 2 × 105 sperm/ml and 8 × 106 sperm/ml on fertilization of cumulus-free, zona-intact F1 (CBA × C57BL) mouse ova by QS and F1 (CBA × C57BL) mouse spermatozoa was studied. The spermatozoa from both strains of mice exhibited optimal fertilization rates at 2 × 106 sperm/ml. However, at sperm concentrations greater than 4 × 106 sperm/ml and less than 1 × 106 sperm/ml, fertilization rates were significantly reduced. F1 spermatozoa were more susceptible to dilution than QS spermatozoa. A significant interaction between strain and sperm concentration indicated that the two strains produced different fertilization rates at different sperm densities.Extracts of epididymal fluid, medium from capacitated spermatozoa, or ampulla fluid did not improve the fertilization rate at 2 × 105 sperm/ml, but retaining the cumulus oophorus did. The decrease in fertilization rate at 8 × 106 sperm/ml can in part be attributed to a nondialysable inhibitor from the neat sperm preparation that appeared to be of epididymal origin.
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    URL: http://dx.doi.org/10.1002/mrd.1120050107
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    Changes in the stainability and sulfhydryl level in the sperm nucleus during sperm-oocyte interaction in mice (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 167-179 
    Details
    ISSN: 0148-7280
    Keywords: sulfhydryl ; Giemsa stain ; mouse sperm ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: During maturation in the epididymis, mouse sperm nuclei become difficult to stain with Giemsa and its component basic dyes. Mature sperm from the cauda epididymis can be stained only after DTT treatment. Stainable sperm such as those from the testis accumulate 3H NEM when examined by autoradiography, while unstainable sperm do not, indicating a close correlation between the basic dye binding capacity and SH levels in the sperm nuclei. During insemination of zonafree ovarian oocytes with a germinal vesicle (GV), mature sperm nuclei become stainable and capable of binding with 3H NEM. At the same time, sperm have established pronase-resistant contact with the oocyte. Similarly, sperm nuclei become stainable during fertilization when the sperm attachment to the egg becomes pronase resistant. However, these changes occur before sperm chromatin decondensation begins. Therefore, it is suggested that S-S bonds in sperm nucleoproteins are reduced when the sperm establish a stable contact with the egg plasma membrane, thus reversing sperm maturational changes. The reduction of S-S bonds may be a prerequisite for sperm chromatin decondensation.
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    URL: http://dx.doi.org/10.1002/mrd.1120050207
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    Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin, Lytechinus variegatus (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 363-377 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; sea urchin ; bioelectric response ; secretion ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation.These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50-400 msec), but minor (≅10 mV), electrical transient that leads to an action potential and then both the Na+-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/mrd.1120050407
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    Architecture of the hamster oocyte-cumulus complex (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 9 (1984), S. 261-272 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; cumulus ; oocyte ; sperm ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The structure of the hamster oocyte-cumulus complex (OCC) has been analyzed to help understand how mammalian sperm penetrate the investing coats of the oocyte. At ovulation, oocytes of most mammalian species are surrounded by a zona pellucida, corona radiata, and cumulus layer. Cells of the cumulus and corona radiata are separated by an extracellular matrix (ECM) that contains hyaluronic acid. In hamsters, the diameter of mature follicular OCCs is 0.61 ± 0.12 mm, whereas in freshly ovulated OCCs in culture medium it is 0.78 ± 0.15 mm. This indicates the OCC expands at or after ovulation. The corona radiata is 1-4 cell layers deep, and corona radiata cells are closely packed (center-to-center distances between adjacent cells in follicular OCCs averaged 14 ± 3 μm). The cumulus layer is 5-8 cell layers deep, and intercellular spaces are much larger (center-to-center distances between adjacent cumulus cells in follicular OCCs averaged 50 ± 20 μm). An ovulated OCC has an approximate volume of 248 × 106 μm3 and was estimated to contain approximately 5,700 cells. Studies with stretched OCCs show that ink particles can readily penetrate the extracellular spaces of the cumulus and corona radiata layers and interact with the ECM, staining it black. At the periphery of the OCC, the ECM appeared discontinuous and formed “cords” containing cumulus cells, whereas closer to the corona radiata the matrix completely filled intercellular spaces. Ink penetrated the corona radiata, but the ECM was difficult to visualize because of the close packing of cells in this layer. Our observations on unfixed OCCs show that cell packing becomes more dense proceeding from the periphery of the OCC to the zona pellucida and that the ECM at the periphery differs from the matrix deeper in the OCC. These data suggest that an incoming sperm would find penetration of the investing coats to increase in difficulty (that is physical resistance would increase) as it got closer to the oocyte. We have also examined the effects of processing for microscopy on the structure of the OCC. Both standard fixation procedures and fixation in the presence of ruthenium red caused condensation of the ECM, especially in the cumulus layer, and thus produced a significant decrease in the diameter of the OCC.
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    URL: http://dx.doi.org/10.1002/mrd.1120090304
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    Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs (1983)
    New York, NY : Wiley-Blackwell
    Gamete Research 8 (1983), S. 65-78 
    Details
    ISSN: 0148-7280
    Keywords: sea urchin ; ammonia-activation ; fertilization ; fertilization cone ; sperm asters ; pronuclear development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated 3H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, 3H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.
    Additional Material: 19 Ill.
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    URL: http://dx.doi.org/10.1002/mrd.1120080108
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    Ultrastructural aspects of in vivo fertilization in the cow (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 10 (1984), S. 241-251 
    Details
    ISSN: 0148-7280
    Keywords: ultrastructure ; fertilization ; cow ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In vivo fertilization of cow eggs has been studied by electron microscopy. Eggs were recovered from intracervically inseminated heifers 30 to 42 hr after the onset of oestrus. The corona cells remained attached to 4 out of the 15 eggs studied, but no sign of sperm phagocytosis was noted.Spermatozoa close to the zona pellucida, but not in contact with it, were not acrosome reacted. In contrast, all sperm penetrating the zona pellucida had completed the acrosome reaction. Vesiculated products of the reaction were present at the zona surface of every penetrated egg, indicating that in this species, the acrosome reaction occurs at the surface of the zona pellucida.During sperm passage through the zona pellucida, the equatorial segment overlaid by its plasma membrane remained intact.Soon after penetration into the ooplasm, the sperm nucleus decondensed; at the same time, the female chromosomes resulting from the second meiotic division aggregated in a few masses of condensed chromatin. A nuclear envelope started to form around the condensed female chromatin, while it was not yet present around the decondensing male nucleus.After swelling, the two pronuclei presented similar ultrastructural morphology; they contained small, compact, agranular nucleoli with a large fibrillar center and unevenly distributed chromatin. The pronuclear envelope contained pores and presented characteristic blebbing. The endoplasmic reticulum was closely apposed to the nuclear envelope and large Golgi structures were proximal to the pronuclei.
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    Gametes and fertilization in the Chinese hamster (1983)
    New York, NY : Wiley-Blackwell
    Gamete Research 8 (1983), S. 97-117 
    Details
    ISSN: 0148-7280
    Keywords: egg ; soernatozoa ; fertilization ; Chinese hamster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Freshly ovulated eggs are each surrounded by a compact cumulus oophorus. The overall diameter of the normal egg (including the zona pellucida) is about 100 μm. Cumulus cells, particularly those near the egg, are arranged redially in a viscous noncellular matrix. The spermatozoon is about 250 μm in length. The head a large acrosome, changes in which can be readily examined with the light (phase- contrast) microsope. When exposed to physiological salt solutions, testicular spermatozoa either were motionless or flexed the posterior half of their tails slowly. Spermatozoa from the caput epididymis were highly motile, flexing the entire tail. A few of them moved progressively. Mature spermatozoa from the vas deferens were highly motile and moved either straightforward or in a circle. They vibrated their tails stiffly without flexing them.In normally mated females, fertilization began sometime between 2 and 3 h after ovulation and was completed within the next 4 to 5 h. Spermatozoa swimming in the ampullary fluid or within the cumulus oophorus about the time of fertilization flexed the anterior half (which roughly corresponds to the midpieac region) of their tails. This peculiar movement may be homologous to the so-called “hyperactivation” of spermatozoa as reported in several other mammalian species. Actively motile spermatozoa within the cumulus or no the zona pellucida had either modified (“collapsed”) or no acrosomal caps. The sperm head usually passed verticually or nearly through the zona, but the path was oblique in some instances. In 54% of the recently fertilized eggs examined, the entire length of the sperm tail was within the perivitelline space; in the other 46% of the eggs varying lenghts of the tail remined the perivitelline space, the tails were extruded from the vitellus of many eggs even before the eggs began their first cleavage.When unfertilized eggs in the cumulus oophorus were inseminated with vas deferens spermatozoa in a modified Tyrode's solution (m-TALP), about 80% of them were ferrtilized by 4-6 h after insemination. The vast majority were monospermic. When eggs were freed from the cumulus prior to insemination, none were fertilized, suggesting that the cumulus cells or their matrix assisted capacitation and/or the acrosome reaction of the spermatozoa under the in vitro conditions employed. No eggs were fertilized by the testicular or caput epididymal spermatozoa regardless of the presence or absence of cumulus oophorus around the eggs at the time of insemination.
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    Induction of polyspermic fertilization in sea urchins by the leukotriene antagonist FPL-55712 and the 5-lipoxygenase inhibitor BW755C (1985)
    New York, NY : Wiley-Blackwell
    Gamete Research 11 (1985), S. 41-50 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; polyspermy ; sea urchin eggs ; leukotrienes ; arachidonic acid ; FPL-55712 ; BW-755C ; 5-lipoxygenase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Arachidonic acid can be oxidized via the cyclooxygenase pathway to produce prostaglandins and via the 5-lipoxygenase pathway to produce leukotrienes. These substances are known to be extremely potent regulators of cellular function in somatic tissues, particularly during inflammatory reactions. Recent studies have implicated cyclooxygenase-derived products in preventing polyspermy in sea urchins [Schuel et al, Gamete Res 10:9-19, 1984]. FPL-55712, a receptor antagonist for leukotrienes in somatic tissues, causes a dose-(1-10 μM) and sperm-density-dependent induction of polyspermic fertilization in sea urchins if added before the egg completes the cortical reaction (elevation of the fertilization envelope). Eggs pretreated with FPL-55712 become polyspermic upon subsequent insemination with untreated sperm in sea water. Sperm pretreated with the drug do not cause polyspermy when used to inseminate untreated eggs. The 5-lipoxygenase inhibitor BW775C also promotes polyspermy. FPL-55712 and BW755C do not retard elevation of the fertilization envelope. These findings imply that (1) leukotrienes may be produced via the 5-lipoxygenase pathway during fertilization in sea urchins, and (2) the reaction of leukotrienes with putative receptors on the egg's surface may modulate its receptivity to sperm during the cortical reaction, and thereby help prevent polyspermy.
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    Effects of surfactants on the fertilizing capacity and acrosome reaction of sea urchin spermatozoa (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 9 (1984), S. 115-125 
    Details
    ISSN: 0148-7280
    Keywords: acrosome reaction ; fertilization ; sea urchin ; spermatozoa ; surfactants ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of seven surfactants on spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, were studied. All these surfactants induced the acrosome reaction and inhibited the fertilizing capacity of spermatozoa. There was a statistically significant correlation between the concentrations that induce the acrosome reaction and inhibit fertilization. The critical micelle concentrations (CMC) of surfactants in sea water were almost even and these values, which are inherent physical properties of surfactants, did not provide a direct measure of their inhibitory effect of fertilization. Among seven surfactants, p-menthanyl-phenol polyoxyethylene (8.8) ether (TS-88) with a characteristic hydrophobes was the most potent both in the induction of acrosome reaction and in the inhibition of fertilization. Various ethylene oxide adducts to p-menthanyl-phenol were also tested for the purpose of comparison. It is suggested that the effects of surfactants on sea urchin spermatozoa at low concentrations reflect their activity associated with the hydrophobic group inherent in each surfactant.
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    Evidence of phospholipase A2 in the sea urchin egg: Its possible involvement in the cortical granule reaction (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 9 (1984), S. 329-338 
    Details
    ISSN: 0148-7280
    Keywords: cortical granule reaction ; phospholipase A2 ; quinacrine ; sea urchin ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Fertilization of the sea urchin egg involves an exocytotic event known as the cortical granule reaction (CGR). In many cell systems, phospholipase A2 is implicated in regulation of the secretory event. Indirect evidence suggests that phospholipase A2 mediates the CGR; however, there has been no direct demonstration of phospholipase A2 activity in the sea urchin egg. We report here evidence of phospholipase A2 activity in egg homogenate of the sea urchin Lytechinus pictus. The enzyme was calcium-dependent and had a pH optimum near the intracellular pH of the unfertilized egg. Neither exogenous calmodulin nor trifluoperazine had any apparent effect on enzyme activity. Quinacrine, a phospholipase A2 inhibitor, blocked the enzyme activity in the egg homogenate. In intact eggs, quinacrine blocked the CGR in a dose-dependent, egg-concentration-dependent manner. The inhibitory effect of quinacrine on the CGR could not be overcome by the phospholipase A2 activator melittin or by the calcium ionophore A23187. Quinacrine did not inhibit sperm-egg binding or sperm incorporation. These results lend further support to the hypothesis that phospholipase A2 is involved in the CGR.
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    URL: http://dx.doi.org/10.1002/mrd.1120090308
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    Developmental capacity of rat embryos produced by in vivo or in vitro fertilization (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 10 (1984), S. 77-82 
    Details
    ISSN: 0148-7280
    Keywords: rat ; fertilization ; development ; in vivo ; in vitro ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This work compares the ability of rat zygotes fertilized in vitro or in vivo to develop into viable embryos. All oocytes were from adult cyclic females. After the first cleavage, the zygotes were transferred to oviducts of pseudopregnant recipients. Their fate was examined on day 13 at laparotomy and again on day 20. Ninety-five of 146 in vivo fertilized zygotes developed into normal sized 13-day fetuses and 72 (55%) to apparently normal near-term fetuses. Forty-six of 135 in vitro fertilized zygotes developed up to day 13, and 30 (24%) developed to term. It appears that the probability that in vitro fertilized rat zygotes will develop into viable embryos is about half the chance of in vivo fertilized zygotes. Since the two types of zygotes were morphologically identical, the morphological appearance of the two-cell stage is not an adequate criterion for judging developmental potential.
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    URL: http://dx.doi.org/10.1002/mrd.1120100109
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    The site of the acrosome reaction during in vivo penetration of the sheep oocyte (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 10 (1984), S. 97-105 
    Details
    ISSN: 0148-7280
    Keywords: ultrastructure ; fertilization ; sheep ; oocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In vivo fertilization of sheep eggs has been studied by electron microscopy. Remnants of the acrosome reaction were present at the zona surface of every penetrated egg, indicating that the acrosome reaction in sheep occurs at the surface of the zona pellucida.To determine whether follicular oocytes could specifically bind spermatozoa, oocytes isolated from different size classes of antral follicles were transferred into the oviducts of mated ewes, recovered 4 hr 30 min later, and analyzed by electron microscopy. Oocytes from follicles up to 1 mm in diameter failed to bind spermatozoa and were not penetrated. In contrast, the zona of oocytes from follicles ≥ 2 mm in diameter induced the acrosome reaction. These oocytes were penetrated but failed to achieve cortical granule exocytosis and so to mount a block to polyspermy. Moreover, sperm nuclei incorporated into the ooplasm did not decondense although the sperm nuclear envelope was dispersed.
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    URL: http://dx.doi.org/10.1002/mrd.1120100202
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    Rat eggs normally exhibit a variety of surface phenomena during fertilization (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 10 (1984), S. 107-118 
    Details
    ISSN: 0148-7280
    Keywords: Rat ; fertilization ; in vivo ; in vitro ; topography ; egg ; actin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The surface topography of the rat egg was examined during fertilization in vitro and in vivo. Using phase optics, 348 in vitro fertilized and 50 in vivo fertilized eggs were continuously monitored throughout the 7-hour period of sperm incorporation. A myriad of different surface configurations were seen, with each egg exhibiting one or more of the following changes. A small number of eggs (4-6%) formed surface elevations over the sperm head after its detachment from the flagellum, 15-30 min after sperm-egg fusion; 1 to 1.5 hr after fusion, 40-50% of the eggs produced the so-called incorporation cone, a prominent surface elevation over the decondensing sperm nucleus. The vast majority of eggs (74-82%) formed surface elevations over the proximal tip of the flagellum 2-3 hr after sperm-egg fusion. These had no association with the decondensing sperm nucleus. A few eggs (11-12%) exhibited multiple protrusions that were distributed randomly about the egg surface, whereas 14-20% did not manifest any surface elevations and remained spherical throughout the sperm incorporation period. Regardless of the type of surface change, all of the eggs resumed a spherical shape by the time sperm incorporation was complete. These observations are in contrast to the conclusions by previous authors that formation of the so-called incorporation cone over the decondensing sperm nucleus is a ubiquitous event.
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    Influence of the concentration of sperm on the percentage of eggs fertilized for three strains of mice (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 10 (1984), S. 415-422 
    Details
    ISSN: 0148-7280
    Keywords: mouse ; fertility ; fertilization ; sperm ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This study was conducted to determine the optimal concentration of sperm to use for the insemination of females to detect differences among strains of mice in the percentage of eggs fertilized. Female ICR mice were inseminated with sperm of concentrations ranging from 0.25 to 8 × 106/50 μl from males of either DBA/2N, CF1, or C57BL/6N strains. Differences among strains were detected only when approximately 50% of the eggs were fertilized but not when each of the strains fertilized either a high or low percentage of eggs. The optimal concentration of sperm therefore was the concentration that gave approximately 50% fertilized eggs.
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    URL: http://dx.doi.org/10.1002/mrd.1120100407
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    An improved method for isolation of fertile zona-free mouse eggs (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 13 (1986), S. 213-222 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; zona-free egg ; chymotrypsin ; plasma membrane ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Studies of sperm-egg fusion using zona-free mouse eggs are impaired by the procedures used for removal of the zona pellucida. Methods involving proteolytic digestion or mechanical aspiration through micropipettes are limited in that proteases can adversely affect fertility and mechanical removal often results in low egg yields. An efficient procedure for preparation of zona-free mouse eggs was developed using a combined enzymatic (chymotrypsin) mechanical approach (CT-M procedure). Zona-intact eggs, obtained after hyaluronidase treatment, were exposed to 0.001% α-chymotrypsin in medium containing 0.5% bovine serum albumin (BSA). Brief (2 minute) exposure to chymotrypsin under these conditions caused pronounced zona distention in a majority (80-90%) of the eggs, facilitating mechanical removal and resulting in a high yield of zona-free eggs. Eggs prepared by the CT-M method displayed identical penetration levels relative to mechanically denuded eggs. CT-M prepared eggs also showed sperm concentration dependent penetration levels and demonstrated a plasma membrane block to polyspermy, qualities previously observed in mechanically prepared eggs [Wolf DP, 1978, Dev Biol 64:1-10]. Eggs could be exposed to 0.001% CT for zona distention over a 2-10-minute time period with no detrimental effects on fertility. The effect of chymotrypsin was also studied by treating zona-free eggs for 30 minutes over a 1-1,000-μg/ml range of enzyme, and a concentration-dependent reduction in penetration levels was observed. These results indicate that the CT-M method is a useful procedure for the isolation of large numbers of zona-free mouse eggs.
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    The cortical reaction in pig oocytes during in vivo and in vitro fertilization (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 13 (1986), S. 241-251 
    Details
    ISSN: 0148-7280
    Keywords: oocyte ; fertilization ; cortical granule ; pig ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The structure of pig oocytes after in vivo and in vitro fertilization and following treatment with the ionophore A23187 with differing levels of calcium are described, with particular reference to the cortical granules.Fertilization in vivo and in vitro resulted in cortical granule exocytosis. Sperm penetration in vivo was more rapid than in vitro and resulted in the dispersal of the cortical granules' contents in the perivitelline space following exocytosis. The contents of the granules remained adjacent to the plasmalemma after in vitro fertilization and suboolemmar vesicles were less numerous than after in vivo fertilization. High calcium levels were necessary to induce the dispersal of the cortical granule contents following treatment with ionophore. The observations are discussed regarding their relevance to the blockage to polyspermy.
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    An in vitro technique to study penetration of hamster oocyte-cumulus complexes by using physiological numbers of sperm (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 13 (1986), S. 293-308 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; oocyte cumulus complex ; acrosome ; hyperactivation of motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have developed an inexpensive in vitro system for studying cumulus penetration and fertilization by using physiological numbers of sperm. This system simulates conditions believed to exist in vivo more closely than any in current usage. In this system, 1-100 hamster sperm are used to challenge fresh hamster oocyte-cumulus complexes (OCC). Only fresh (nonoviducal) OCC are used, as they present the most stringent challenge to sperm. Because sperm numbers are low, OCC do not disperse, and sperm can be studied microscopically during penetration of the cumulus oophorus and corona radiata. These conditions permit microscopic assessment of the sperm acrosome. Video tapes of experiments allow easy review and analysis of experiments. Results obtained employing this technique show that, in vitro, (1) capacitated, acrosome-intact hamster sperm can penetrate the extracellular matrix between cumulus cells and bind to the zona pellucida; (2) the “figure-eight motility” characteristic of hyperactivated hamster sperm swimming in culture medium is suppressed when sperm swim in the extracellular matrix between cumulus cells; and (3) fertilization occurs in capillary tubes when low numbers of sperm are used. The in vitro system that we have described will be useful in analyzing the mechanisms used by sperm to penetrate the cumulus and corona radiata and to clarify the role of the acrosomal enzymes in fertilization.
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    URL: http://dx.doi.org/10.1002/mrd.1120130404
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    Artificial insemination of the American lobster (Homarus) (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 14 (1986), S. 25-31 
    Details
    ISSN: 0148-7280
    Keywords: artificial insemination ; crustaceans ; lobsters ; fertilization ; spermatophore ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A technique has been developed for artificially inseminating freshly molted female lobsters (Homarus). Using the criterion that eggs were fertilized if they showed development of normal cleavage patterns, at least 49.5% of the inseminated females extruded fertilized eggs. Sixty-two percent of the fertilized eggs developed to the eyespot stage, and 26.6% of these subsequently hatched. Eggs were not fertilized in 7.7% of the extrusions. In 42% of the samples, it was not possible to establish whether fertilization had occurred because either samples were collected prior to initiation of cleavage or eggs were too poorly preserved to assess cleavage with confidence. It is probable that many of the eggs that were evaluated as equivocal were also fertilized. This technique should be valuable in the experimental and commercial aquaculture of lobsters and might be directly applicable to other crustaceans.
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    Changes in elemental composition of fertilized and unfertilized northern pike (Esox lucius) eggs incubated in buffer with and without dimethyl sulfoxide: An X-ray microanalysis study (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 14 (1986), S. 91-106 
    Details
    ISSN: 0148-7280
    Keywords: fish ; eggs ; fertilization ; incubation ; dimethyl sulfoxide ; cryoprotectant ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Fertilized and unfertilized eggs from the northern pike (Esox lucius) were incubated 2 hr in buffer with 0 and 10% (v/v) dimethyl sulfoxide and then quickly frozen in the wells of aluminum blocks submerged in liquid nitrogen. Control eggs and ovarian fluid were similarly frozen immediately after collection. The frozen eggs were sectioned, freeze dried, mounted on stubs, and carbon coated. X-ray microanalysis was used to determine changes in element levels and dimethyl sulfoxide (Me2SO) penetration in the zona radiata, cytoplasm, cortical alveoli, and egg yolk. Unfertilized eggs incubated without Me2SO showed decreased levels of Na, Cl, and K in the zona radiata; fertilized eggs, incubated without Me2SO showed decreased levels of Na, P, and Cl in the zona radiata and increased levels of K in the cytoplasm; unfertilized eggs, incubated with 10% Me2SO showed decreased Na and Cl in the zona radiata, decreased K in the cytoplasm and increased K in the cortical alveoli; fertilized eggs incubated in buffer with 10% Me2SO showed decreased levels of Na, P, Cl, and K (zona radiata), P, Cl, and K (cytoplasm), Na (yolk), and increased Cl in the yolk (all P〈.01). Me2SO (v/v) levels reached 1.5-3.1% in the zona radiata, 0-3.2% in cytoplasm, 2.3-8.7% in cortical alveoli, and 0-1.6% in the yolk. Unfertilized eggs showed more Me2SO penetration than fertilized eggs.
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    Sperm-egg interactions in the shrimp Rhynchocinetes typus (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 14 (1986), S. 171-180 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; Crustacea ; spermatozoon ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sperm-egg interaction in Rhynchocinetes typus was studied with the phase-contrast and scanning electron microscopes. R typus spermatozoa present in the vas deferens have the shape of a round-headed nail. After contact with seawater it is possible to observe the unfolding of the rays or stellate arms, giving the spermatozoon the appearance of an inverted umbrella. From the center of the flat face of the umbrella emerges a spike with longitudinal striations. Ovarian eggs and spermatozoa were mixed in vitro by agitating them for two minutes in Millipore-filtered seawater. The first gamete contact was established by the spermatozoon through the tip of the spike, which exerted a lytic action on the egg envelopes. After the rigid spike was completely inside the egg, the rays became aligned parallel to each other and began to enter the eggs. Toward the final stages of ray entry, it was possible to observe fusion of the ray membranes with one another, and later the fusion process continued toward the tip of the radial spines. Concomitantly, the egg surface that surrounds the sperm swelled in a circular fashion and formed a fertilization cone. After the spermatozoon entry was complete, a scarlike mark appeared at the place on the egg surface through which penetration occurred. The whole penetration process was completed within 45-60 minutes.
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    Difference of fertilizing capacity between testicular sperm and vas deferens sperm in Cynops pyrrhogaster (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 14 (1986), S. 209-214 
    Details
    ISSN: 0148-7280
    Keywords: amphibia ; sperm maturation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The fertilizing capacity was compared between testicular and vas deferens sperm in Cynops pyrrhogaster. The testicular sperm was not capable of fertilizing jelly eggs. In contrast, the vas deferens sperm was already capable of fertilizing the newt jelly eggs. There was no inhibitory factor for fertilizing jelly eggs in the testis. These results suggest that the testicular sperm is immature as to the fertilizing capacity. The testicular sperm gained the fertilizing capacity for the jelly eggs by treatment with Holtfreter's solution or 1/20 strength Holtfreter's solution. The treatment may promote the step of maturation to achieve the fertilizing capacity. The treated testicular sperm did not fertilize dejellied eggs, although vas deferens sperm fertilized dejellied eggs. Therefore, the maturation state of the treated testicular sperm is different from that of vas deferens sperm. Newt sperm may be matured within the vas deferens, as the newt does not have an organ like the mammalian epididymis.
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    Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova:Relevance to sperm nucleus transformation in the fertilized egg (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 14 (1986), S. 265-275 
    Details
    ISSN: 0148-7280
    Keywords: buthionine sulfoximine ; ovary ; ovum ; fertilization ; sperm nucleus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Glutathione (GSH), the major low-molecular-weight thiol in mammalian cells, is believed to be a necessary factor for the transformation of the disulfide-stabilized sperm nucleus into the male pronucleus after fertilization. Its concentration in mouse ova, isolated from the ampulla of the oviduct after hormone-induced superovulation of 3-4-week-old mice, has been determined by an enzymic cycling microassay. The level found was 1.80 pmol per ovum. Mean ovum diameter was estimated as 71-72 μm, indicating a GSH concentration of 9-10 mM in the mouse egg. Administration of L-buthionine S, R-sulfoximine, an inhibitor of GSH biosynthesis, during the 2 days preceding ovulation, reduced ovum GSH content below 0.20 pmol (〈1.0 mM). The mean GSH concentration of the hormone-stimulated ovaries was reduced from 3.2 mM to 0.2 mM under these conditions.It has also been demonstrated that measurement and manipulation of ovum and ovarian levels of GSH can aid in studying its function in ovaries, ova, and early embryos. Hormone-induced superovulation was achieved in BSO-treated prepuberal C57B1/6 X SJL mice whose ovaries contained less than 10% of control levels of GSH. Over 50% of the isolated ova were fertilized in vitro. However, abnormal one-cell embryos resulted in which the maternally derived pronucleus coexisted with an unchanged sperm nucleus, thus confirming that adequate levels of GSH are necessary for initiating transformation of the fertilizing sperm nucleus.
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    Protamine in the rat, its fate in vivo, and its degradation in vitro by egg homogenate (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 14 (1986), S. 293-306 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; proteolysis ; nuclear proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Spermatogenesis in the male rat involves chemical and physical transformation of chromatin. There is a shift in the chemical composition of rat testicular sperm chromatin containing histones to nucleoprotamines in the mature sperm. Rat protamine contains a high percentage of arginine. The biosynthesis and kinetics of H3-arginine incorporation into rat sperm protamine was at its peak in the testis 2 days after intratesticular injection. The specific activity of protamine in the testis was reduced to 50% 5 days after injection. Specific activity of protamine in the epididymis was maximal at 9 days after injection and remained at a similar level for 12 days. The kinetics of H3-arginine and H3-thymidine in ejaculated sperm were compared. It was found that maximal specific activity of H3-arginine (H3-Arg/No. of sperm) in the spermatozoa was obtained 20-22 days following its intratesticular injection. The peak of H3-thymidine specific activity (H3-thymidine/No. of sperm) was obtained 53-59 days following the injection.H3-Arg labeled sperm were capacitated in vitro and were allowed to penetrate zona free rat eggs in vitro. The disappearance of the label from the sperm head was observed 1-2 hr after penetration. Protamine labeled in vitro with I125 incubated with extracts of freshly ovulated rat eggs shows that protamine could be degraded by the cytoplasmic extract (10-30%). The proteolytic activity is maximal at pH 7.0-8.0. Cytoplasmic extracts from cumulus cells incubated with I125 rat protamine under identical conditions did not show any significant proteolytic activity. Trypsin degradation of rat protamine shows a different pattern of degradation. It is assumed that the H3-labeled protamine in the sperm chromatin is removed and probably degraded early after penetration, making possible the remodeling of the sperm chromatin by embryonic histones.
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    URL: http://dx.doi.org/10.1002/mrd.1120140403
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    Microfilaments during sea urchin fertilization: Fluorescence detection with rhodaminyl phalloidin (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 14 (1986), S. 277-291 
    Details
    ISSN: 0148-7280
    Keywords: rhodaminyl-labeled phalloidin ; cytochalasin E ; microfilament distribution ; fertilization ; cell division ; sea urchin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Rhodaminyl-labeled phalloidin is used to demonstrate the distribution of microfilaments during fertilization and early development in eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The surface of unfertilized eggs have numerous punctate fluorescence sites at which rhodaminyl phalloidin binds, indicating the presence of actin oligomers or polymers. During fertilization this punctate pattern of fluorescence begins to change. Within thirty seconds of insemination, the fertilization cone is first detectable with this technique as an erect structure on the surface of the egg. The fertilization cone grows to a maximum size by 8-9 minutes, and is resorbed by 16 minutes after insemination. The surface of the fertilized egg displays numerous fluorescent fibers by 10 minutes after insemination rather than the punctate fluorescence observed in unfertilized eggs, indicative of the burst of microfilament assembly resulting in microvillar elongation. The elongated microfilaments persist through cytokinesis. Staining is also detected throughout the cortices of unfertilized, fertilized, and cleaving eggs. Cytochalasin E (10 μM, 30 min) prevents microfilament elongation and cytokinesis and reduces the cortical staining intensity after fertilization. At cleavage, contractile rings, appearing as narrow equatorial bundles of fibers, have been detected in Lytechinus variegatus as transient structures.
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    The evolution of hamster sperm motility during capacitation and interaction with the ovum vestments in vitro (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 14 (1986), S. 333-346 
    Details
    ISSN: 0148-7280
    Keywords: spermatozoon ; motility ; capacitation ; hamster ; fertilization ; acrosome reaction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Movement characteristics of golden hamster spermatozoa were studied upon collection from the cauda epididymis, during an incubation which capacitates the spermatozoa in vitro, during penetration of the cumulus, and during attachment to and penetration of the zona pellucida. High-speed videomicrography was employed to quantitate flagellar beat frequency and shape. The status of the acrosome was also assessed. During capacitation, hamster spermatozoa become increasingly invigorated before the onset of hyperactivated motility. Within the cumulus, beat frequency and curvature are reduced, apparently in response to the physical resistive properties of the matrix material. These properties appear to vary within the cumulus. Initial attachment to the zona precedes completion of the acrosome reaction, is non-rigid, and is accompanied by increased beat frequency and curvature. Subsequently, the onset of rigid binding to the zona, completion of the acrosome reaction, and increased flagellar beat frequency are very closely associated in time. The latter produces an increase in thrust against the zona. Preliminary results indicate that ensuing zona penetration requires not more than five minutes, is at oblique angles, and is associated with a continuation of vigorous flagellar beating.
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    Follicular factors influence oocyte fertilizability by modulating the intercellular cooperation between cumulus cells and oocyte (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 21 (1988), S. 223-232 
    Details
    ISSN: 0148-7280
    Keywords: pig ; oocyte maturation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to investigate whether the follicular tissue influences cumulus-oocyte interaction and, consequently, the fertilizability of the egg, four experiments were carried out. In the first, cumulus-enclosed pig oocytes were cultured for 44 h in control medium (modified TCM-199) or in follicle-conditioned medium, and the intercellular coupling was studied by measuring 3H-uridine uptake. In control medium the intercellular cooperation started to decline immediately, and at 24-32 h the uncoupling was almost complete. By contrast, in follicle, conditioned medium, it remained at high levels until 24-32 h. In the second experiment protein synthesis patterns of oocytes were studied. Oocytes cultured in conditioned medium were characterized by a 45-kD protein band, while those maturing in control medium were identifiable by a marked 56-kD band. In the third experiment mature oocytes were fertilized in vitro. The percentage of penetrated egg was higher in oocytes matured in conditioned medium than in control medium. In addition, only oocytes matured in conditioned medium could consistently decondense spermatozoa and form male pronuclei. Metabolic cooperation, protein synthesis patterns, and fertilizability were also studied in oocytes matured in control medium supplemented with either 17β-estradiol or progesterone or testosterone or dihydrotestosterone or androstenedione or ether extract of conditioned medium. Only ether extract and progesterone stimulated cumulus oocyte interaction and sperm decondensation. In the last experiment oocytes denuded at different stage of their maturation in conditioned medium were fertilized in vitro. The longer the eggs were cultured with the cumulus, the higher was their penetrability. Moreover, only oocytes denuded after 40 h of culture could, once fertilized, promote the formation of male pronuclei.These data demonstrate that follicular secretions are fundamental for the maintenance in vitro of a functional intercellular coupling between cumulus and oocyte, which is necessary for the egg to become penetrable by spermatozoa and to acquire the conditions required for the formation of male pronuclei.
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    Capacitation potential of the fallopian tube: A study involving surgical insemination and the subsequent incidence of polyspermy (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 21 (1988), S. 255-266 
    Details
    ISSN: 0148-7280
    Keywords: pig ; fertilization ; vitellus ; chromatin aggregates ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In an attempt to demonstrate limitations in the capacitating potential of the Fallopian tube, ejaculated boar spermatozoa were introduced directly into the isthmus at varying intervals before ovulation. The incidence and degree of polyspermy subsequently observed were taken as indicators of the population of capacitated spermatozoa confronting the newly ovulated eggs: the more extensive the condition of polyspermy, the greater the number of capacitated spermatozoa presumed to have been available at the site of fertilization. Results are based on 673 eggs from 53 animals.When suspensions containing 2.21-3.87 × 108 sperm per ml were introduced 36-40 hours and 26-30 hours before ovulation, 85% and 61% respectively of the eggs were polyspermic, such eggs exhibiting mainly dispermy and trispermy. By contrast, when comparable sperm suspensions from the same boar were instilled 17-18 hours before ovulation, 70% of the eggs were polyspermic but the degree of polyspermy had increased dramatically: most eggs contained 40 or more sperm heads in the vitellus, invariably forming swollen chromatin aggregates rather than male pronuclei. Surgical insemination at times closer to ovulation significantly reduced the incidence and degree of polyspermy, reaching a low of 2% with insemination 1-2 hours before ovulation. These results therefore support the concept of a limited capacitation potential of the Fallopian tube.In a separate series of observations, mating animals shortly before surgical insemination with sperm suspensions from the same boar markedly reduced the incidence of polyspermy. This latter observation may be of clinical significance in procedures of laparoscopic or transcervical insemination into the tubes to alleviate human infertility. The manner whereby myosalpingeal physiology could be modified in response to coital stimulation is discussed.
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    Inhibition of adenosine-metabolizing enzymes modulates mouse sperm fertilizing ability: A changing role for endogenously generated adenosine during capacitation (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 21 (1988), S. 267-276 
    Details
    ISSN: 0148-7280
    Keywords: adenosine ; adenosine deaminase ; adenylate cyclase ; capacitation ; cyclic AMP ; fertilization ; 5′-nucleotidase ; spermatozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of inhibiting adenosine-metabolizing enzymes on sperm fertilizing ability was studied to investigate a possible role for endogenously generated adenosine in the regulation of capacitation. The compounds used have been shown to be effective inhibitors of the relevant enzymes in similarly incubated mouse sperm suspensions. Inhibition of 5′-nucleotidase activity with α,β-methylene adenosine 5′-diphosphate (AMPCP), to reduce available endogenous adenosine, caused a dose-dependent inhibition of the fertilizing ability of partially capacitated spermatozoa, which was significant with 100 and 250 μM AMPCP. Conversely, inhibition of adenosine deaminase with 100 nM coformycin, to increase available endogenous adenosine, promoted the fertilizing ability of partially capacitated spermatozoa when the fertilization rate of control suspensions was low. However, coformycin had no effect on sperm suspensions with moderate fertilizing ability, and it inhibited fertilizing ability when added to capacitated spermatozoa. These data are consistent with a promotion of the early stages of capacitation by endogenously generated adenosine and suggest that sensitivity to adenosine changes as capacitation proceeds. Because the majority of adenosine-metabolizing enzyme activity resides in or is directed toward the extracellular compartment in such suspensions, these effects of adenosine may be mediated at the outer surface of the cell. By interacting with receptors on adenylate cyclase, externally produced adenosine could modulate intracellular levels of cyclic adenosine monophosphate (cAMP), thereby influencing fertilizing ability.
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    Role for fucose-sulfate-rich carbohydrates in the penetration of zona-pellucida-free hamster eggs by hamster spermatozoa (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 21 (1988), S. 353-358 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; sperm-egg fusion ; cell-surface carbohydrates ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The interaction of acrosome-reacted hamster spermatozoa and zona-pellucida-free hamster eggs was investigated by incubating gametes in the presence of a variety of simple and complex carbohydrates. Significant inhibition of gamete fusion was achieved only in the presence of fucoidan and ascophyllin, two algal polysaccharides containing fucose sulfate. These compounds did not interfere with sperm motility, capacitation, or acrosome reactions. It is concluded that these two compounds share common structural features with putative cell-surface carbohydrates involved in sperm-oolemmal interaction.
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    URL: http://dx.doi.org/10.1002/mrd.1120210404
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    Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro, in relation to the timing of the acrosome reaction (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 15 (1986), S. 187-212 
    Details
    ISSN: 0148-7280
    Keywords: hyaluronidase ; capacitation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Hamster spermatozoa were tested for their ability to penetrate the intact cumulus matrix at low sperm:egg ratios (approximately 3:1). Uncapacitated spermatozoa attached to the surface of the cumulus and could not penetrate. Spermatozoa capacitated in vitro began to be able to penetrate after about 2 hr of preincubation, coincidentally with the first appearance of hyperactivation and spontaneous acrosome reactions. In all, 628 in vitro incubated spermatozoa were evaluated on and in cumuli: 270 could penetrate, but only ten of these were judged to have intact, “unmodified” acrosomes. Almost all spermatozoa capable of penetrating showed optically “modified” and swelling acrosomal caps, and this confirms previous observations on cumulus penetration in vivo. Penetration appeared limited to a phase in capacitation prior to completion of the acrosome reaction, as spermatozoa that had lost the acrosomal cap penetrated poorly and showed reduced viability. Penetration of the cumulus was inhibited by the hyaluronidase inhibitor sodium aurothiomalate. Cumulus penetrating ability could result either from a change in surface properties of the sperm at capacitation, which renders them less “sticky” to the matrix, or from release or activation of a “cumulus lysin.” We conclude that the ability to enter the cumulus matrix coincides with physiological changes in spermatozoa that occur during a terminal phase of capacitation preceding complete loss of the acrosomal cap, and that initiation of this process in vivo must precede sperm-zona contact.
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    Sea urchin egg-cortical granule protease has arginyl proteolytic specificity (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 15 (1986), S. 227-236 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; gamete enzymes ; reverse-phase high-performance liquid chromatography (RP-HPLC) ; Strongylocentrotus purpuratus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83-88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP-HPLC analysis with a spectrophotometric Nα-benzoyl-L-arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin-like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.
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    Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′-dihexyloxacarbocyanine iodide) (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 15 (1986), S. 267-283 
    Details
    ISSN: 0148-7280
    Keywords: fluorochromes ; fertilization ; nuclear events ; mitochondria ; surf clam ; sea urchin ; hamster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′-dihexyloxacarbocyanine iodide (DIOC6) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae-free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC6 staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC6 (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC6-positive organelles. Sea urchin and surf clam sperm stained with DIOC6 fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC6 were less motile than unstained sperm, they were capable of inseminating only zonae-free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.
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    Characterization of cell surface polypeptides of unfertilized, fertilized, and protease-treated zona-free mouse eggs (1989)
    New York, NY : Wiley-Blackwell
    Gamete Research 23 (1989), S. 91-101 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; zona-free egg ; membrane polypeptides ; radioiodination ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The polypeptide composition of unfertilized, fertilized, and protease-treated zona-free mouse eggs was evaluated in this study. Zona-free eggs were radioiodinated by an Iodogen-catalyzed reaction. Light microscopic autoradiography of egg sections revealed that labeling was restricted to the cell surface. Labeled eggs were solubilized, and cell surface polypeptides were identified by one-dimensional SDS polyacrylamide gel electro-phoresis and autoradiography. The unfertilized egg demonstrated 8-10 peptides that incorporated125I, with major bands observed at approximately 145-150, 94, and 23 kilo-daltons (kD). Zona-free eggs fertilized in vitro and then radiolabeled demonstrated several new bands in comparison to unfertilized eggs, with a major band appearing at approxi mately 36 kD. Treatment of radiolabeled unfertilized eggs with either trypsin or chymo-trypsin (1 mg/ml for 5-20 min) caused enzyme-specific modifications in labeled polypep tides. Trypsin (T) treatment resulted in time-dependant modification of the three major peptides at 145-150,94, and 23 kD. Chymotrypsin (CT) treatment, in contrast, was asso ciated with loss or modification of the 94 kD band, with no apparent effect on either the 145-150 or 23 kD band. Taken together with previous data indicating that T or CT egg treatment interferes with sperm-egg attachment and fusion (Boldt et al.: Biol Reprod 39:19-27, 1988), these results suggest a possible role for the 94 kD protein in sperm-egg interaction.
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    Purification of the sperm-binding factor and identification of a sperm attack molecule from the egg of the sea urchin, Hemicentrotus pulcherrimus (1987)
    New York, NY : Wiley-Blackwell
    Gamete Research 18 (1987), S. 1-16 
    Details
    ISSN: 0148-7280
    Keywords: Fab fragments ; fertilization ; sperm-egg binding ; species-specificity ; polyspermy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A sperm-binding factor, which seems to have a primary role in binding sperm to the egg, was isolated from the egg surface of Hemicentrotus pulcherrimus and purified by monitoring the neutralization of the fertilization inhibition exerted by Fab fragments against crude sperm-binding factor. An improved purification for this sperm-binding factor is described in the present paper. The preparation of purified sperm-binding factor revealed one major protein band with an apparent molecular weight of 61,000 after sodium dodecylsulfate-polyacrylamide gel electrophoresis.A substance with the fertilization inhibitory effect on sperm, was isolated by diethylaminoethyl (DEAE)-Sephadex column chromatography in the course of purification of the sperm-binding factor and termed “sperm attack molecule.” One precipitin line was formed between the sperm attack molecule and anti-crude sperm-binding factor serum in a double-immunodiffusion test. Fab fragments were prepared against partially purified sperm-binding factor or sperm attack molecule, and the effect of these Fab fragments on eggs was investigated. Anti-sperm-binding factor Fab fragments inhibited the fertilizability of eggs, whereas anti-sperm attack molecule fab fragments did not. However, anti-sperm attack molecule Fab fragments impaired elevation of the vitelline layer. It is possible that the sperm attack molecule prevents polyspermy.Sperm attack molecule contains 3.7% neutral sugars. Its inhibitors effect was cancelled by trypsin or heat.
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    A computer-assisted assay for mouse sperm hyperactivation demonstrates that bicarbonate but not bovine serum albumin is required (1987)
    New York, NY : Wiley-Blackwell
    Gamete Research 18 (1987), S. 121-140 
    Details
    ISSN: 0148-7280
    Keywords: sperm motility ; capacitation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mammalian sperm hyperactivation (HA) is a change in motility that accompanies capacitation (CAP) and is dependent on calcium (Ca) (Yanagimachi and Usui, Exp Cell Res 89:161, 1974). HA may be important for transport through the female tract and/or for fertilization. To develop an objective and quantitative assay for HA in individual mouse sperm, a computer-assisted motion-analysis system was used to describe sperm trnaslational movements. To determine which movements were characteristic of HA, Ca-dependent motility was identified. This was done by incubating sperm with or without calcium (Ca+ or Ca- sperm, respectively), and determining the range of values for each motility parameter that was present only among Ca+ sperm. To do this, we compared frequency distributions of motility parameter values at the time of maximal CAP (90 min). CAP was monitored by measuring the level of in vitro fertilization and by evaluating the pattern of chlortetracycline binding to individual sperm heads [Ward and Storey, Dev Biol 104:287, 1984]. Two Ca-dependent motility subgroups were apparent: 1) a “slow-speed” subgroup with a curvilinear velocity (Vc) 〈 169 μm/sec that had none of the characteristics expected of HA sperm; and 2) a subgroup with higher speeds (Vc 〉 169 μm/sec) and wider-amplitude head movements as measured by curvilinear progressiveness ratio (PRc 〈 0.56). The latter subgroup was selected as HA, since the frequencies and time course were similar to those for CAP in the same population. Two media components known to be important for CAP, bicarbonate and bovine serum albumin (BSA) were then tested to determine whether they were necessary for HA. Incubation of sperm without bicarbonate prevented HA, but omitting BSA did not affect HA during the first 3 hrs. These data suggest that HA is not tightly coupled with CAP.
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    Ultrastructural mapping of a sperm plasma membrane autoantigen before and after the acrosome reaction (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 19 (1988), S. 387-399 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; zona pellucida ; label-fracture ; antigen localization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The rabbit sperm plasma membrane autoantigen, RSA, is a zona binding protein that binds the spermatozoon to the zona pellucida both before and after the acrosome reaction. In the present study rabbit spermatozoa undergoing the acrosome reaction in vitro are described and monospecific polyclonal mouse anti-RSA and protein A-gold label is used with the label-fracture technique (Pinto de Silva and Kan, J Cell Biol, 99:1156-1161, 1984) to map the location of RSA at the ultrastructural level before and after the acrosome reaction. RSA is most concentrated in the plasma membrane over the postacrosomal-equatorial region border. The label appears to cluster over the anterior aspects of the postacrosomal region's tooth-like projections. Following the acrosome reaction, RSA is still present in the postacrosomal region and often appears clustered in the medial aspects of the equatorial region.
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    Effect of follicle somatic cells during pig oocyte maturation on egg penetrability and male pronucleus formation (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 20 (1988), S. 177-183 
    Details
    ISSN: 0148-7280
    Keywords: pig ; oocyte ; spermatozoa ; maturation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to investigate the effect of the somatic cells of the follicle on the preparation of the oocyte for fertilization three experiments were carried out. In the first, pig oocytes, cultured for 46 h in the presence of extroverted follicles (follicle oocytes) or surrounded by the cumulus (cumulus oocytes), were exposed to sperm in an in vitro fertilization system. Follicle oocytes showed a higher rate of fertilization than that recorded in cumulus oocytes (80% vs. 47%). In addition, significantly more sperm penetrated into the ooplasm of follicular oocytes (3.77/egg) than into that of cumulus oocytes (1.42/egg). To investigate the reason for the observed fertilization difference zona-free oocytes were studied in the second experiment. Significantly more spermatozoa were recorded in the ooplasm of follicle oocytes than in that of cumulus oocytes, thus suggesting that the effect of the follicle on fertilizability may partly depend on an action on the plasma membrane of the oocyte. A further effect of the follicular tissue was on cytoplasmic maturation: only follicular oocytes were capable of consistently promoting male pronucleus formation. In cumulus oocytes, sperm that entered the cytoplasm remained in a condensed form near the female pronucleus. In the third experiment cumulus oocytes and denuded oocytes were matured in medium that had been previously used to mature follicle oocytes. This conditioned medium was alone able to affect sperm penetration and male pronucleus formation in cumulus oocytes, but it did not exert any influence on denuded oocytes. This suggests that the observed effect of the follicle is mediated by soluble factors that, however, cannot influence the oocyte without some direct cell-oocyte contact.
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    Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 20 (1988), S. 185-201 
    Details
    ISSN: 0148-7280
    Keywords: acrosome reaction ; fertilization ; lipoproteins ; lipids ; albumin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [3H]cholesterol and of [3H]cholesteryl sulfate from labeled spermatozoa. Efflux of [3H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [3H]cholesterol and [3H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (VLDL), high-density lipoproteins (HDL), as well as the albumin fraction (d 〉 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [3H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60-70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [3H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [3H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [3H]sterol binding capacity that is 2 - 3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.
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    Genetic analysis of sperm function in fertilization (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 20 (1988), S. 241-264 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; genetic analysis ; sperm function ; T-complex ; sperm morphology ; acrosomal enzymes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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    Classification, inhibition, and specificity studies of the vitelline coat lysin from toad sperm (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 20 (1988), S. 287-300 
    Details
    ISSN: 0148-7280
    Keywords: sperm lysin ; vitelline coat ; serine protease ; acrosome reaction ; fertilization ; Bufo japonicus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The sonicated supernatant of the sperm of the toad, Bufo japonicus, can digest easily the vitelline coat (VC) of uterine eggs, and to a lesser extent the VC of coelomic eggs, but not that of activated eggs. The VC lysis and fertilization were competitively inhibited in the presence of t-butyloxycarbonyl-L-Gln-L-Arg-L-Arg-4-methylcoumaryl-7-amide (Boc-Gln-Arg-Arg-MCA), suggesting the involvement of proteases in the fertilization process. Starting from a sonicated supernatant, a potent VC lysin, possessing hydrolytic activity on Boc-Gln-Arg-Arg-MCA, was obtained by anion-exchange chromatography and gel filtration. The activity of the partially purified lysin was inhibited by diisopropyl fluorophosphate (DFP) and by such trypsin inhibitors as soybean trypsin inhibitor, leupeptin, and (p-amidinophenyl) methanesulfonyl fluoride hydrochloride, but not by chymostatin, E-64, and ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. The molecular weight of the lysin was estimated to be 32K, based on the fluorographic image of 3H-DFP binding to the lysin on sodium dodecyl sulfate gel electrophoresis. The VC lysin was most active at pH 7.0-7.6 and under low ionic strength equivalent to fresh water. The release of the VC lysin was induced upon incubation of sperm with the contents of oviducal pars recta granules (PRG), which are known to induce the acrosome reaction. We conclude that the protease studied here represents the VC lysin of toad sperm that is involved in fertilization by digesting the VC of uterine eggs, probably released as a result of the acrosome reaction induced by PRG.
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    Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 20 (1988), S. 365-376 
    Details
    ISSN: 0148-7280
    Keywords: sperm nuclear decondensation ; oocyte chromosomes ; colcemid ; fertilization ; secondary condensation ; MPF ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona-free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.
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    Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 20 (1988), S. 413-420 
    Details
    ISSN: 0148-7280
    Keywords: sperm ; capacitation ; acrosome reaction ; cholesterol ; bovine ; ovum ; lysophosphatidylcholine ; in vitro ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol-to-phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona-free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39°C) in a modified Tyrode's solution (TALP) containing (1) Chol-free liposomes ( - Chol, 50 × 106 sperm and 600 nmol phospholipid/ml); (2) liposomes containing 30 mol% Chol (+ Chol, 2 × 108 sperm and 300 nmol total lipid/ml); or (3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with  - Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 × 106) were incubated immediately with either zona-free hamster ova (HO) or in vitro matured bovine ova (BO) in 50-μl droplets of TALP under medical fluid in an atmosphere of 5% CO2 in air (3 h, 39°C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined. Percent penetration (%P) of HO (X ± SEM) for 30 and 40 μg of LPC/mg BSA was 59.4 ± 5.3 and 82.9 ± 5.4; 38.5 ± 5.6 and 52.3 ± 4.7; and 16.0 ± 4.6; and 23.2 ± 5.6 for  - Chol, Control, and + Chol treatments, respectively (n = 3). %P of BO (X ± SEM) for 30, 35, and 40 μg of LPC/mg BSA was 43.3 ± 5.4, 70.7 ± 7.5, and 81.5 ± 5.1 for  - Chol and 16.4 ± 6.9, 36.2 ± 6.9, and 44.2 ± 8.6 for Control treatments, respectively (n = 3). In a second set of experiments %P of BO (X ± SEM) was 63.6 ± 6.8, 31.8 ± 4.9, and 10.5 ± 3.4 for  - Chol, Control, and +Chol treatments, respectively, when 40 μg LPC/mg BSA was added (n = 2). %P and the number of sperm per fertilized ovum were consistently higher for the  - Chol treatment for both HO and BO (P 〈 .01). These results demonstrate that Chol removal from bovine sperm facilitates penetration of ova in vitro suggesting a potential role in bovine sperm capacitation.
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    Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 21 (1988), S. 29-40 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; sperm receptor ; vitelline envelope ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.
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    Sperm entry into zona-free oocytes in the hamster oviduct: Implications for the mechanisms of acrosome reaction induction (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 21 (1988), S. 85-91 
    Details
    ISSN: 0148-7280
    Keywords: zona pellucida ; oviduct ; fertilization ; acrosome reaction induction ; capacitation ; mammals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The possible importance of the zona pellucida for induction of the acrosome reaction (AR) and establishment of sperm/egg associations in the fallopian tube was investigated by instilling zona-free eggs into the oviductal ampulla of hamsters that had been inseminated with epididymal spermatozoa 6-7 hours previously. The eggs were recovered only 60-90 minutes later because of increasing difficulty with time of collecting zona-free eggs from the oviduct. In the zona-free group, 41 (4%) of 1,101 transferred eggs were recovered, of which 20% contained spermatozoa with decondensing nuclei (mean 4.4/egg). A similar (22%) fertilization rate (mean 3.2 spermatozoa/egg) was found among intact (control) eggs recovered after instillation into the contralateral oviduct.Mammalian spermatozoa are not incorporated even into zona-free eggs before AR occurs. These results thus demonstrate that an AR in functional hamster spermatozoa in vivo and establishment of sperm/egg associations in vivo require no interaction with the zona pellucida nor with other products of ovulation.
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    Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa (1989)
    New York, NY : Wiley-Blackwell
    Gamete Research 24 (1989), S. 403-413 
    Details
    ISSN: 0148-7280
    Keywords: heparin ; fertilization ; dextran sulfate ; fucose sulfate glycoconjugate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P 〈0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.
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    URL: http://dx.doi.org/10.1002/mrd.1120240407
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    Developmental capability of rat oocytes matured in vitro in defined medium (1985)
    New York, NY : Wiley-Blackwell
    Gamete Research 12 (1985), S. 255-263 
    Details
    ISSN: 0148-7280
    Keywords: oocyte ; maturation ; fertilization ; fetal development ; cumulus-free ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The normality of in vitro matured oocytes was compared to that of in vivo matured (ovulated) oocytes at the following stages of development: germinal-vesicle breakdown, first polar body formation, fertilization (two polar bodies and two pronuclei with a sperm tail or first cleavage), and fetal development (day 20 fetuses). At all points, the in vitro oocytes exhibited a reduced ability, with oocytes matured cumulus-free having the poorest. The exposure of oocytes to human chorionic gonadotropin (hCG) for 2 hr before collection or during incubation improved their rates of maturation and development to day 20 fetuses but not their ability to undergo fertilization. While beneficial, the exposure to gonadotropins before or during maturation was not essential, as evidenced by the production of two day 20 fetuses matured and fertilized in vitro without any gonadotropin (luteinizing hormone or hCG) treatment in vivo or in vitro. These data demonstrate that in the population of in vitro matured oocytes there exist individuals wholly competent of complete normal development, albeit in a reduced proportion in comparison to normally matured and ovulated oocytes. That the in vitro handling, treatment, and culture of the oocytes may be responsible for some of the reduced developmental ability observed is suggested by the developmental abilities of ovulated oocytes under different conditions. Ovulated oocytes fertilized in the donor had the highest rates of development (46%), followed by those fertilized after transfer into mated recipients' oviducts (20%). The lowest rate was achieved with in vitro fertilized oocytes (7%), which represented the group subject to the greatest degree of manipulation and distinction from the normal in vivo process.
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    N-acetyl-β-D-glucosaminidase activity in the cortical granules of Xenopus laevis eggs (1985)
    New York, NY : Wiley-Blackwell
    Gamete Research 12 (1985), S. 305-312 
    Details
    ISSN: 0148-7280
    Keywords: glucosaminidase ; cortical granules ; egg enzymes ; fertilization ; glycosidases ; frogs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The glycosidase activities associated with Xenopus laevis eggs were determined using p-nitrophenyl glycosides as substrates. The egg lysate contained significant amounts of α-fucosidase, N-acetyl-β-D-galactosaminidase, and α-mannosidase activities with smaller amounts of other glycosidase activities, including N-acetyl-β-D-glucosaminidase. A cortical granule exudate obtained from ionophore-activated dejellied eggs contained predominantly glucosaminidase activity with only trace amounts of other glycosidase activities. Perivitelline space material obtained from activated or fertilized jellied eggs contained only glucosaminidase activity. Using cytochemical staining procedures, glucosaminidase activity was present in the perivitelline space and the inner aspect of the jelly coat after fertilization or activation of the egg, but not before. The rate of glucosaminidase activity released from activated eggs occurred with the same kinetics as the cortical reaction. The cortical granule and noncortical granule glucosaminidase activities had different electrophoretic mobilities as determined by disc gel electrophoresis. Thus, the Xenopus laevis egg has two N-acetyl-β-D-glucosaminidase activities, one associated with the cortical granules and the other associated with the noncortical granule compartment of the cell. The cortical granule enzyme released from the egg at fertilization may function in altering the egg's penetrability to supernumerary sperm.
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    URL: http://dx.doi.org/10.1002/mrd.1120120309
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    Observation on the incorporation cone in the rat (1978)
    New York, NY : Wiley-Blackwell
    Gamete Research 1 (1978), S. 27-37 
    Details
    ISSN: 0148-7280
    Keywords: oocyte ; fertilization ; incorporation cone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: At the time of in vivo sperm-egg fusion in the rat, a small region of the oolemma under the head of the fertilizing sperm is observed to be free of microvilli. The microvilli-free region increases in area, and by one hour after sperm-egg contact extends over an area 20-30 μ in circumference and bulges out to form an “incorporation cone” visible by light microscopy. The microvilli-free incorporation cone reaches its maximum size at about two hours after sperm-egg interaction. It soon becomes smaller and has disappeared three to four hours after sperm-oocyte fusion. The cone cytoplasm is characterized by a 0.1 μ zone of thin filaments below the plasma membrane. Cytochalasin-B, 2.5 μg/ml, prevents formation of the cone or destroys the intact cone. It is suggested that micro filaments may be involved in the formation of the incorporation cone.
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    URL: http://dx.doi.org/10.1002/mrd.1120010106
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    Isolation of sperm bindin from the oyster (Crassostrea gigas) (1978)
    New York, NY : Wiley-Blackwell
    Gamete Research 1 (1978), S. 89-99 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; acrosome ; bindin ; oyster ; sperm-egg interaction ; cell surface ; intercellular adhesion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A method was devised for isolating the insoluble content of the acrosome granule of sperm of the oyster Crassostrea gigas. The method involves the dissolution of the entire cell, except for the acrosome granule, in the detergent sodium lauroyl sarcosinate (sarcosy I). The isolated acrosome granule content is ring-shaped and is 84% protein by weight. SDS-polyacrylamide gel electro-phoresis of this material yields from 1 to 4 bands of 65,000; 53,000; 43,000 and 34,000 apparent molecular weight, all of which stain with the PAS reaction indicating the material is a glycoprotein. The 65,000 molecular weight component is always present, but the presence of the other three bands varies with each preparation. The isolated acrosome granules agglutinate formaldehyde-fixed oyster eggs. A trypsin-generated glycopeptide digest of oyster egg surfaces inhibits the agglutinin activity of the isolated acrosome granules. We propose that the acrosomal glyco-protein material is oyster sperm bindin which functions as the adhesive substance bonding the sperm to the egg during fertilizaion.
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    The effect of in vitro storage of hamster sperm on fertilization and early development (1978)
    New York, NY : Wiley-Blackwell
    Gamete Research 1 (1978), S. 235-245 
    Details
    ISSN: 0148-7280
    Keywords: hamster ; sperm storage ; fertilization ; development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of storage of hamster sperm in vitro at temperatures above freezing on fertilization and early development were studied. Initially, female hamsters were inseminated artificially with epididymal sperm stored up to five days at 5°, 16°, or 23-25°C. Eggs were examined at the pronuclear stage for evidence of fertilization. The best conditions for the storage of hamster sperm were found to be 16°C in a 5% CO2 in air gas phase. Pre- and postimplantation development were then assessed following insemination of females with sperm stored at 16°C. An increased frequency of eggs with three pronuclei, a decrease in the number of pregnant females, and an increase in the number of resorption sites were found with increasing sperm storage time. Chromosome mosaicism and triploidy were encountered in 9.5-day embryos resulting from eggs fertilized by stored sperm. No chromosome abnormalities were found in a control group of embryos. Thus, storage of hamster epididymal sperm resulted in decreased fertility and an increased frequency of abnormal development.
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    Release of acrosomal enzymes following in vitro capacitation of ejaculated rabbit spermatozoa (1979)
    New York, NY : Wiley-Blackwell
    Gamete Research 2 (1979), S. 1-13 
    Details
    ISSN: 0148-7280
    Keywords: capacitation ; acrosomal enzymes ; rabbit sperm ; acrosomal membranes ; fertilization ; lysosomal enzymes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Significant release of the acrosomal enzymes arylsulfatase, β-N-acetylhexosaminidase and hyaluronidase was observed following the treatment of ejaculated rabbit spermatozoa for 12 hours in 20% rabbit serum for inducing in vitro capacitation, and these sperm were capable of in vivo fertilization; however, the treatment of sperm for 15 minutes in high ionic strength (380 mOsm/kg) or low ionic strength medium (305 mOsm/kg) for in vitro capacitation did not result in any significant release of the above enzymes nor were the sperm capable of in vivo fertilization. Serum-treated spermatozoa remained significantly motile following the 12 hour treatment, 51% underwent the acrosome reaction and were capable of fertilizing 66% of the ova in vivo. Identical serum treatment of lysosomes from rabbit liver resulted in a comparable release of the lysosomal enzymes. Serum treatment for in vitro capacitation resulted in vesiculation of the anterior margin of half the spermatozoa, but left their inner acrosomal membranes and equatorial segments intact. A biochemical relationship between the release of acrosomal enzymes and capacitation is suggested.
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    Fertilizability of cryopreserved zona-free hamster ova (1979)
    New York, NY : Wiley-Blackwell
    Gamete Research 2 (1979), S. 357-366 
    Details
    ISSN: 0148-7280
    Keywords: ova ; spermatozoa ; cryopreservation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mature unfertilized ova from superovulated hamsters were freed from all investments and frozen at -50°C. They were cooled at about 1°C/min to 0°C then at 0.8° to 0.6°C/min to -50°C. At 0°C, dimethyl sulfoxide was added to a final concentration of 1.25 M. The ova were stored at -50°C for up to four months. Thawing was performed at 2-4°C/min and followed by several washes with insemination medium. Approximately 90% of the ova were normal in appearance after thawing. The frozen and thawed ova with normal appearance could be penetrated by hamster or human spermatozoa at a rate comparable to unfrozen controls. The ability of hamster ova to tolerate storage at a relatively convenient temperature (-50°C) for long periods (tested for up to four months) makes possible their shipment at low cost to institutions lacking this resource. There they can be used for basic biological studies of sperm-egg interaction or in the clinical assessment of human sperm quality.
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    URL: http://dx.doi.org/10.1002/mrd.1120020408
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    Partial activation of sea-urchin eggs by bonellin (1980)
    New York, NY : Wiley-Blackwell
    Gamete Research 3 (1980), S. 309-316 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; membrane potential ; bonellin ; amino acid incorporation into proteins ; DNA synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We here describe further studies on the action of bonellin on sea-urchin eggs. Bonellin brings about Some of the changes that are known to occur in the egg upon fertilization. In particular, it appears to cause the increased rate of incorporation of amino acids into proteins, the increase of the voltage noise, and the exocytosis of some of the cortical granules. A comparison with the effect of ammonia is discussed.
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    Inhibition of acrosin by oviductal secretory immunoglobulin A (1981)
    New York, NY : Wiley-Blackwell
    Gamete Research 4 (1981), S. 15-23 
    Details
    ISSN: 0148-7280
    Keywords: secretory IgA ; oviduct fluid ; acrosin ; spermatozoa ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A major inhibitor of acrosin in rhesus monkey and rabbit oviduct fluid, isolated by isoelectrofocusing in sucrose gradients, displayed a broad peak in the acidic region of the column and was demonstrated to contain secretory IgA specific for acrosin. Its identity was established by immunodiffusion, by the removal of acrosin inhibition with antisera to IgA (α-chain), and by its correct molecular weight during ultracentrifugation. Purified human serum IgA also inhibited rabbit, rhesus monkey, and human acrosins, but neither purified human IgG nor IgM had any inhibitory effect on these acrosins. Neither oviduct fluid secretory IgA nor purified human serum IgA inhibited the activity of bovine pancreatic trypsin. The high specificity of secretory IgA for acrosin and its presence in every rabbit and rhesus monkey oviduct fluid specimen examined suggests a possible regulatory role for this antibody in reproduction.
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    Temperature dependence of capacitation in bat sperm monitored by zona-free hamster ova (1981)
    New York, NY : Wiley-Blackwell
    Gamete Research 4 (1981), S. 525-533 
    Details
    ISSN: 0148-7280
    Keywords: capacitation ; fertilization ; spermatozoa ; in vitro ; Little Brown Bat ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The temperature dependence of capacitation in bat sperm (Myotis lucifugus lucifugus) was studied by monitoring fertilizations rates of zona-free hamster ova at different temperatures. Spermatozoa were cultured in BWW medium at temperatures 4°C, 24°C, 32°C, 42°C, and 55°C from 0-24 hr. Activation of sperm could be determined visually due to the change in movement seen through light microscopy. Activation was later confirmed by higher rates of fertilization. Preincubation of the bat sperm was found to have a direct effect on the success of penetration of the zona-free hamster ova. Holding bat spermatozoa at low temperature for long intervals allowed them to remain motile but unable to fertilize. Sperm are not irreversibly damaged, however, and activation, when the temperature is increased to 32°C, is faster than when sperm are intitially put at 32°C, resulting in good fertilization rates.
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    Unilateral tunicamycin sensitivity of gametogenesis in dioecious isogamous chlamydomonas species (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 1-9 
    Details
    ISSN: 0148-7280
    Keywords: Chlamydomonas ; tunicamycin ; gametogenesis ; fertilization ; glycoproteins ; speciation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sex cell adhesion in isogamous chlamydomonads is caused by a complementarity between sex-specific mating type substances, glycoproteins anchored in the flagella membrane of (+) and (\documentclass{article}\pagestyle{empty}\begin{document}$ \mathop - \limits^. $\end{document}) gametes. The systems of mating type substances are species-specific and condition, by their individuality, gametic incompatibility between species. The adhesion systems of several species share one common feature: the attainment of the agglutination capacity is sensitive to tunicamycin, but in one sex only. The effect is interpreted as due to the interference of tunicamycin with the synthesis of the mating type substances by blocking of their glycosylation in one but not in the other sex. It is postulated that the tunicamycin-sensitive gametic adhesiveness depends, within the mating-type-specific glycoprotein, on an N-glycosidically bound ligand of carbohydrate nature. A concept on the origin of sibling species by mutative modulations within the proper ligands of the glycoproteinaceous mating type substances is developed.
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    Reaction of sperm with egg-derived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin (1981)
    New York, NY : Wiley-Blackwell
    Gamete Research 4 (1981), S. 365-377 
    Details
    ISSN: 0148-7280
    Keywords: block to polyspermy ; hydrogen peroxide ; sperm peroxidase ; sperm catalase ; cortical reaction ; fertilization ; phenylhydrazine ; 3-amino-1,2,4-triazole ; ovoperoxidase ; sea urchin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Recent evidence suggests roles for egg derived hydrogen peroxide (H2O2) and ovoperoxidase (secreted by cortical granules) in both fertilization envelope hardening and the block to polyspermy in sea urchins. Strongylocentrotus purpuratus eggs were found to release H2O2 during the cortical reaction at fertilization. Treatment of sperm with equivalent concentrations of H2O2 resulted in a rapid loss of sperm fertilizing ability. Attempts were made to induce polyspermy by utilizing ovoperoxidase inhibitors at concentrations known to inhibit fertilization envelope hardening. Eggs fertilized in phenylhydrazine became polyspermic, while 3-amino-1,2,4-triazole-treated eggs did not. These data suggested that a sperm peroxidase might be involved in preventing polyspermy. This hypothesis was tested by the addition of phenylhydrazine or 3-amino-1,2,4-trizaole to H2O2-treated sperm. Phenylhydrazine acted to protect sperm fertility from H2O2, while 3-amino-1,2,4-triazole increased the adverse effect of H2O2. Simultaneous addition of both inhibitors to sperm incubated in H2O2 gave an intermediate value of sperm fertility. These data indicate that (1) H2O2 generated by sea urchin eggs during the cortical reaction at fertilization is used for two separate processes, fertilization envelope hardening and the prevention of polyspermy; (2) ovoperoxidase is probably not involved in preventing polyspermy; and (3) egg-derived H2O2 reacts directly with sperm enzymes to prevent polyspermy. The phenylhydrazine-sensitive enzyme in the sperm is probably a peroxidase that acts to inactivate sperm, while the 3-amino-1,2,4-triazolesensitive enzyme is probably a catalase which protects sperm from H2O2. This hypothesis is consistent with model experiments on horseradish peroxidase and bovine liver catalase.
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    Chromatin transitions in the fertilizing sperm nucleus of the sea urchin, strongylocentrotus purpuratus (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 181-190 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; sperm ; chromatin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Electron microscopic analysis of fertilization in the sea urchin, Strongylocentrotus purpuratus, has been carried out in an effort to establish the sequence of events involving dispersion of the paternal chromatin. Subsequent to loss of the nuclear envelope the condensed sperm chromatin begins to disperse under the influence of egg cytoplasmic factors. However, this process does not proceed at a uniform rate as is observed in other species examined to date. Portions of the paternal genome rapidly transform into dispersed chromatin while other adjacent regions disperse at a reduced rate. This variation in the time sequence of dissociation of the paternally derived chromosomes results in a reticulum of electron lucent and electron dense chromatin within the developing male pronucleus. As the paternally derived chromatin is dispersing and migrating centrad, membranous vesicles of maternal origin become aligned along the peripheral aspect of the chromatin. Deposition of a continuous bilaminar nuclear envelope around the dispersing sperm chromatin results in the formation of the definitive male pronucleus. At the time the male pronucleus is formed the paternally derived chromosomes have not completely dispersed and are visualized as a reticulum of condensed and dispersed chromatin. These results indicate that not all the paternally derived chromatin is modified in the same manner during pronuclear development.
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    Sperm-egg ratios and the site of the acrosome reaction during in vivo fertilization in the hamster (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 239-256 
    Details
    ISSN: 0148-7280
    Keywords: acrosome reaction ; fertilization ; cumulus oophorus ; hamster spermatozoon ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Little is known about the timing of the mammalian sperm acrosome reaction during fertilization in vivo. To study this problem, female hamsters were inseminated at about the time of ovulation, and the contents of the ampullary regions of their oviducts were subsequently examined at various intervals. No living spermatozoa were recovered from ampullae earlier than 4 hr after insemination. The first appearance of living spermatozoa coincided closely with the first appearance of fertilized eggs in the same oviduct. The total numbers of living spermatozoa did not start to exceed the number of eggs in the same ampulla, until after 50% or more of the eggs had been fertilized. Hamster spermatozoa are highly efficient at making contact with eggs, and the fertilizing spermatozoon probably spends no more than 2½ -5½ min in penetrating the cumulus oophorus. Spermatozoa that enter the ampulla appear to be ready to undergo the acrosome reaction, and complete it while they are passing through the cumulus or shortly before, or after, contacting the surface of the zona pellucida.
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    Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 323-344 
    Details
    ISSN: 0148-7280
    Keywords: calcium ; fertilization ; spermatozoa ; eggs ; hamster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Using a semi-chemically defined medium, the requirement of extracellular Ca2+ for survival, capacitation, and acrosome reaction of spermatozoa as well as various stages of fertilization in the hamster was studied. A Ca2+-deficient environment is unfavorable for long-term survival of spermatozoa. Sperm capacitation may occur in Ca2+-deficient media, but not as efficiently as in normal media. The acrosome reaction definitely requires extracellular Ca2+. Other processes or phenomena that require extracellular Ca2+ are initiation and maintenance of hyperactivated motility of spermatozoa, penetration of acrosome-reacted spermatozoa into the zona pellucida, fusion of the spermatozoa with eggs, and the development of pronuclear eggs into two-cell embryos. Extracellular Ca2+ is apparently unnecessary for the attachment of spermatozoa to the zona and egg surfaces, decondensation of the sperm nucleus, and the development of sperm and egg pronuclei within the egg. These results were compared with data obtained in other species such as the sea urchin, mouse, rat and guinea pig.
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    Use of zona-free hamster ova to assess sperm fertilizing ability of bull and stallion (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 217-227 
    Details
    ISSN: 0148-7280
    Keywords: spermatozoa ; bull ; stallion ; vitelli ; hamster ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Zona-free hamster ova interacted with bull and stallion spermatozoa after treatment of ejaculated semen to capacitate the sperm cells. Sperm conditioning by prolonged incubation in BWW medium (18-26 hr) prior to insemination was effective for capacitation of bull and stallion sperm. Preincubation of bull sperm for 70 or 105 min in defined medium (DM) with NaCl content elevated to result in 350 m0sM/kg also led to penetration of hamster vitelli. More rapid sperm conditioning was possible and higher proportions of interacting vitelli followed insemination with bull or stallion sperm exposed to high ionic strength DM (380-390 m0sM/kg) for 10 min before incubation in isotonic DM prior to insemination, the treatment adopted in subsequent work.Initial efforts to assess relative fertilizing ability of freshly ejaculated semen from two fertile bulls (A and B) in A1 usage resulted in uniformly high ( 〉 90%) levels of sperm-vitelli interaction (for both) when the hamster ova employed resulted from superovulation with PMSG and HCG. Following use of ova from untreated hamsters sperm samples of bull A and bull B interacted with 53.8% and 84.9% (P 〈 0.05) of zona-free hamster ova, respectively. Conception data (60-90 day nonreturn rates) resulting from A1 with semen collected during the same interval but processed and stored in liquid nitrogen prior to use revealed an inverse relationship to proportions of vitelli interacting with fresh sperm; nonreturn rates were 69.3% and 66.3% for bull A and bull B, respectively. A similar treatment effected capacitation of frozen-stored bull semen to enable sperm-vitelli interaction. These findings encourage additional efforts to correlate testing of processed semen with fertility.
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    URL: http://dx.doi.org/10.1002/mrd.1120050302
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    Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian, halocynthia roretzi (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 291-301 
    Details
    ISSN: 0148-7280
    Keywords: ascidian ; sperm ; acrosin-like enzyme ; protease ; lysin ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl2. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH2Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH2Cl, and tosyl-Phe-CH2Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs.Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.
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    URL: http://dx.doi.org/10.1002/mrd.1120050309
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    Novel procedures for collection of sea urchin egg cortical granule exudate: Partial characterization and evidence for postsecretion processing (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 6 (1982), S. 39-52 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; sea urchin ; Strongylocentrotus purpuratus ; cortical granule exudate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have developed two procedures to collect total cortical granule exudate in a soluble form from eggs of the sea urchin Strongylocentrotus purpuratus. Egg suspensions were either treated with dithiothreitol to disrupt the vitelline envelope or divalent cations were removed postinsemination to prevent the normal vitelline-to-fertilization envelope transition. Rapid acidification of the insemination mixture (dithiothreitol-treated eggs) to pH 6.0 prevented precipitation of the paracrystalline protein fraction described by Bryan [1970a]. Exudate was partitioned into three fractions. The pH 8.0-insoluble fraction appeared to be identical to the paracrystalline protein fraction. The pH 8.0-soluble fraction was separated into pH 4.0-soluble and-insoluble fractions. Analysis for peroxidase and protease activities showed that peroxidase activity was localized in all three fractions whereas protease activity was restricted to the pH 4.0 insoluble fraction as reported [Carroll and Epel, 1975]. A minimum of six major proteins were detected on native polyacrylamide gels of total exudate. Under reducing and denaturing conditions, 12 polypeptides ranging from 19,000 to 165,000 in molecular weight were detected in total exudate; six polypeptides were recovered in the pH 8.0-insoluble fraction. To test the hypothesis that protease and peroxidase activities process cortical granule proteins after secretion, we inseminated eggs in solutions containing peroxidase and protease inhibitors. The paracrystalline protein fraction crystallized slowly from insemination mixtures containing both inhibitors compared to controls and there were dramatic differences in exudate electrophoretic patterns. We suggest that cortical granule protease and peroxidase activities process the exudate so that the paracrystalline protein fraction rapidly crystallizes during normal fertilization.
    Additional Material: 3 Ill.
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    URL: http://dx.doi.org/10.1002/mrd.1120060106
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    Sperm tail entry into the mouse egg in vitro (1982)
    New York, NY : Wiley-Blackwell
    Gamete Research 6 (1982), S. 215-223 
    Details
    ISSN: 0148-7280
    Keywords: spermatozoon ; egg ; fertilization ; in vitro ; incorporation ; cincmatography ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cumulus-free mouse eggs were placed on microscope slides and inseminated with capacitated mouse spermatozoa. Fertilization could then be observed through the phase contrast microscope and recorded by time-lapse cinematography. Following the penetration of the fertilizing spermatozoon through the zona pellucida and the fusion of the sperm head with the vitelline membrane, the entire sperm tail gradually entered the vitellus. The time required for tail incorporation into the vitellus as measured in 49 eggs varied from 3 h 3 min to 5 h 49 min, with a mean time of 4 h 23 min. When tail incorporation began, the greater part of the flagellum was still outside the zona pellucida; occasionally it slipped into the perivitelline space, but generally it remained outside the zona and shortened by degrees as incorporation proceeded. The motility of the fertilizing spermatozoon declined abruptly very soon after fusion of the sperm head with the vitellus and remained at a very low level during the 3-6 h required for tail incorporation. Sperm motility, therefore, does not appear to be the main determinant in tail incorporation and the primary mechanism responsible for it remains unclear. As the sperm tail slowly entered the vitellus, the second meiotic division was completed with concomitant extrusion of the second polar body. Key stages in second polar body formation were correlated with events in tail incorporation. Differences between fertilization in vitro and in vivo are discussed.
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    URL: http://dx.doi.org/10.1002/mrd.1120060305
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    The effect of catecholamines and their antagonists on the fertilization of cumulus-free mouse ova in vitro at a suboptimal spermatozoal density (1983)
    New York, NY : Wiley-Blackwell
    Gamete Research 7 (1983), S. 111-122 
    Details
    ISSN: 0148-7280
    Keywords: catecholamines ; antagonists ; mouse ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A suboptimal sperm concentration was used to assess the capacity of catecholamines to stimulate the fertilization of cumulus free F1,(C57BL × CBA) mouse ova in vitro. At a concentration of 50 μM, (L) epinephrine significantly increased the proportion of ova fertilized at 2 × l05 spermatozoa/ml. However, when (D, L) propranolol at an equimolar concentration was tested for inhibition of the (L) epinephrine effect, fertilization was inhibited in both the test and control dishes. At l0μM, propanolol by itself or in the presence of 50μM (L) epinephrine significantly increased the number of ova fertilized at 2 × l05 sperm/ml. Norepinephrine (50 μM) and phentolamine (50 μM), either alone or together, were also slightly stimulatory. Some data are presented to suggest that propranolol may act in a nonadrenergic manner to precipitate the acrosome reaction and that the stimulatory effect is maximised when it is added to spermatozoa at the same time as ova addition. It was suggested that propranolol may act to trigger calcium influx by a nonspecific alteration in membrane function for example in (Ca + Mg) ATPase activity. It was concluded that spermatozoa at suboptimal densities are capable of achieving fertilization and that sperm concentration dependency in fertilization in vitro may be a reflection of the proportion of spermatozoa achieving capacitation.
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    A separate catalase and peroxidase in sea urchin sperm (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 10 (1984), S. 267-281 
    Details
    ISSN: 0148-7280
    Keywords: sea urchin sperm ; catalase ; peroxidase ; phenylhydrazine ; 3-amino-1,2,4-triazole ; azide ; fertilization ; polyspermy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The release of hydrogen peroxide (H2O2) by the fertilized sea urchin egg has been shown to assist in the prevention of polyspermy [Coburn et al, 1981; Boldt et al, 1981]. Physiological data suggested that egg-derived H2O2 reacts with a phenylhydrazine-sensitive sperm peroxidase to inactivate sperm, while a 3-amino-1,2,4-triazole-sensitive catalase acts to protect sperm from H2O2 [Boldt et al, 1981]. Strongylocentrotus purpuratus sperm contain heat and pronase labile catalase and peroxidase activities. Differential extraction of sperm (hypotonic phosphate buffer for catalase and Triton X-100 at high ionic strength for peroxidase) results in complete separation of these enzyme activities. The catalase is highly sensitive to inhibition by azide and 3-amino-1,2,4-triazole, and less sensitive to inhibition by phenylhydrazine. The peroxidase is highly sensitive to inhibition by phenylhydrazine and relatively insensitive to 3-amino-1,2,4-triazole and azide. These results show that two distinct H2O2 reactive enzymes, catalase and peroxidase, are present in sea urchin sperm, and are consistent with our hypothesis concerning the biological functions of these enzymes in fertilization.
    Additional Material: 4 Ill.
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    The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 9 (1984), S. 1-19 
    Details
    ISSN: 0148-7280
    Keywords: spermatozoa ; acrosome ; fertilization ; hamster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Adult female golden hamsters were induced to superovulate. When they were mated several hours prior to ovulation or artificially inseminated about the time of ovulation, nearly 100% of their eggs were subsequently fertilized monospermically. During the progression of fertilization when the eggs were still surrounded by compact cumulus oophorus, the contents of the ampullary region of the oviducts were collected and spermatozoa moving in the ampullary fluid, within the cumulus and on/in the zonae pellucidae of unfertilized eggs, were examined by light and electron microscopy to evaluate the status of their acrosomal caps.Most spermatozoa swimming in the ampullary fluid had apparently intact acrosomal caps, while the vast majority moving within the cumulus had distinctly modified acrosomal caps. Most spermatozoa that had passed through the cumulus and reached the zona surfaces had remnants of their acrosomal caps (“acrosomal ghosts”). When the ghosts were present around the sperm heads on the zona, the heads pivoted about a point roughly corresponding to the places where the ghosts were located. The ghosts seemed to firmly attach to the zona surfaces, then were split open by the sperm heads and left behind as the sperm heads advanced into the zona. A few spermatozoa on the zona surfaces had no acrosomal ghosts (at least not detectable by light microscopy). In this case, the sperm head pivoted about either the inner acrosomal membrane or the equatorial segment of the acrosome. In no instance were spermatozoa with intact acrosomal caps found on zona surfaces.We infer from these observations that most spermatozoa in vivo initiate their acrosome reactions while they are advancing through the cumulus. When they arrive at the zona surfaces, acrosomal ghosts are generally present on the sperm heads. These ghosts appear to hold sperm heads to zona surfaces as well as to restrict the direction of advancement of sperm head through the zona. In a minority of cases, ghostless spermatozoa reach the zona surfaces. As these spermatozoa appear to be able to penetrate the zona successfully, structures other than the acrosomal ghost (ie, the inner acrosomal membrane and the plasma membrane over the equatorial segment of the acrosome) may also attach to zona surfaces before spermatozoa penetrate into the zona.
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    Effects of egg aging on in vitro fertilization and first cleavage division in the hamster (1983)
    New York, NY : Wiley-Blackwell
    Gamete Research 8 (1983), S. 219-230 
    Details
    ISSN: 0148-7280
    Keywords: egg aging ; fertilization ; cleavage anomalies ; hamster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The postovulatory fertile life of mammalian eggs is remarkably short (approximately 6-36h). Anomalies of embryogenesis may result from fertilization of aged, defective eggs. Attempts to study this problem using whole-animal models are complicated by chances in the natural milieu of the gametes. In the present study, postovulatory hamster eggs were allowed to agein vivo then fertilized in vitro. Cumulus-intact eggs recovered from superovulated hamsters either 2 or 9 h after the estiated time of ovulation (12 h postHCG) were incubated for 4 h with preincubated sperm suspentions in a modified Tyrode's solution devised for in vitro fertilization. Eggs were either fixed or cultured for another 20h in fresh medium to allow cleavage to occur, then examined by light microscopy (phase and interference-contrast). No significant difference was found in the ablities of fresh and aged eggs to be penetrated by spermatozoa (94% vs 90%, respectively; 8 replicated experiments), but only 59% of penetrated aged eggs were found to undergo morphologically normally fertilization (2 polar bodies, 2 prounclei) compared with 75% of fresh eggs (difference significant, P〈 0.01). About 13% of eggs were polyspermic in both categories. The most common anomaly in aged fertilied egges was failure to extrude the second polor body (23% off eggs vs 8% of fresh eggs, P 〈 0.01). Only 21% of aged eggs underwent first cleavaage, and only 74% of these appeared morphologically normal, compared with value of 68% and 98%, respectively, for fresh eggs. These data show that in the hamster, abnormal fertilization and cleavage failure can, in part, be directly attributed to postovulatory deterioration of eggs. We also infer that the apparently very short penetrable life of hamster eggs in vivo shown by previous investigators is an indirect effect of postovulatory changes in the female reproductive tract that are unfavorable for sperm-egg interactions.
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    Acrosomal reaction and early events at fertilization in Marthasterias glacialis (Echinodermata: Asteroidea) (1985)
    New York, NY : Wiley-Blackwell
    Gamete Research 11 (1985), S. 157-167 
    Details
    ISSN: 0148-7280
    Keywords: acrosomal reaction ; fertilization ; Asteroidea ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: When the spermatozoon of M glacialis contacts the mature oocyte jelly it adheres to it. Following this, there is a slight tumefaction of the acrosome, which is followed by the disruption of the apical acrosomal vesicle and cytoplasmic membranes. Acrosomal vesicle contents are liberated and spread along the outer surface of the oocyte jelly. Meanwhile, the acrosomal process begins to extend, penetrates all the jelly extension, then the vitelline layer, and finally contacts the cytoplasmic egg membrane. Nevertheless, the sperm cell continues lying at the outer border of the jelly. From the beginning of the acrosome reaction the dense and finely fibrillar subacrosomal material is connected, by some expansions, to the basal acrosomal vesicle membrane. Both nuclear and mitochondrial diameters have diminished.
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    URL: http://dx.doi.org/10.1002/mrd.1120110206
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    Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments (1989)
    New York, NY : Wiley-Blackwell
    Gamete Research 22 (1989), S. 443-469 
    Details
    ISSN: 0148-7280
    Keywords: sperm ; transport ; mucus ; cumulus ; zona pellucida ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mechanisms of mammalian sperm migration through the female reproductive tract and ovum vestments are described. The perspective is biophysical as well as biochemical and morphological, and the focus is upon the role of sperm motility in these processes. Sperm forward progression is characterized as an interactive process between the the cell and its environment, and the mediation of flagellar bend propagation by the physical properties of its surroundings is described. These properties, together with flagellar beat kinematics, sperm morphology, and surface properties, determine the magnitude of the forces generated by sperm and their consequent rate of progression. Sperm interactions with the cervical mucus, the cumulus oophorus, and the zona pellucida are described. The poorly understood affinity of the sperm surface for the macromolecules of the mucus, cumulus, and zona is stressed, as is the viscoelastic structural mechanical resistance of these biopolymers to sperm motion. The kinematics and consequences of hyperactivated sperm motion are presented, with emphasis on objective characterization of such motion (as a biomarker), along with analysis of the mechanical advantage that such motion may confer on spermatozoa during egg-vestment interaction.
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    URL: http://dx.doi.org/10.1002/mrd.1120220410
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    Identification, isolation, and properties of a plasma membrane protein involved in the adhesion of boar sperm to the porcine zona pellucida (1989)
    New York, NY : Wiley-Blackwell
    Gamete Research 23 (1989), S. 103-118 
    Details
    ISSN: 0148-7280
    Keywords: gamete-interactions ; fertilization ; mammals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Boar sperm plasma membranes contain an integral protein (Mr 55 kDa) that apparently functions in the adhesion of sperm to the zona pellucida (Peterson and Hunt: J Cell Biol 105:170a, 1987.) In experiments described in this report, the protein is identified after additional steps of purification involving lectin affinity chromatography and preparative PAGE. An active form of the adhesion protein (APz) develops or becomes first exposed in the corpus epididymis and is fully active in the cauda epididymis; a significant portion of this conformationally labile protein, while integral to the plasma membrane, cannot be solubilized by nonionic detergents and may be associated with the membrane skeleton. APz does not exhibit enzymatic properties thought possibly to be involved in sperm-zona interaction in this and other species. Galactosyltransferase substrates and inhibitors and anliproteases including soybean trypsin inhibitor, pepstatin, leupeptin, and p-aminoben-zamidine failed to block sperm from binding to porcine eggs. Boar sperm proacrosin and antiproacrosin antibody failed to inhibit sperm-egg binding. When plasma membranes or fractions containing APz that bind to dextran sulfate agarose were chromatographed on L-fucose agarose, a sugar which binds proacrosin, plasma membrane proteins that bound to the column failed to absorb anti-APz antibody, Anti-APz was absorbed by fractions that did not contain proacrosin. These data indicate that APz is not proacrosin. Since anti-APz monovalcnt antibody raised from whole cauda or corpus sperm plasma membranes or from chromatographic fractions containing APz completely block capacitated sperm from binding to eggs, and since the ability of this antibody to be absorbed develops as sperm become capable of binding to eggs, we view AP, to be the major and perhaps only plasma membrane protein involved in the adhesion of capacitated boar sperm to eggs prior to the acrosome reaction.
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    Ethanol inhibits human and hamster sperm penetration of eggs (1987)
    New York, NY : Wiley-Blackwell
    Gamete Research 16 (1987), S. 97-107 
    Details
    ISSN: 0148-7280
    Keywords: human spermatozoa ; fertilization ; hamster spermatozoa ; acrosome reaction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of alcohol on the fertilizing ability of both human and hamster spermatozoa was examined by an in vitro fertilization assay using hamster ova. Spermatozoa were incubated in capacitating media for 3 hr (hamster sperm) and 4 hr (human sperm). Hamster ova were inseminated with preincubated sperm and were examined after 2 to 3 hr. Ethanol was added to the capacitating media at concentrations of 25, 50, 100, 200, and 400 mg%. Fertilization of zona-free hamster eggs by human spermatozoa was reduced from 49.6% in no alcohol to 16.7% in 400 mg% ethanol. Fertilization of hamster eggs by hamster sperm revealed a reduction from 63.6% to 33.7% in cumulus-intact eggs and from 65.8% to 10.8% in cumulus-free eggs in the presence of ethanol at 400 mg%. Hamster sperm acrosome reaction was reduced from 47% to 12%. When these hamster sperm with reduced acrosome reaction were placed with zona-free hamster eggs, the 100% fertilization rate was not reduced; however, the fertilization index, which reflects the number of swelling sperm heads per egg, was reduced from 8.5 to 1.8. This suggests that as little as 12% of the sperm with an acrosome reaction is sufficient to fertilize 100% of the zona-free eggs. If ethanol was added to the insemination media only, there was no inhibition of fertilization by human sperm or hamster sperm that had been previously capacitated in an ethanol-free media. Removal of the ethanol from the preincubated sperm produced fertilization at control levels; thus the inhibitory effect is reversible. These results indicate that ethanol may affect fertilization by an inhibition of the capacitation and/or acrosome reaction process.
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    Effects of glutathione (GSH and GSSG) and glutathione reductase (GR) on zona-free hamster oocyte ability to decondense human sperm (1989)
    New York, NY : Wiley-Blackwell
    Gamete Research 24 (1989), S. 21-30 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; chromatin decondensation ; egg activation ; cortical organization ; calcium regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Effects of reduced glutathione (GSH), oxidized glutathione (GSSG), or glutathione reductasc (GR) supply were studied on the ability of hamster oocytes to be fertilized by human sperm. Zona-free oocytes were pretreated with these compounds prior to sperm insemination. Oocyte pretreatment with high concentrations of GSH or GSSG (50 or 100 mM. 30 min) significantly increased the penetrated oocyte rate (PR). Polyspermy was not increased except when high concentrations of GSH (100 mM) were used. Incubation of oocytes with GR (1 or 10IU/ml) prior to sperm insemination induced increasing dose-dependent PR. Polyspermy increased significantly with 10 mM GR in oocyte incubation medium. Oocyte incubation for 30 min with the sulfhydryl blocking agent iodoacetamide (1 mM) led to a drastic decrease in oocyte penetration and in polyspermy.Our results demonstrate an original way to increase the efficacy of human-hamster heterospecific fertilization. Various hypotheses are discussed explaining these observations which open new investigations for heterospecific and homospecific in vitro fertilization.
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    Isolation of 125I-concanavalin A-labeled plasma membrane from unfertilized mouse eggs (1987)
    New York, NY : Wiley-Blackwell
    Gamete Research 16 (1987), S. 303-310 
    Details
    ISSN: 0148-7280
    Keywords: Zona-free mouse egg ; plasma membrane ; concanavalin A ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A procedure was developed for isolation of plasma membrane (PM) preparations from unfertilized mouse eggs. Zona-free mouse eggs prepared by the method of Boldt and Wolf (Gamete Res 13:213-222, 1986) were labeled with 125I-concanavalin A (ConA) prior to sonication and fractionation on iso-osmotic self-generated Percoll density gradients. Experiments using the ConA-specific sugar α-methylmannoside (αMM) indicated that 125I-ConA bound specifically to the egg PM. Greater than 95% of 125I-ConA binding to zona-free eggs was blocked in the presence of 0.1 M αMM, and incubation of eggs in αMM after 125I-ConA labeling caused release of 85-90% of bound label. Fractionation of 125I-ConA-labeled eggs by Percoll density gradient centrifugation yielded a single radioactive peak at density = 1.025, corresponding to egg PM material. Prolonged incubation of 125I-ConA-labeled eggs or egg sonicates prior to fractionation did not alter the location of the radioactive peak, indicating that 125I-ConA did not label other organelles. As a control, human erythrocytes were labeled with 125I-ConA and fractionated under identical experimental conditions and yielded a single radioactive peak at density (1.020) comparable to that observed for 125I-ConA-labeled eggs. These results indicate that 125I-ConA can be used as a specific marker to support PM isolation from small numbers of zona-free mouse eggs.
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    Synthetic organic pH buffers can support fertilization of guinea pig eggs, but not as efficiently as bicarbonate buffer (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 19 (1988), S. 123-129 
    Details
    ISSN: 0148-7280
    Keywords: spermatozoa ; fertilization ; bicarbonate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Bicarbonate ions (HCO3-) in the medium are not absolutely essential for the fertilization of guinea pig eggs. However, fertilization takes place most efficiently in HCO3--buffered medium. Capacitation, the acrosome reaction, and zona penetration by spermatozoa can occur in HCO3--free media with synthetic organic buffers (i.e., MOPS, TES, HEPES, Tris, and TAPSO) but not as efficiently as in the HCO3--buffered medium. It appears that HCO3- functions as more than just a pH-buffering molecule.
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    In vitro and in vivo studies of mammalian sperm nuclear decondensation (1985)
    New York, NY : Wiley-Blackwell
    Gamete Research 11 (1985), S. 349-365 
    Details
    ISSN: 0148-7280
    Keywords: spermatozoa ; mammalian ; fertilization ; mammalian ; nucleus ; spermatozoon ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 22 Ill.
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    URL: http://dx.doi.org/10.1002/mrd.1120110403
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  • 96
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    Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 15 (1986), S. 161-175 
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    ISSN: 0148-7280
    Keywords: sperm enzymes ; acrosome ; fertilization ; testis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH4)2SO4 precipitation, Con A-Sepharose 4B chromatography, and Sephadex G-200 chromatography to a specific activity of 27.4 μmol Ala3 hydrolyzed min-1 mg-1 protein. Chromatography on a calibrated G-200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64-66,000 daltons. The enzyme had optimal activity toward hydrolysis of L-alanyl-alanyl-alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin-layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH4)2SO4 fraction were reacted with the specific DPP II substrate, lysyl-alanyl-4-methoxy-2-napthylamide. Analytical isoelectric focusing of the G-200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl-alanyl-7-amino-4-trifluoromethylcou-marin, revealed multiple isoforms focusing at pI = 4.8-5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120150207
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  • 97
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    In vitro fertilization with normal development in the sheep (1987)
    New York, NY : Wiley-Blackwell
    Gamete Research 16 (1987), S. 159-170 
    Details
    ISSN: 0148-7280
    Keywords: ovine ; oocyte ; spermatozoa ; maturation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two-to six-cell stage within 40 h (75. 8% for ovulated and 62. 6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20-22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two- to six-cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61. 9%) and 10 (66. 6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained ( 〉 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two- to four-cell) from tubal oocytes and ten embryos (two- to six-cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress ( 〉 3 months).
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120160207
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  • 98
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    Incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro (1987)
    New York, NY : Wiley-Blackwell
    Gamete Research 16 (1987), S. 205-216 
    Details
    ISSN: 0148-7280
    Keywords: cryopreservation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To assess the effect of low temperature storage on mouse oocytes we (1) examined the capacity for normal development of embryos derived from frozen oocytes fertilized in vitro after transfer to pseudopregnant foster mothers and (2) analyzed the chromosome complement at the first cleavage division. Fewer frozen than control oocytes were fertilized (36% vs 66%), but after embryo transfer the proportion of fertilized eggs that implanted (67-68%) and formed normal foetuses (50-53%) was similar in the two groups. Freezing did not affect the observed incidence of aneuploidy (1.5-3.3%). The frequency of polyploid embryos derived from frozen oocytes was almost double that of controls (15.8% vs 8.5%), but it is unclear whether this is a real effect of freezing or is an artifact produced by the chromosome preparation technique.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120160303
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  • 99
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    Superovulation in immature and mature Chinese hamsters (1987)
    New York, NY : Wiley-Blackwell
    Gamete Research 16 (1987), S. 281-290 
    Details
    ISSN: 0148-7280
    Keywords: eggs ; superovulation ; fertilization ; Chinese hamster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mature female Chinese hamsters ovulate an average of 8.8 ± 1.0 (mean ± SD) eggs per female in each estrous cycle. Superovulation can be induced in both immature and mature females by subcutaneous or intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and either human chorionic gonadotropin (hCG) or pituitary luteinizing hormone (PLH). The best superovulation in immature females was induced by the administration of 15 IU of PMSG followed 72 hr later by injection of 15 IU of hCG (about 25 eggs per female) or 0.2 mg (200 IU) PLH (about 46 eggs per female). Ovulation started about 13-15 hr after administration of hCG (or PLH) and was completed during the next 5-6 hr. Superovulation in mature females could be induced by injecting PMSG any day of the estrous cycle, but the best superovulation (about 39 eggs per female) was induced by injecting 15 IU of PMSG on day 1 (day of ovulation) followed by the injection of 0.4 mg of PLH 72 hr later. When immature females treated with the best superovulatory protocol were mated on the evening of PLH injection, only 5% of the eggs were found fertilized 50 hr after PLH administration. On the other hand, about 60% of the eggs were found fertilized in mature females mated following treatment with the best superovulatory protocol. The majority (83-85%) of superovulated eggs obtained from both immature and mature females were normally fertilized in vitro.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120160402
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  • 100
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    Impaired ability of human spermatozoa to penetrate zona-free hamster oocytes: Is a postacrosomal sheath anomaly involved? (1987)
    New York, NY : Wiley-Blackwell
    Gamete Research 17 (1987), S. 145-156 
    Details
    ISSN: 0148-7280
    Keywords: fertilization ; postacrosomal sheath ; sperm ; zona-free hamster egg penetration ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We selected 17 infertile men whose sperm ultrastructural study revealed at least 70% of spermatozoa with postacrosomal sheath (PAS) anomalies. Among the other sperm head defects, those affecting the nuclear shape were most frequently encountered and were highly correlated with PAS anomalies (r = +0.71; P 〈 .01). PAS anomalies were also correlated with chromatin condensation defects (r = +0.67; P 〈 .01) and acrosome anomalies (r = +0.53; P 〈.05). Those spermatozoa were tested for their ability to penetrate zona-free hamster oocytes and were compared to a control sperm population. It was shown that sperm head morphological anomalies impaired the ability of spermatozoa to attach to and penetrate the oocyte. The highest significant and negative correlations were found between the penetration rate and 1) the percentage of spermatozoa with PAS anomalies (r = -0.81; P 〈 .01) and 2) the percentage of spermatozoa with nuclear shape anomalies (r = -0.66; P 〈 .01). The effect of PAS anomalies on human fertilization process are discussed.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120170206
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