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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 3 (1983), S. 513-524 
    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.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 1 (1980), S. 31-40 
    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.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 3 (1983), S. 213-226 
    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.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 22 (1983), S. 235-244 
    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.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 3 (1980), S. 211-216 
    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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  • 6
    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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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 3 (1980), S. 45-57 
    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.
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  • 8
    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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  • 9
    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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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 61-70 
    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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  • 11
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 5 (1982), S. 167-179 
    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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  • 12
    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.
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  • 13
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 9 (1984), S. 261-272 
    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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  • 14
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 8 (1983), S. 65-78 
    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.
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  • 15
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 10 (1984), S. 241-251 
    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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  • 16
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 8 (1983), S. 97-117 
    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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  • 17
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Gamete Research 9 (1984), S. 115-125 
    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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  • 18
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 9 (1984), S. 329-338 
    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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  • 19
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    Gamete Research 10 (1984), S. 77-82 
    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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  • 20
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    Gamete Research 10 (1984), S. 97-105 
    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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  • 21
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    Gamete Research 10 (1984), S. 107-118 
    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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  • 22
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    Gamete Research 10 (1984), S. 415-422 
    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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  • 23
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    Gamete Research 3 (1980), S. 309-316 
    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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  • 24
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    Gamete Research 4 (1981), S. 15-23 
    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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  • 25
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    Gamete Research 4 (1981), S. 525-533 
    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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  • 26
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    Gamete Research 5 (1982), S. 1-9 
    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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  • 27
    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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  • 28
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    Gamete Research 5 (1982), S. 181-190 
    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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  • 29
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    Gamete Research 5 (1982), S. 239-256 
    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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  • 30
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    Gamete Research 5 (1982), S. 323-344 
    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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  • 31
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    Gamete Research 5 (1982), S. 217-227 
    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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  • 32
    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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  • 33
    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.
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  • 34
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    Gamete Research 6 (1982), S. 215-223 
    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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  • 35
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    Gamete Research 7 (1983), S. 111-122 
    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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  • 36
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    Gamete Research 10 (1984), S. 267-281 
    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.
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  • 37
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    Gamete Research 9 (1984), S. 1-19 
    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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  • 38
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    Gamete Research 8 (1983), S. 219-230 
    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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  • 39
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    Gamete Research 4 (1981), S. 25-34 
    ISSN: 0148-7280
    Keywords: fertilization ; pronuclei ; chromatin decondensation ; protamine ; kinase ; glutathione reductase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to derive information about possible mechanisms by which the sperm head is converted into the male pronucleus during fertilization in the rabbit, unfertilized egg homogenate was assayed for two enzyme activities. Protamine was extracted from rabbit sperm, purified, and labelled with [14C] in an in vitro reaction and used as a probe to assay for a protein kinase which could transfer [32P]PO4 from [γ-32P]ATP onto the substrate. A kinase with a pH optimum of approximately 8.0 to 8.5 is described. Assays for the enzyme glutathione reductase were performed using homogenates from eggs or embryos at three early stages of development. Results suggest that oocytes can oxidize 2.58 × 10-6 μmol NADPH per minute per oocyte, unfertilized eggs 5.16 × 10-7 μmol NADPH per minute per ovum, and 20- to 24-hour postcoitus fertilized eggs 2.30 × 10-6 μmol NADPH per minute per ovum. The relevance of these observations to male pronuclear formation is discussed.
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  • 40
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    Gamete Research 4 (1981), S. 219-229 
    ISSN: 0148-7280
    Keywords: fertilization ; sea urchin ; Arbacia punctulata ; Erythrosin B ; dyes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Fertilization and ionophore activation of the sea urchin Arbacia punctulata were inhibited in the presence of six analogs of the dye fluorescein. The concentration of any one dye needed for blockage of sperm or Ca-ionophoremediated activation in 50% of the eggs (I50) was a function of the dye's lipid solubility. Substantially higher concentrations of each dye were required to block activation by Ca-ionophore (A23187) than were needed to inhibit sperm activation. A detailed study of the action of Erythrosin B (tetraiodofluorescein) showed that its effects were readily reversible. The I50 concentration of Erythrosin B increased as temperature increased from 10 to 25°C. The kinetics of blockage indicated that Erythrosin B blocked some early step in the program of fertilization. The results suggest that these anionic dyes may inhibit fertilization by preventing successful sperm-egg fusion.
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  • 41
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    Gamete Research 5 (1982), S. 161-166 
    ISSN: 0148-7280
    Keywords: fertilization ; bonellin ; porphyrin ; crosslinking of membrane proteins ; cholesterol oxidation ; cleavage formation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In this paper we report on the mechanism of action of bonellin on sea urchin eggs. Bonellin acts in the same way as porphyrins, ie, by causing the crosslinking of membrane proteins and the peroxocompounds formation. A possible role of membrane organization related to cleavage formation is discussed.
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  • 42
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    Gamete Research 6 (1982), S. 199-207 
    ISSN: 0148-7280
    Keywords: hamster ; sperm aging ; fertilization ; ova ; zygotes ; triploidy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of aging golden hamster spermatozoa in the female reproductive tract on the percentage of ova fertilized, the stage of development, and the chromosome complement of the resulting zygotes were studied. Females were inseminated artificially with cauda epididymal sperm at 6 h (control), 10, 15,18, or 21 h before the estimated time of ovulation. A decrease in the percentage of ova fertilized was found as the time spermatozoa were aged in utero prior to ovulation increased. The zygotes collected at 56 h postovulation from females inseminated 15 or 18 h prior to ovulation were delayed in development, as judged by the number of blastomeres. Although an increase of chromosomally abnormal zygotes was not found, a possibility exists that mosaicism may have been present, as evidenced by zygotes with unequal sized blastomeres, and went undetected.
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  • 43
    ISSN: 0148-7280
    Keywords: egg fusion ; sperm ; capacitation ; fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4-4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 104 per ml and an optimum was attained at a concentration of 5.0 × 105 per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.
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  • 44
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    Gamete Research 6 (1982), S. 305-313 
    ISSN: 0148-7280
    Keywords: albumin ; capacitation ; fertilization ; spermatozoa ; zona pellucida ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Serum albumin is an obligatory component of the incubation medium for the fertilization of mouse ova. Normal, untreated bovine serum albumin supports high rates of fertilization of cumulus-free ova both with and without their zonae pellucidae. Heat-treated or trichloroacetic acid-extracted bovine serum albumin is unable to support the fertilization of a majority of zona-intact ova but fertilization of zona-free ova is unimpaired. Spermatozoa incubated in medium containing heated bovine serum albumin fertilize zona-intact ova when 2 mM caffeine is present but the progress of sperm head decondensation is delayed when compared to normal controls. Trichloroacetic acid extracted BSA preferentially and irreversibly inhibits zona penetration by spermatozoa, but this effect is not mediated by an inhibition of spermatozoal motility or zona-binding ability. This effect occurs after only a 10-min preincubation of the spermatozoa in the extracted BSA or when the medium contains only a 10% (v/v) proportion of this albumin. It is estimated that mouse spermatozoa under the conditions used take 2 hr to penetrate the zonae pellucidae of 50% of ova and effect fertilization.
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  • 45
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    Gamete Research 7 (1983), S. 103-109 
    ISSN: 0148-7280
    Keywords: mouse ; strains ; media ; fertilization ; in vitro ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Success rates of superovulation in response to gonadotropic hormone treatment and in vitro fertilization (ie, mitotic cleavage following insemination) of mouse eggs from outbred CD-1, hybrid CB6Fl, or hybrid B6CBAF1, mice were compared using either a mouse inseminationmedium, modified Krebs-Ringer-bicarbonate (m-KRB), or a human insemination medium, Ham's F10 nutrient mixture. Inseminations were performed in either organ culture dishes or screw-top, flat-side tissue culture tubes. Mean superovulation rates (± SD) were 24.2 (5.1) for CD-1, 33.0 (5.8) for CB6F1, and 16.3 (6.6) for B6CBAF1 mice. For in vitro cleavage the best combination of mouse strain, insemination medium, and culture container was achieved using CB6F1, mice, m-KRB medium, and culture tubes. However, Ham's medium used with either hybrid mouse strain was shown to be employable for fertilization of mouse eggs in vitro as a quality control assay and/or experimental model system for testing the human in vitro fertilization procedure.
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  • 46
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    Gamete Research 9 (1984), S. 399-408 
    ISSN: 0148-7280
    Keywords: sperm ; fertilization ; decondensation ; nuclear protein ; mouse ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Zona-free hamster eggs have been fertilized in vitro with boar spermatozoa in a medium enriched by arginine-3H and the sites of localization of newly synthesized arginine-3H-labeled proteins have been investigated using fine-structure autoradiography. It was confirmed that such proteins are synthesized during fertilization and that they accumulate to a notable degree in decondensing sperm chromatin as well as in the chromatin of the female pronucleus and also of the second polar body. A similar process did evidently take place also in defective pronuclei, characterized by a core of a still condensed chromatin and by remaining nuclear membrane. In such male pronuclei the highest concentration of the label was seen just on the border of the condensed chromatin, on the expected site of nuclear protein exchange. It is supposed that, at least in this experimental system, any morphologically detectable sperm decondensation is accompanied immediately by a shift from sperm basic nuclear proteins to other nuclear proteins.
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  • 47
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    Gamete Research 10 (1984), S. 9-19 
    ISSN: 0148-7280
    Keywords: fertilization ; polyspermy ; sea urchin eggs ; sperm peroxidase ; anti-inflammatory drugs ; cyclooxygenase ; prostaglandins ; arachidonic acid cascade ; indomethacin ; flufenamic acid ; meclofenamate ; aspirin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sea urchin eggs are known to release H2O2 during the cortical reaction at fertilization to help prevent polyspermy by inactivating excess sperm in the vicinity. This process resembles the peroxidatic killing of bacteria by phagocytic leukocytes during inflammation. Associated with these reactions in leukocytes, arachidonic acid is released from phospholipids and can be oxidized via the cyclooxygenase pathway to produce prostaglandins. Nonsteroidal anti-inflammatory drugs (NSAID) that are cyclooxygenase inhibitors in somatic cells were used to determine whether Arbacia punctulata and Strongylocentrotus purpuratus eggs use these processes to help prevent polyspermy. The potent cyclooxygenase inhibitor indomethacin causes a dose (10-100 μM) and sperm density dependent induction of polyspermy if added before the egg completes the cortical reaction. It does not retard elevation of the fertilization envelope and does not promote polyspermy by protecting sperm from peroxidatic inactivation by egg-derived H2O2. Other potent cyclooxygenase inhibitors, flufenamate and meclofenamate, also induce polyspermy at 10-60 μM. Aspirin, a weak cyclooxygenase inhibitor in somatic cells, does not cause polyspermy at 5 mM. These findings provide evidence that prostaglandins or other cyclooxygenase-derived metabolites may help assure monospermic fertilization in sea urchins.
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  • 48
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    Gamete Research 8 (1983), S. 279-293 
    ISSN: 0148-7280
    Keywords: fertilization ; jelly coat ; species specificity ; sperm enzyme ; sea urchin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A species-specific factor capable of disersing the jelly coat surrounding eggs has been purified from sperm of the sea urchin, Anthocidaris crassisina. It does not exert its effect on the vitelline layer. The purification has been accomlished by a four-step procedure involving ammonium sulfate fractionation, gel filtration on Sepharose CL-4B, ion-exchange column chromatography on DEAE-cellulose, and affinity column chromatograhy on heparin-Seharose CL-6B. The isolated factor is homogenous in sodium dodecyl sulfate polyacrylamide gel electrohoresis in the presence or absence of β-mercatoethanol, estimated molecular weight being about 140,000.The jelly dispersion by the present factor is activated by CaCl2, and inhibited by KCl, MnCl2, EDTA, and EGTA, and by sulfated saccharides such as chondroitin sulfate A and C, heparin, and glucose-6-sulfate, Inorganic sulfated such as (NH4)2SO4 and Na2SO4 have no effect on jelly dispersion. This factor is heat-labile, its activity in 30 min at 50°C.The present factor is found also in the seminal Plasma, and released from sperm themselves by treatment with Triton X-100 .These results suggest that this factor is loosely bound to the serm surface. Although glycosidase and arylsulfatase activities are detectable in the seminal plasma, these enzyme activities are not detectable in the purified jelly disersing factor.Only trypsin and α chymotrysin among commercial enzymes tested dispersing activity is inhibited neither by trypsin inhibitors such as N-α-p-tosyl-L-lysine-chloromethyl ketone, soybean trypsin inhibitor, ovomucoid trypsin inhibitor, nor by chymotrypsin inhibitors such as L-1-tosylamide-2 pheny-ethylcholoromethyl ketone and chymostatin Participation of trysin-like and chymotrypsin-like enzymes in jelly dispersion seems unlikely.
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  • 49
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    Bioelectromagnetics 4 (1983), S. 295-301 
    ISSN: 0197-8462
    Keywords: fusion reactors ; magnetic fields ; biological effects ; fertilization ; fish ; Life and Medical Sciences ; Occupational Health and Environmental Toxicology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Physics
    Notes: The sensitivity of trout ova and sperm to 1-T magnetic fields was investigated. It was determined that (1) overall test results combining seven independent Z-statistics demonstrated a significant (α 〈 0.0001) enhancement of fertilization when ova alone were exposed to the magnetic field prior to fertilization; (2) similarly, overall test results combining Z-statistics from eight independent experiments indicated a significant (α 〈 0.0004) enhancement when sperm alone were exposed; and (3) statistical analysis of nine independent experiments confirmed enhanced fertilization (α 〈 0.0001) when both ova and sperm were exposed to the magnetic field prior to fertilization. Although these data indicated that both ova and sperm were sensitive to magnetic fields, simultaneous exposure of both gametes did not have a greater total effect on fertilization rate than the sum of their individual effects.
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