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  • Cell & Developmental Biology  (6,449)
  • 1990-1994  (6,449)
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  • 1
    ISSN: 0886-1544
    Keywords: cleavage furrows ; cytokinesis ; actin ; phalloidin ; myosin ; filamin ; talin ; attachment plaques ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: PtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows. © 1994 Wiley-Liss, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 161-168 
    ISSN: 0886-1544
    Keywords: fluorescent nucleotide analogs ; methylanthraniloyl ATP ; anthraniloyl ATP ; Chlamydomonas ; axonemal mutants ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Substrate analogs are useful for studying the structures of active sites and for distinguishing between similar enzyme activities. Fluorescent ribose-modified ATP analogs were used to investigate the functional differences between dynein ATPases. These analogs reactivate (support the movement of) sea urchin sperm axonemes, yet they do not reactivate wild-type Chalmydomonas axonemes. Surprisingly, the analogs reactivate the axonemes of mutants completely missing the outer arm dyneins. Competition experiments using ATP and these analogs provide strong evidence that the analogs bind to all dynein active sites but fail to release a subset of dyneins from rigor. We suggest that this subset of Chlamydomonas outer arm dyneins unable to use the analogs remains in rigor in the presence of the analogs and paralyzes the axoneme. © 1994 Wiley-Liss, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 4
    ISSN: 0886-1544
    Keywords: sliding movement ; 22S dynein ; Tetrahymena cilia ; dynein-track ; singlet microtubule ; ATP ; polarity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chlamydomonas and Tetrahymena axonemal dyneins have previously been found to bind to porcine brain microtubules to produce a microtubule-dynein complex. At appropriate microtubule:dynein concentration, microtubules in the complex became covered to saturation by dynein arms of the same polarity and at a spacing of 24 nm [Haimo et al., 1979; Haimo and Fenton, 1988; Haimo, 1989; Porter and Johnson, 1983a].In the present study, two different types of microtubule-dynein complexes (α-and β-complexes) were prepared from Tetrahymena ciliary 22S dynein and porcine brain tubulin. The characteristics of the adenosine triphosphate (ATP)-induced extrusion of microtubules from these complexes were analyzed, as a simple and direct in vitro assay for the ATP-induced extrusion of single microtubules. The α-complex prepared by adding dynein to microtubules showed an interrupted sliding movement, which would stop and start several times following the addition of ATP. In the β-complex, prepared by adding dynein bound to DEAE-tubulin to pre-assembled microtubules, microtubules became covered with dynein molecules whose orientation and binding were uniform with respect to microtubule polarity. The microtubules in the β-complex extruded at 12 μm/second following the addition of ATP. Dark-field and electron microscopy indicated that the extruded microtubules had undergone sliding on a dynein-track that had become detached from the complexes and had been absorbed onto the surface of the glass slide. At higher light intensity under a dark-field microscope, the dynein-track was seen to be composed of rows of dynein molecules arranged densely. The orientation of dynein molecules in rows appeared to be uniform considering the images of bound dynein in the β-complex under electron microscope. The higher sliding velocity of the microtubules on these dynein-tracks compared to that seen on slides coated at random with dynein [Vale and Toyoshima, 1988, 1989], may be due to more efficient force generation by this dense arrangement of dynein molecules with the same polarity on the tracks. © 1994 Wiley-Liss, Inc.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 206-218 
    ISSN: 0886-1544
    Keywords: sperm motility ; sperm maturation ; flagella ; protein kinases ; protein kinase inhibitor ; cGMP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ejaculated ram sperm were demembranated with Triton X-100, separated from the detergent-soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264-273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was ∼100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP-dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5-24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5-24) prior to reactivation. The cytosol-free models retained 〉90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X-100 alone, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selective inhibitor of cGMP-dependent protein kinase. © 1994 Wiley-Liss, Inc.
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  • 6
    ISSN: 0886-1544
    Keywords: microtubule-associated protein 2 ; neurons ; microtubule-associated proteins ; cytoskeleton ; dendrites ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule-associated protein 2 (MAP-2) is an abundant component of the cytoskeleton present in dendrites and cell bodies of neurons of the CNS. To examine the biological function of MAP-2, two MAP-2 antisense (AS) oligonucleotides complementary to the 5′ region of the rat MAP-2 cDNA were added to rat primary embryonic day 17-18 (E17-18) cultured cortical neurons 24 h after plating and neurite outgrowth and morphology studied. The treatment of primary cortical cultures with either of the two MAP-2 AS oligonucleotides resulted in decreased MAP-2 and reduction in the number of neuritic processes relative to the control or MAP-2 sense-treated cultures. By immunostaining and light microscopy the AS-treated neurons appeared smaller, more rounded, and less intensely stained for MAP-2 than the untreated or the MAP-2 sense-treated cultures. By electron microscopy disorganized microtubules and a reduction in the number of microtubules within neurites of the AS-treated cultures were observed. We conclude that MAP-2 continues to be required for microtubule spacing and stability within neurites once they have formed. © 1994 Wiley-Liss, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 1-24 
    ISSN: 0886-1544
    Keywords: myofibrillogenesis ; directionality ; non-muscle myosin II ; myosin ; α-actinin ; Z-bodies ; zeugmatin ; titin ; C-protein ; premyofibril ; nascent myofibril ; mature myofibril ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When cardiac muscle cells are isolated from embryonic chicks and grow in culture they attach to the substrate as spherical cells with disrupted myofibrils, and over several days in culture, they spread and extend lamellae. Based on antibody localizations of various cytoskeletal proteins within the spreading cardiomyocyte, three types of myofibrils have been identified: 1) fully formed mature myofibrils that are centrally positioned in the cell, 2) premyofibrils that are closest to the cell periphery, and 3) nascent myofibrils located between the premyofibrils and the mature myofibrils. Muscle-specific myosin is localized in the A-bands in the mature, contractile myofibrils, and along the nascent myofibrils in a continuous pattern, but it is absent from the premyofibrils. Antibodies to non-muscle isoforms of myosin IIB react with the premyofibrils at the cell periphery and with the nascent myofibrils, revealing short bands of myosin between closely spaced bands of α-actinin. In the areas where the nascent myofibrils border on the mature myofibrils, the bands of non-muscle myosin II reach lengths matching the lengths of the mature A-bands. With the exception of a small transition zone consisting of one myofibril, or sometimes several sarcomeres, bordering the nascent myofibrils, there is no reaction of these non-muscle myosin IIB antibodies with the mature myofibrils in spreading myocytes. C-protein is found only in the mature myofibrils, and its presence there may prevent co-polymerization of non-muscle and muscle myosins. Antibodies directed against the non-muscle myosin isoforms, IIA, do not stain the cardiomyocytes. In contrast to the cardiomyocytes, the fibroblasts in these cultures stain with antibodies to both non-muscle myosin IIA and IIB. The premyofibrils near the leading edge of the lamellae show no reaction with antibodies to either titin or zeugmatin, whereas the nascent myofibrils and mature myofibrils do. The spacings of the banded α-actinin staining range from 0.3 to 1.4 μm in the pre- and nascent myofibrils and reach full spacings (1.8-2.5 μm) in the mature myofibrils. Based on these observations, we propose a premyofibril model in which non-muscle myosin IIB, titin, and zeugmatin play key roles in myofibrillogenesis. This model proposes that pre- and nascent myofibrils are composed of minisarcomeres that increase in length, presumably by the concurrent elongation of actin filaments, the loss of the non-muscle myosin II filaments, the fusion of dense bodies or Z-bodies to form wide Z-bands, and the capture and alignment of muscle myosin II filaments to form the full spacings of mature myofibrils. © 1994 Wiley-Liss, Inc.
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  • 8
    ISSN: 0886-1544
    Keywords: cytoskeleton ; microtubule-associated protein (MAP) ; marine egg extracts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Alkaline pH favors the assembly of microtubules (MTs) in marine egg extracts [Suprenant and Marsh, 1987: J. Cell Sci. 184:167-180; Suprenant, 1989: Exp. Cell Res. 184:167-180; 1991: Cell Motil. Cytoskeleton 19:207-220] and mammalian brain extracts [Tiwari and Suprenant, 1993: Anal. Biochem. 215:96-103], even though the assembly of purified microtubule protein (MTP) from both of these sources is favored at slightly acidic pH. The present investigation examines whether alkaline pH has a direct or indirect effect on MT nucleation and growth in soluble brain extracts. Cell-free extracts were prepared from bovine cerebral cortex, and a nucleated assembly assay was used to demonstrate that MT assembly in brain extracts is favored at slightly acidic pH. The increase in MT mass found at alkaline pH is due to an increase in the solubility of tubulin not an increase in the extent of assembly On average, 47.7 ± 11.3% of the total tubulin is soluble at pH 7.2, while only 30.9 ± 8.9% of the tubulin is soluble at pH 6.8. A model is proposed that indicates how microtubule proteins from both mammalian brain and marine eggs may be associated with pH-dependent factors. © 1994 Wiley-Liss, Inc.
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  • 9
    ISSN: 1040-452X
    Keywords: Xenopus ; SP5 ; cDNA cloning ; Translational control ; Poly(A) tail ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: As part of our continuing program to understand the molecular mechanisms controlling the synthesis of sperm-specific nuclear proteins (SPs1-6) during spermatogenesis in Xenopus, we report here on the isolation of a cDNA clone for SP5, the partial sequencing of the amino acids in the SPs, and the expression of the mRNA for SP5. A cDNA clone (pXSP633) was isolated from a cDNA library, previously prepared from poly (A)+ mRNA obtained from Xenopus round spermatids. Determination of the amino acid sequence of the N-terminal regions of all the SPs(1-6) suggested that pXSP633 encodes SP5, whereas SPs3, 4, and 6 are derived from a second mRNA species, and SPs1 and 2 from a third mRNA species. Thus it seems likely that the six SPs are derived from three different mRNA species. Northern blot analyses of RNA, extracted from primary spermatocytes and round spermatids, was performed with oligonucleotide probes specific for SPs4 and 5 mRNAs. The results showed that whereas both SPs4 and 5 mRNAs are expressed in primary spermatocytes, the amount of SP5 mRNA is only about one-fifth of that of SP4 mRNA. However, both mRNA species undergo a similar size change in the length of their poly (A) tracts during spermatogenesis: the size of the mRNA in cultured round spermatids on day 0 was longer than that in primary spermatocytes, but the size of the mRNA in round spermatids on day 6 was shorter than that in round spermatids on day 0. © 1994 Wiley-Liss, Inc.
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  • 10
    ISSN: 1040-452X
    Keywords: Follistatin ; cDNA ; Bovine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Bovine follistatin cDNA sequences were derived using a cloning strategy based entirely on reverse transcription and polymerase chain reaction amplification of RNA derived from bovine ovarian and testicular tissues. Complete bovine follistatin cDNA coding sequences are presented including 1,029 bases of open reading frame, the 5′ translational start codon, and the 3′ translational stop codon. Homologies of bovine follistatin cDNA with pig, human, rat, and partial sheep sequences are 94.3%, 92.4%, 89.9%, and 98.4% at the nucleic acid level and 98.3%, 97.1%, 95.6%, and 100% at the deduced amino acid level, respectively. Northern blot analysis on a survey of bovine reproductive tissues showed strongest expression in ovaries collected from superovulated cows and major RNA species at 2.8 Kb and 1.75 Kb. © 1994 Wiley-Liss, Inc.
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  • 11
    ISSN: 1040-452X
    Keywords: Sex determination ; Sex determining region Y ; Postmeiotic expression ; HMG box containing proteins ; Interstitial cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Although its expression in adult testis was immediately apparent, the role for Sry (sex determining region, Y) in testicular function remains elusive. We have performed transcriptional studies in an effort to elucidate potential roles of Sry by studying the time and location of its transcription in mouse testes. Northern analyses and more sensitive nuclease protection assays detected transcripts in 28-day-old testes and beyond. The highly sensitive technique of reverse transcription polymerase chain reaction (RTPCR) could not detect Sry expression in 14-day testes when primers for the most conserved portion of the gene, the high mobility group (HMG) box, were used, but primers for the circular form detected Sry transcription at all postnatal stages studied. The same HMG box primers were able to detect expression of Sry in XX, Sxra or Sxrb testes. This suggested that Sry is expressed in cells other than germ cells, which was confirmed with studies on fractionated cells - RTPCR detected transcription of Sry in the highly pure interstitial cell fraction. However, Leydig cells and a Leydig cell tumor were negative for Sry expression. We performed in situ studies in an attempt to localize the expression of Sry in the testes. Abundant expression of an Sry cross-hybridizing transcript was found in spermatogonia, in early spermatocytes, and in some interstitial cells with antisense probes to the HMG box or a more specific, 3′ region, whereas the sense probe gave little or no hybridization. It is probable that the circular transcripts, which are seen in reverse transcriptase positive (RT+) and RT- reactions by PCR because of the RT activity of Taq polymerase, are responsible for the hybridization seen in spermatogonia and spermatocytes, whereas linear and circular forms are detected later. Thus Sry is expressed in pre- and postmeiotic germ cells and in somatic cells of the testes. © 1994 Wiley-Liss, Inc.
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  • 12
    ISSN: 1040-452X
    Keywords: Insulin-like growth factors ; Diabetes ; Embryos ; Mouse ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Insulin-like growth factors (IGF-I and IGF-II) play an important regulatory role in fetal growth and development. Alterations in expression of these growth factors may result in developmental abnormalities, macrosomia, and intrauterine growth retardation, which occur with a higher incidence in diabetic pregnancies. In situ hybridization histochemistry was employed to investigate the distribution and abundance of IGF-I and IGF-II in peri-implantation and postimplantation conceptuses from normal and streptozotocin-treated diabetic mice. Animals were sacrificed on gestational days 5, 6, 7, 8, and 9. The entire uterine horn was prepared for hybridization with antisense and sense α-35S-dATP labeled oligonucleotide probes for IGF-I, IGF-II, and mouse β-actin. IGF-I transcript was apparent only in myometrium at 6 days of gestation in normal and diabetic mice. IGF-II transcripts were restricted to trophoectoderm cells within the implantation chamber on day 5. Following implantation, IGF-II transcripts were found in trophoectodermal derivatives, primitive endoderm, mesoderm, heart, walls of the foregut, and mesenchyme in normal and diabetic postimplantation conceptuses. There were no apparent differences between normal and diabetic samples in the distribution and abundance of the IGF-II transcript from gestational days 7, 8, and 9. The embryos from the diabetic mother at day 6 were growth retarded and had a significant decrease in the expression of IGF-II. These results suggest that maternal hyperglycemia may retard development of the early implanting conceptus in a narrow window around day 6 through a mechanism involving decreased IGF-II expression. Fetuses from diabetic pregnancies that escape this critical period appear to develop and express IGF-II in an equivalent manner to those of the control group. © 1994 Wiley-Liss, Inc.
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  • 13
    ISSN: 1040-452X
    Keywords: Embryo culture ; Blastocyst formation ; Galactose ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: One-cell mouse embryos that block at the 2-cell stage can progress to the morula stage in CZB medium, but fail to cavitate and then swell and lyse. A 1-min exposure to 27 mM glucose at the 4-cell stage (∼42 hr) will support a high frequency of development to the blastocyst stage (75%) in the same medium. A glucose exposure is beneficial anytime between 30 and 54 hr of culture (67-73% blastocysts). Of a group of additional sugars and glucose analogues tested for their ability to replace glucose, only galactose was equivalent in promoting embryo development to the blastocyst stage (64% blastocysts). © 1994 Wiley-Liss, Inc.
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  • 14
    ISSN: 1040-452X
    Keywords: Preimplantation embryo ; Insulin ; IGFs ; Protein synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Previously constructed protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage, have been used to analyze the effects of insulin, IGF-I, and IGF-II on protein synthesis in these developmental stages. Proteins were labeled by placing, for 2 hr, synchronous cohorts of 35-50 embryos into human tubal fluid (HTF) medium containing L-[35S]-methionine (1 mCi/ml) in the presence or absence of one of the growth factors. The embryos were then washed with medium and lysed. Samples were processed for 2-D gel analysis. For each embryonic stage and each growth factor, four or five experimental replicates were done and the gel images were compared using the PDQUEST system. Using the computer-assisted analysis, we were able to identify proteins that showed a statistically significant (P 〈 0.05) change in synthesis. At the eight-cell stage of development insulin caused increased synthesis of two proteins and decreased synthesis in three proteins. Insulin-treated blastocyst stage embryos exhibited an increased synthesis in eight proteins and decreased synthesis for one protein. The effect of IGF-I at the eight-cell stage of development was mostly inhibitory; the synthesis of only one protein increased and the synthesis of five proteins showed a decrease. Similar results were obtained with blastocyst stage embryos; four proteins demonstrated an increase in synthesis while 14 proteins showed a decrease. Eight-cell stage embryos incubated with IGF-II had seven proteins with a decreased synthesis, although in blastocyst stage embryos, nine proteins showed increased synthesis. However, seven IGF response proteins were found to be proteins that showed significant changes in isotope incorporation during the eight-cell to blastocyst stage of development (Shi et al., 1993). In all, 54 proteins were affected, and these were unique; thus, protein synthesis in preimplantation mouse embryos is influenced by insulin and the IGFs, and further, each growth factor affects specific proteins. © 1994 Wiley-Liss, Inc.
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  • 15
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 37 (1994), S. 413-424 
    ISSN: 1040-452X
    Keywords: Primordial germ cell ; Oogonia ; Fetus ; Cattle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Gonadal cell suspensions were made from bovine fetuses of 35-55-, 56-80-, and 80-130-day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads.In suspensions from 35-55-day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56-80-day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80-130-day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid-phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation. © 1994 Wiley-Liss, Inc.
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  • 16
    ISSN: 1040-452X
    Keywords: Seminal fluid proteins ; Isoelectric focusing ; Molecular heterogeneity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (Mr) between 6 and 100 kDa. Many isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a Mr of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its Mr to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their Mr nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content. © 1994 Wiley-Liss, Inc.
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  • 17
    ISSN: 1040-452X
    Keywords: Boar ; Spermatozoa ; Anti-agglutinin ; Epididymal protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The present report identifies epididymal boar anti-agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs-Ringer bicarbonate at 37°C (5% CO2 in air). In the samples washed three or five times and then incubated for 3-5 h, higher rates (72-79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2-5 cells (head-to-head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head-to-head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti-agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti-agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro. © 1994 Wiley-Liss, Inc.
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  • 18
    ISSN: 1040-452X
    Keywords: In vitro fertilization ; Fertilization-specific antibody ; Immunohistochemistry ; Mouse-hamster-human cross-reacting antibodies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Several intrasplenic immunizations with batches of ∼15 or ∼30 zona-free, unfertilized mouse oocytes resulted in 200-300 hybrids, respectively, among which about 20 positive clones were selected from each fusion between splenic plasma cells and SP2/0 myeloma cells. When nonimmunized splenic plasma cells were used, only one antibody, showing weak immunoreaction, was obtained from ∼370 hybrids collected from 2 fusions. From one immunization with a total of 12 zona-free, unfertilized mouse oocytes, 15 positive clones were selected for further study. Eleven of these 15 antibodies reacted with antigens only in unfertilized oocytes but not in fertilized, pronuclear stage oocytes. Three antibodies, which recognized antigens in paraffin-embedded oocyte sections, did not label growing ovarian oocytes, indicating that the antibodies were specific to ovulated, unfertilized oocytes. These antibodies did not detect any antigen epitopes in the panel of tissues examined. The molecular weight of one antigen, corresponding to a IgM antibody that is present both in ooplasma and zona pellucida, was ∼116 kDa. Cross-reactivity to blots of unfertilized zona-free hamster oocytes was demonstrated by 6 antibodies and to unfertilized human oocytes by 7 antibodies. Three antibodies cross-reacted with both hamster and human oocytes. The study indicates that the intrasplenic immunization is an appropriate means of raising antibodies against unfertilized, zona-free mouse oocytes and that the method applied offers an easy way to select antibodies against human oocytes for functional studies. © 1994 Wiley-Liss, Inc.
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  • 19
    ISSN: 1040-452X
    Keywords: Frozen-thawed spermatozoa ; Capacitation ; Acrosome reaction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozenthawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca2+. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine-containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs-free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca2+ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca2+ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca2+ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2. These results indicate that CPPs promote Ca2+ uptake by spermatozoa and are effective for capacitation and/or acrosome reaction of spermatozoa leading to sperm penetration when caffeine is present in the medium and that the effect is reduced by dephosphorylation of CPPs. © 1994 Wiley-Liss, Inc.
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  • 20
    ISSN: 1040-452X
    Keywords: Canine sperm ; Pyospermia ; Ultrastructure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The red wolf (Canis rufus) is an endangered species with 194 individuals remaining in the wild and in various captive facilities. Breeding efforts at the Graham, WA site (Point Defiance Zoo and Aquarium) have involved artificial insemination with fresh or frozen semen in an effort to increase population and maximize the genetic potential of the stock. Electron microscopic observations were made in semen specimens obtained by electroejaculation from mature males prior to their use in an effort to determine semen parameters that might be useful in guiding breeding procedures. Sperm samples were either fixed immediately or treated with capacitating media and fixed after 4 to 7 hr of incubation. Many of the specimens examined were pyospermic (white cell in semen) and showed evidence of spermophagy, primarily by neutrophils. Of the six animals surveyed, only one showed little evidence of spermophagy, and three had extensive pyospermia and spermophagy but this finding was not correlated with fertility. Samples fixed immediately as well as those incubated for several hours showed evidence of spermophagy, indicating that the phagocytosis was not the result of culture. Gene pool restriction and/or captive stress may be contributing factors of reduced semen quality. © 1994 Wiley-Liss, Inc.
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  • 21
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 37 (1994), S. 477-477 
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 22
    ISSN: 1040-452X
    Keywords: Sulfogalactoglycerolipid ; Insulin-like growth factor 1 ; Spermatogenesis ; Phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The synthesis of sulfogalactosyl-glycerolipid (SGG) is a differentiation marker in spermatogenesis restricted to the zygotene and early pachytene spermatocytes. The galactolipid sulfotransferase responsible for the synthesis of SGG is regulated by a phosphorylation mechanism. The activity of this enzyme is reduced in cells later in spermatogenesis by a low molecular weight inhibitor, which can be extracted in organic solvents and purified by reverse phase high pressure liquid chromatography (HPLC). This purified inhibitor is a potent postreceptor insulin-mimetic, which stimulates adipocyte lipogenesis more effectively than does insulin. Phosphoinositol (PI) glycolipids have been proposed as second messengers of the insulin phosphorylation cascade. These species contain a nonacetylated glucosamine, which renders them liable to cleavage by deamidation. The activity of the sulfotransferase inhibitor was lost following nitrous acid deamidation and was labile to PI specific phospholipase C digestion. Insulin and insulin-like growth factor I were found to inhibit germ cell synthesis of SGG in vitro to some degree but had no direct effect on the testicular galacto-lipid sulfotransferase assay. These results indicate that the sulfotransferase inhibitor is a glycosyl phosphoinositide similar to the lipid species, which mediate insulin signal transduction and suggest that germ cell SGG biosynthesis may be regulated by a receptor-mediated phosphorylation pathway. © 1994 Wiley-Liss, Inc.
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  • 23
    ISSN: 1040-452X
    Keywords: Fertilization channels ; Ascidian oocytes ; Gating ; Second messengers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Neomycin, injected into ascidian oocytes to a final concentration of 10-50 mM, inhibits both the fertilization current and the surface contraction, showing that phosphoinositide hydrolysis is required for these early activation events. Sperm-activated fertilization currents are not inhibited in the presence of 100 μg/ml intracellular heparin, suggesting that these currents are not directly gated by InsP3. The sulfhydryl reagent thimerosal at 100 μM, in contrast, significantly increases the fertilization current presumably by sensitizing the channel receptor. Since heparin inhibits the surface contraction, InsP3 receptors are shown to play a role in the propagation of the activation response in ascidian oocyte. Depleting intracellular calcium stores by microinjecting 50 mM EGTA into oocytes does not activate fertilization channels; however, subsequent fertilization of these EGTA loaded oocytes leads to a significantly larger and faster fertilization current. Thus in contrast to somatic cells studied to date, second messenger operated plasma membrane channels in ascidian oocytes are not gated by calcium released from intracellular stores. © 1994 Wiley-Liss, Inc.
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  • 24
    ISSN: 1040-452X
    Keywords: Oocyte maturation ; Germinal vesicle breakdown ; Polar body ; LH/FSH ; Macaque ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus-enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 μg/ml human follicle-stimulating hormone (hFSH), (2) 10 μg/ml human luteinizing hormone (hLH), (3) 1.0 μg/ml hFSH and 10 μg/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3-hr intervals from 0 to 21 hr and at 4-hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P 〉 0.05) among treatments. The time to GVB was accelerated (P 〈 0.05) by gonadotropins (hFSH: 10.8 ± 1.7 hr; hLH: 10.1 ± 1.8 hr; both gonadotropins: 8.8 ± 1.1 hr) when compared to controls (17.4 ± 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P 〉 0.05) among treatments (hFSH: 32.3 ± 1.2 hr; hLH: 35.1 ± 1.4 hr; both gonadotropins: 35.2 ± 1.3 hr; no gonadotropins: 34.1 ± 1.2 hr). The mean interval to extrusion of PB1 was 34.1 ± 0.6 hr. In conclusion, GVB and PB1 extrusions appear to be, in part, independently regulated events in macaque oocytes matured in vitro since the timing of PB1 extrusion is not tightly coupled with the onset of GVB. Although the developmental potential of oocytes may be enhanced by gonadotropins, alternative approaches must be developed to improve the poor competence of oocytes from nonstimulated monkeys to mature in vitro. © 1994 Wiley-Liss, Inc.
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  • 25
    ISSN: 1040-452X
    Keywords: Mouse embryos ; Nuclear antigens ; Cell proliferation ; sn-RNP ; Transcriptional activity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have systematically analyzed by indirect immunofluorescence the subcellular distribution of nuclear antigens in relation to developmental stages of maturing mouse oocytes and developing embryos. Antigens were of two types: (1) a protein whose nuclear localization in interphase somatic cells depends on their proliferative state protein recognized by a monoclonal antibody 43B1N, and (2) snRNP polypeptides recognized by autoimmune sera of anti-Sm and anti-RNP type. The protein recognized by 43B1N was present in the germinal vesicle of oocytes from antral follicles, but absent from the nuclei during the first hours of embryonic life up to the middle to late 2-cell stage. Starting from this stage, it was always found in nuclei of interphase blastomeres, where its “speckles”; co-localized with the speckles containing high concentrations of snRNP polypeptides. SnRNP polypeptides recognized by anti-Sm and anti-RNP sera were in turn found in nuclei of all developmental stages. When embryos were treated with aphidicolin or cytochalasin D to arrest cell division, the 43B1N reacting protein was again localized in the pronuclei at 42 hr post-hCG, i.e., slightly later than the onset of transcriptional activity. These results suggest a progressive building up of nuclei during embryonic development, which could influence gene expression. © 1994 Wiley-Liss, Inc.
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  • 26
    ISSN: 1040-452X
    Keywords: Micromanipulation ; Zona pellucida ; DNA repair ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The treatment of male factor-related infertility has been approached with the advent of several methods for microsurgical fertilization, such as the partial dissection of the zona pellucida (PZD) and the injection of sperm into the perivitelline space (PVSI) of oocytes. These techniques are designed to increase sperm-oolemma interaction by circumventing passage of the sperm through the zona pellucida. The present study was performed to evaluate the influence of PZD and PVSI on the in vitro development of mouse embryos by assessing the rate of sister chromatid exchange (SCE). SCE is considered to be a sensitive indirect indicator of DNA lesions due to various conditions. Oocytes were cultured in vitro after PZD or PVSI and then examined for SCE. There was no significant difference in SCE between control and treatment groups of embryos and the values were similar to those reported by Saito et al. (Fertil Steril 41:460-464, 1984). The rate of SCE was low during the first two mitotic cycles, then increased from cycle two to three before declining slightly between the 3rd and 4th cycles of cell division. These data demonstrate that the direct interaction of sperm and oocyte by PZD or PVSI did not have an adverse effect on the development of mouse embryos as assessed by the rate of SCE. © 1994 Wiley-Liss, Inc.
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  • 27
    ISSN: 1040-452X
    Keywords: Mouse preimplantation development ; Reverse transcription-PCR ; Culture median ; RNA synthesis ; mRNA ; Gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Results of previous studies indicate that culture of preipmlantation mouse embryos in SOM medium containing 85 mM NaCl promotes better development in vitro, as well as supporting higher rates of protein synthesis, when compared to culture in SOM containing 125 mM NaCl (Anbari and Schultz, 1993, Mol Reprod Dev 35:24-28; Biggers et al., 1993, Mol Reprod Dev 34:380-390). In the present study we compare the effect of culturing embryos in these 2 media on several aspects of RNA synthesis and gene expression in order to determine whether the reduced development in SOM containing 125 mM NaCI and lower rates of protein synthesis are correlated with decreases in RNA synthesis and stability and changes in gene expression. Although no apparent differences in the metabolism of [3H]uridine to UMP, UDP, and UTP and its incorporation into total RNA are observed when 2-cell embryos are cultured to the morula stage in either medium, a 20% decrease in the rate of mRNA synthesis is found when embryos are cultured in SOM containing 125 mM NaCl. In addition, pulse-chase experiments reveal that total mRNA is less stable when the embryos are cultured in SOM containing 125 mM NaCl. Using a reverse transcription-polymerase chain reaction to assay for changes in the relative amounts of specific mRNAs, the relative amounts of mRNAs for IGF-I and IGF-II and their cognate receptors are dramatically reduced in embryos cultured in SOM containing 125 mM NaCl, whereas only a mild reduction is observed in the relative amount of actin mRNA. In contrast, when freshly isolated morulae are cultured to the blastocyst stage in either of these 2 media, similar amounts of these mRNAs are observed. Last, high-resolution, 2-dimensional gel electrophoresis reveals significant changes in the pattern of protein synthesis when the embryos are cultured in SOM containing 125 mM NaCl. Results of these experiments suggest that culture of embryos in medium containing lower concentrations of NaCl that are normally present in various culture media results in higher rates of mRNA synthesis and greater mRNA stability. These changes in RNA synthesis may underlie, at least in part, the improved development in vitro that is fostered by SOM containing 85 mM NaCl. © 1994 Wiley-Liss, Inc.
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  • 28
    ISSN: 1040-452X
    Keywords: Leukemia inhibitory factor ; Rabbit ; Endometrium ; Blastocyst implantation ; Pregnancy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Leukemia inhibitory factor (LIF) has been shown to play an important role in the implantation of mouse blastocysts. The present study was designed to document the appearance of LIF in the rabbit uterus during early pregnancy and to determine whether changes just prior to implantation, similar to those in mice, occurred. LIF was localized in endometrial epithelium, myometrium, and endometrial glands. A low level of LIF was detected in the uterus of nonestrous and estrous females. LIF expression reached its highest level on day 5 of pregnancy and declined on days 6 and 7. By day 13 of pregnancy, little endometrial LIF was apparent. The expression of LIF on day 5 of pseudopregnancy was similar to that on day 5 of pregnancy. LIF expression was much higher at implantation sites than that at nonimplantation areas on day 7 of pregnancy. It is concluded that LIF may be important for the implantation of rabbit blastocysts. © 1994 Wiley-Liss, Inc.
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  • 29
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 153-159 
    ISSN: 1040-452X
    Keywords: TGF-α ; TGF-β ; Oocytes ; Cumulus cells ; Gap junctions ; Maturation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Growth factors are known to regulate ovarian function. In the present study, effects of these growth factors, TGF-α, TGF-β, and activin-A were tested on spontaneous porcine oocyte maturation. Cumulus-oocyte complexes (COC) were cultured in the presence of TGF-α, TGF-β, and activin-A for 48 hr. Stages of meiotic maturation were assessed by staining with acetic orcein. Among these factors, only TGF-α significantly enhanced the maturation rate, whereas TGF-β suppressed the spontaneous maturation rate. The site of action of TGF-α on COC and the interaction between TGF-α and EGF receptor was also examined. Denuded oocytes, alone or in coculture with cumulus cells, were cultured in the presence of TGF-α for 48 hr. TGF-α did not have any significant effect on denuded oocyte maturation. Heptanol was employed to investigate the role of gap junctions on TGF-α-induced oocyte maturation in COC. Although heptanol did not have any significant effect in the control medium, heptanol reversed the stimulatory effect of TGF-α on porcine oocyte maturation. TGF-α was able to displace 125I-EGF binding on COC. In conclusion, TGF-α enhances the spontaneous maturation of porcine oocytes by generating positive signal(s) in cumulus cells that are transferred to the oocyte via gap junctions. TGF-α shares the same receptor with EGF on porcine COC. TGF-β, in contrast, inhibits porcine oocyte maturation. © 1994 Wiley-Liss, Inc.
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  • 30
    ISSN: 1040-452X
    Keywords: Calmodulin ; Actin ; Human sperm ; Golden Syrian hamster oocyte ; Fertilization ; Immunogold electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In the present study, immunogold labeling of ultrathin sections of human sperm, before and after incorporation into hamster oocyte, was used to obtain insight into the ultrastructural localization and possible function of calmodulin during fertilization. In heads of ejaculated, capacitated, and acrosome-reacted fixed human sperm, calmodulin was mainly found in two compartments, the subacrosomal layer and the postacrosome. After sperm-egg fusion, the subacrosomal calmodulin was unaltered and surrounded by the fertilization cone in which actin was abundant. There was no co-localization of calmodulin and actin. In contrast, postacrosomal calmodulin disappeared as soon as the sperm head was incorporated into egg cytoplasm. These unique localizations and redistributions are in agreement with the concept of a calmodulin targeting from acrosome toward postacrosome through the subacrosomal layer during spermatogenesis (Weinman et al., 1986b: J Histochem Cytochem 34:118). Moreover, they strongly suggest a role for calmodulin both in sperm-egg fusion and in the initial pulse of Ca2+ occurring during fertilization. © 1994 Wiley-Liss, Inc.
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  • 31
    ISSN: 1040-452X
    Keywords: Oviductal glycoprotein ; Gametes ; Immunofluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The objective of this study was to determine if human oviduct specific glycoprotein (huOGP) would associate with hamster ovarian oocytes and human sperm during in vitro incubation. The huOGP used in these studies was partially purified from human hydrosalpinx fluid. Hamster ovarian oocytes and human sperm samples were incubated in culture medium with and without huOGP. Association of huOGP was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against huOGP. Intense fluorescence of the zona pellucida, and bright but uneven fluorescence of the perivitelline space, were observed in hamster ovarian oocytes following incubation in the presence of huOGP. A similar but more uniform pattern of fluorescence was observed when hamster oviductal oocytes (positive controls) were incubated in culture medium alone. Fluorescence was absent when oocytes were assayed with preimmune serum. The association of huOGP with the zona pellucida and perivitelline space appeared to be specific since thyroglobulin, a large molecular weight glycoprotein, and human serum albumin, the major protein in oviduct fluid, did not associate with the hamster oocytes nor inhibit huOGP association when included in the culture medium. Fluorescence was absent when human sperm incubated with huOGP were assayed with antiserum to huOGP. However, human sperm fluoresced when incubated with a uterine glycoprotein, CUPED, which had previously been shown to bind to cat sperm during in vitro incubation. Sperm also fluoresced brightly when human sperm antibody was used as a positive control. Solubilization of sperm membrane proteins postincubation and analysis of these proteins by 1-D SDS-PAGE followed by immunoblotting also failed to show an association of huOGP with human sperm. Electron microscopy of sperm both pre- and postsolubilization confirmed that the sperm membranes were removed by this process. In conclusion, the association of huOGP with hamster oocytes in vitro suggests that huOGP may associate with human oocytes in vivo, whereas that may not be true for human sperm in vivo. The association of huOGP with oocytes may serve to facilitate the process of fertilization and early embryonic development within the oviduct. © 1994 Wiley-Liss, Inc.
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  • 32
    ISSN: 1040-452X
    Keywords: Immunofluorescence ; Cytoskeltal proteins ; Antibody ; Gel electrophoresis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The cytoskeletal protein actin was identified in the mature spermatozoon of the European edible crab, Cancer pagurus Linnaeus, by indirect immunofluoresce with monoclonal and polyclonal anti-actin antibodies, fluorescent phalloidin, and DNAase I. The actin was localized in two distinct concentric rings within the acrosome vesicle of the spermatozoon and appeared to correlate with the internal zonation of the vehicle. Modifications of the fluorescent pattern for actin were observed in sperm cells which were undergoing changes associated with the acrosome reaction. In these cases, fluorescent staining was observed in the nucleocytoplasm immediately subjacent to the perforatorial column and sometimes in the perforatorial column within the acrosome vesicle. Equally intense fluorescence was observed in an apical perforatorial projection. SDS-PAGE of C. pagurus sperm confirmed the presence of actin in the cells. A single band of actin (approximately 43 kDa) comigrated with rabbit muscle actin when immunoblotted onto nitrocellulose with mouse monoclonal anti-actin. The actin-associated cytoskeletal proteins α-actinin, tropomyosin, and spectrin were also identified within the spermatozoon of C. pagurus using specific polyclonal antibodies, but their presence was not confirmed by SDS-PAGE and immunoblotting. © 1994 Wiley-Liss, Inc.
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  • 33
    ISSN: 1040-452X
    Keywords: Mouse oocyte ; Meiosis ; Lithium ; InsP3 ; myo-inositol ; Chromatin ; Microtubules ; Cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The incubation of mechanically denuded mouse oocytes in medium containing LiCl delayed both germinal vesicle breakdown (GVBD) and polar body extrusion in a dose-dependent and reversible manner. When myo-inositol alone was added to the culture medium, we observed that it accelerated GVBD and increased the rate of polar body extrusion, whereas, when combined with LiCl, the normal timing of GVBD was recovered. In the same way, when inositol trisphosphate (InsP3) was microinjected into the ooplasma, we observed an important improvement of the rate of GVBD, as compared to control oocytes, and prevention of lithium inhibition. However, neither myo-inositol nor InsP3 were able to rescue totally the oocytes from the negative effect of lithium on polar body extrusion. Moreover, lithium induced some important changes in microtubule and chromosome organizations. Before extrusion of the first polar body, the reduction of the spindle size or the appearance of short individualized chromosomes dispersed around a large aster of microtubules were often observed, whereas, after polar body extrusion, the spindle appeared smaller and chromosomes were often trapped in the midbody. Thus lithium affects mouse oocyte maturation at two different levels: GVBD and polar body extrusion. Whereas the former seems to be affected via polyphosphoinositide turnover, the latter is InsP3-independent and seems to be influenced negatively via underdevelopment of microtubular structures. © 1994 Wiley-Liss, Inc.
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  • 34
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    ISSN: 1040-452X
    Keywords: FGF-4 ; Polarizing activity ; Limb development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The apical ectodermal ridge plays a central role in limb development through its interactions with the underlying mesenchyme. Removal of the AER results in cessation of limb outgrowth and leads to truncation of the limb along the proximo-distal axis. The many functions attributed to the ridge include maintenance of the progress zone mesenchyme. Here, cells are stimulated to proliferate, are maintained in an undifferentiated state, and are assigned progressively more distal positional values as the limb grows. The AER also functions to maintain the activity of the polarizing region, a region of mesenchyme which is thought to provide the primary signal for patterning along the antero-posterior axis.We have begun to explore the function of fibroblast growth factor-4 (FGF-4) during limb development. FGF-4, which encodes an efficiently secreted protein, is expressed in the AER. We have previously demonstrated that FGF-4 protein can stimulate limb mesenchyme proliferation and can induce the expression of a downstream homeobox gene, Evx-1 (homologue of the Drosophila even-skipped gene), that is normally regulated by a signal from the AER. To determine to what extent FGF-4 protein can substitute for the AER to allow normal limb outgrowth, we performed experiments on the developing chick limb in ovo. Remarkably, we find that after AER removal, the FGF-4 protein can provide all the signals required for virtually normal outgrowth and patterning of the limb. Further studies indicate that proliferation of progress zone cells is not sufficient, and that an additional signal is produced by the posterior mesenchyme in response to FGF-4 which enables progress zone cells to acquire progressively more distal fates. Thus FGF-4 maintains progress zone activity through a combination of at least two signals - one that acts directly on progress zone cells to stimulate their proliferation, and one that acts indirectly by maintaining the production of patterning signal(s) by the posterior mesenchyme. We further show that failure of the posterior mesenchyme to produce this signal correlates with failure to maintain polarizing activity. This raises the possibility that the signal produced by the posterior mesenchyme and required for progressive proximo-distal limb patterning is identical to the polarizing activity. Further experiments demonstrate that retinoic acid, which mimics the activity of the polarizing region, can supply this signal. In conclusion, the finding that a single growth factor can serve as both the direct and indirect signals required to maintain progress zone activity provides a simple mechanism for ensuring that growth and pattern formation are linked in the developing limb. © 1994 Wiley-Liss, Inc.
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  • 35
    ISSN: 1040-452X
    Keywords: Fibroblast growth factors ; Receptors ; Skeletal Muscle ; Limb Development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling.We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridgederived factor that participates in limb outgrowth and patterning. © 1994 Wiley-Liss, Inc.
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  • 36
    ISSN: 1040-452X
    Keywords: Nuclear localization ; Initiation codons ; Glycosaminoglycans ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Multiple forms of FGF-2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF-2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF-2 as the AUG codon is 3′ to the CUG codons. Although the 18 kDa form FGF-2 is localized primarily in the cytosol, a significant fraction of the HMW FGF-2 has a nuclear location. The nuclear localization of HMW FGF-2 is determined by amino acid residues in the amino-terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino-terminal extension of HMW FGF-2. The nuclear localization of HMW FGF-2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF-2, or all forms of FGF-2, we have found that HMW FGF-2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF-2. These cells are transformed by what appears to be the intracellular action of HMW FGF-2. The interaction of FGF-2 with heparin has also been examined. Contrary to other reports claiming that FGF-2 required heparin or heparan-sulfate for interaction with its high-affinity receptor, we have found that FGF-2 binds to its receptor in the absence of glycosaminoglycans, and that this binding activates the receptor. © 1994 Wiley-Liss, Inc.
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  • 37
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 39 (1994), S. 106-111 
    ISSN: 1040-452X
    Keywords: Fibroblast growth factor ; Transcription ; Embryonal carcinoma cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Embryonal carcinoma (EC) cells provide a useful model system for studying the roles of growth factors during early mammalian development. In 1988, we determined that EC cells express a member of the fibroblast growth factor (FGF) family that cannot be detected after EC cells undergo differentiation. Attempts to understand how differentiation regulates the production of FGFs led to the finding that EC cells express the fibroblast growth factor k-FGF (FGF-4), whereas there is a large decrease in the steady state levels of k-FGF mRNA when EC cells differentiate. This suggested that transcription of the k-fgf gene is repressed when EC cells differentiate. To investigate this possibility, we prepared a series of reporter gene constructs containing various regions of the murine k-fgf gene. These constructs were transfected into two mouse EC cell lines and one mouse embryonic stem (ES) cell line. We determined that the mouse 5′ flanking region cannot support expression of the reporter gene. In both EC and ES cell lines, expression of the reporter gene is elevated greatly by the addition of a 316 bp region from the third exon of the murine k-fgf gene. Sequence analysis of the 316 bp region identified one and possibly two conserved octamer binding motifs. These sequences are likely to be involved in regulation of the k-fgf gene, because differentiation of EC cells is known to reduce the expression of octamer binding proteins, including Oct-3. To test the possible role of octamer binding proteins, we examined the expression of our reporter gene constructs in F9-differentiated cells and in PYS-2 cells. In these cells, the expression of our k-fgf/reporter gene constructs is very low. However, expression of the reporter gene is elevated significantly when these cells are cotransfected with a construct that contains an Oct-3 cDNA under the control of a strong viral promoter. To test further the importance of the octamer motifs found in the 316 bp enhancer-like region, we replaced the 316 bp region in our constructs with a smaller region (48 bp) that contains the downstream octamer motif and flanking sequences. Like the 316 bp region, this 48 bp region elevated the expression of the reporter gene, but it does so at a much lower level. Thus, it appears that the downstream octamer motif may be involved in the regulation of the k-fgf gene, but that at least one other cis-regulatory element present in the 316 bp enhancer is likely to be involved in the expression of the k-fgf gene in EC cells. Lastly, we have identified two potentially important regulatory regions upstream of the transcription start site. One appears to regulate positively the expression of the murine k-fgf gene in EC cells and in ES cells, and the other appears to regulate negatively the expression of the k-fgf gene in these cells. © 1994 Wiley-Liss, Inc.
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  • 38
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    New York, NY [u.a.] : Wiley-Blackwell
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 39
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    Molecular Reproduction and Development 39 (1994), S. 118-125 
    ISSN: 1040-452X
    Keywords: Xenopus ; Mesoderm induction ; FGF ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To assess the potential role of a molecule in development we need to know three things: 1) what are the biological activities of the molecule, 2) what is its expression pattern, and 3) what are the consequences of removing it from the embryo?In the case of the FGF family in Xenopus embryos we have quite a lot of information about all three questions. Most members of the family can induce mesoderm from isolated animal caps, thus mimicking the natural “ventral vegetal” inducing signal operative in the blastula. This activity can be exerted on isolated, disaggregated cells and does not involve a change in division rate. When overexpressed from injected mRNA, the activity of FGFs depends largely on whether or not they possess a signal sequence, showing the importance of secretion in the inductive process. In addition to the mesoderm-inducing activity, there are effects of overexpression on whole embryos which lead to a suppression of anterior structures.Three types of FGF have so far been cloned from Xenopus: direct homologs of each of the mammalian types FGF-2 and FGF-3, and eFGF (“embryonic FGF”), which is equidistant in sequence from mammalian FGF-4 and FGF-6. Attempts to find homologs of mammalian FGF-5 and FGF-7 in Xenopus have proved unsuccessful. All three types of Xenopus FGF are expressed in early development. FGF-2 and eFGF are present in the oocyte and fertilized egg, and are thus both available at the time of mesoderm induction. FGF-3 and eFGF are both expressed from the embryonic genome during gastrulation and concentrated in the forming mesoderm. FGF-2 is expressed from the embryonic genome during neurulation in the brain, and a little later in the branchial arch mesenchyme and in the forming myotomes. These expression patterns suggest that there are several functions for the FGFs.The most successful strategy for inhibition of the FGF system has been the use of a dominant negative receptor construct introduced by Kirschner and colleagues. Overexpression of this construct can abolish the FGF responsiveness of animal caps. In whole embryos, the absence of FGF signaling causes a reduction, although not a total ablation, of mesoderm formation. There is also a severe effect on axis formation in which formation of the posterior parts is reduced consequent on an inhibition of invagination and elongation of the dorsal mesoderm.Thus, the present evidence suggests that the FGF system contributes to, although is not solely responsible for, mesoderm induction in vivo. It is also necessary for normal gastrulation movements, particularly in the dorsal mesoderm, and is likely to have several later functions, particularly in development of the central nervous system and the myotomes. © 1994 Wiley-Liss, Inc.
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  • 40
    ISSN: 1040-452X
    Keywords: Receptor ; Follicle-stimulating hormone ; Granulosa cells ; Follicle ; Bovine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We report the complementary DNA structure obtained by reverse transcription and polymerase chain reaction amplification encoding the complete amino acid sequence for the bovine follicle-stimulating hormone receptor (bFSHr). The deduced amino acid sequence for the cDNA revealed a mature polypeptide consisting of 678 amino acids (theoretical weight of 76.4 kDa) and a 17 amino acid putative leading signal peptide. The receptor consists of a large NH2-terminal extracellular membrane domain of 349 aa with 3 potential N-linked glycosylation sites, a transmembrane domain (264 aa) consisting of 7 putative membrane spanning segments, and an intracytoplasmic COOH-terminal domain (65 aa). Four potential phosphorylation sites were found in the transmembrane domain and the COOH-terminal domain. The amino acid sequence is 97%, 89%, and 88% homologous to the ovine, human, and rat FSHr respectively, with complete conservation of the 22 cysteine residues in the whole protein and the 3 N-linked glycosylation sites on the extracellular membrane domain. Northern blot analysis of total mRNA in bovine tissues revealed a major mRNA transcript of 2.55 kb for the bFSHr in the ovary without corpus luteum, and in the testis. No expression was found in other tissues analyzed. Total RNA from bovine granulosa cells collected from pregnant mare serum gonadotropin (PMSG)-treated prepubertal heifers showed 2 major mRNA transcripts of 6.8 and 2.55 kb, and 3 minor transcripts of 3.8, 3.3, and 1.6 kb. Bovine granulosa cells cultured with porcine FSH (0, 2, 10 ng/ml) for 4 days showed a decrease in the steady state level of the FSHr mRNA. This decrease was shown to be independent of the size of the transcript. Therefore, expression of the bovine FSHr by bovine granulosa cells is downregulated at the message level when exposed to constant concentrations of FSH. ©1994 Wiley-Liss, Inc.
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  • 41
    ISSN: 1040-452X
    Keywords: Actin ; Adenovirus ; Dominant-negative receptors ; Serum response factor ; Transcription factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Skeletal α-actin (SkA) is representative of the cardiac genes that are expressed at high levels in embryonic myocardium, downregulated after birth, and reactivated by trophic signals including basic fibroblast growth factor (FGF-2) and type β transforming growth factors (TGFβ). To investigate the molecular basis for cardiac-restricted and growth factor-induced SkA transcription, we have undertaken a mutational analysis of the SkA promoter in neonatal ventricular myocytes, with emphasis on the role of three nominal serum response elements. Serum response factor (SRF) and the bifunctional factor YY1 are the predominant cardiac proteins contacting the proximal SRE (SRE1). Mutations of SRE1 that prevent recognition by SRF and YY1, or SRF alone, virtually abolish SkA transcription; mutation of distal SREs was ineffective. A mutation which selectively abrogates YY1 binding increases expression, substantiating the predicted role of YY1 as an inhibitor of SRF effects. SkA transcription requires combinatorial action of SRE1 with consensus sites for Sp1 and the SV40 enhancer binding protein, TEF-1. As an isolated motif, SRE1 can confer responsiveness to both FGF-2 and TGFβ to a heterologous promoter. Whether TEF-1 binding sites likewise can function as FGF response elements is unknown.Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has largely been undertaken to date in neonatal ventricular myocytes, as the adult ventricular myocyte has been refractory to conventional procedures for gene transfer. To circumvent expected limitations of other methods, we have used replication-deficient adenovirus to achieve efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a CMV-IE promoter-driven lacZ reporter gene, and were assayed for the presence of β-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 ×105 PFU, and approached 90% at 1 × 108 PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. The β-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the presence of exogenous lacZ gene. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus.We have constructed virus conferring luciferase activity driven by the SkA promoter (Ad5/SkA/luc) to test for potential developmental control of growth factor responses in cardiac muscle. In adult ventricular myocytes, the construct remains inducible by TGFβ, but little or no response is seen to FGF-2 or FGF-1, which is consistent with prior reports that the FGF receptor is downregulated in terminally differentiated ventricular muscle cells. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of FGF receptors and FGF signaling proteins upon the endogenous genes and gene products of virally modified adult ventricular muscle cells. © 1994 Wiley-Liss, Inc.
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  • 42
    ISSN: 1040-452X
    Keywords: Marker gene ; Preimplantation embryos ; IVF ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We investigated the onset of paternal gene expression in the early mouse embryo. We obtained transgenic mouse embryos by fertilizing BD (C57BL/6N x DBA) F1 hybrid female oocytes in vitro, with sperm from homozygous transgenic males carrying integrated chicken β-actin promoter-driven firefly luciferase cDNA. We then examined the RNA and protein synthesis of the luciferase gene in embryos from the 1- to 2-cell stage. RNA transcripts of the luciferase gene were first detected in the 1-cell stage embryos as early as 13 hr postinsemination, just prior to elongation. By photon-count imaging, functional luciferase was identified at the 2-cell stage 23 hr postinsemination. These findings indicate that the paternal endogenous gene is already transcribed in the late 1-cell embryos, although paternally derived protein is not synthesized until the 2-cell stage. Therefore, these results suggest that the embryonic gene is activated as early as the late 1-cell stage. ©1994 Wiley-Liss, Inc.
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  • 43
    ISSN: 1040-452X
    Keywords: In vitro fertilization ; Preimplantation embryos ; Morphologic grading ; Chromosomal abnormalities ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In in vitro fertilization (IVF) procedures, morphologic embryo grading is the sole criteria for selection of embryos transferable in utero. Cytogenetic analysis of preimplantation embryos was performed to investigate the relationship between chromosomal status and morphologic quality of preimplantation eggs. Aneuploidy was the most frequently observed abnormality. In addition, various types of aberrations such as polyploidy, haploidy, mosaicism, and fragmentation were also found. Our results, pooled with data drawn from previous reports, demonstrated the prognostic value of the embryo grading system as a means for eliminating chromosomally abnormal embryos. In contrast, data suggested that some aspects of the IVF process might be responsible for the occurrence of these abnormalities. © 1994 Wiley-Liss, Inc.
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  • 44
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    Molecular Reproduction and Development 39 (1994), S. 162-165 
    ISSN: 1040-452X
    Keywords: Meiotic arrest ; In vitro maturation ; Cow ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cumulus oocyte complexes (COCs) were cocultured with parts of the follicular wall. Coculture conditions were such that the COCs were 1) in continuous contact with the follicular wall (FWC), 2) separated from the follicular wall at collection but in contact with it during culture (FWR), and 3) separated from the follicular wall, but cultured in its vicinity (FWNR). Oocytes cultured for 24 hr under FWC conditions maintained the germinal vesicle stage. Under FWR conditions the germinal vesicle stage was not maintained, but an arrest at metaphase I of meiosis occurred in mostof the oocytes. When COCs were cultured in the vicinity of the follicular wall (FWNR), meiosis was resumed and similar numbers of oocytes progressed to metaphase II of meiosis as compared to cultures of COCs without coculture with parts of the follicular wall. When COCs were isolated from the follicular wall after 24 hr of culture and additionally cultured for another 24 hr, the oocytes showed the same capability of resuming meiosis as fresh, isolated cumulus oocyte complexes.It is concluded that maintenance of contact with the follicular wall is necessary to maintain meiotic arrest. When COCs restore a physical contact with the follicular wall during culture, an arrest at metaphase I occurs. © 1994 Wiley-Liss, Inc.
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  • 45
    ISSN: 1040-452X
    Keywords: Mitosis ; Synchronization ; Mice ; Blastomere ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mouse 2-, 4-, 8-, and 16-cell embryos were exposed to nocodazole in M16 culture medium. The effect of different concentrations and exposure times on the efficiency of cell cycle synchronization and the development of the treated embyros after release from the drug was determined. The minimum effective concentration (95% of arrested nuclei) for 4-, 8-, and 16-cell embryos was 5μM nocodazole. The effect upon subsequent development of mouse embryos depended upon both the stage of development of the embryo at treatment (P 〈 0.001) and the length of exposure to nocodazole (P 〈 0.001). Exposure to any concentration of nocodazole within the range 2.5-10 μM for 12 hr caused a reduction in the proportion of embryos that formed blastocysts. As the period of exposure to 5μM nocodazole increased from 12 to 24 hr, the proportion of embryos developing to the blastocyst stage decreased. The lower proportion of embyros developing to the blastocyst stage and to term (P 〈 0.01) suggests that the more advanced stages were more susceptible to damage as a result of exposure to nocodazole. The rate of development of 4-cell embryos to blastocysts was not affected when an exposure time of 9 hr was used. Together these results show that it is possible to use nocodazole to arrest mouse embryonic cells in mitosis but that it is not appropriate to culture the embryos in the presence of this drug for prolonged periods. Individual blastomeres completed mitosis at 60-90 min and started DNA synthesis at 120-150 min after release from nocodazole. Nuclei from blastomeres thus synchronized were used to conduct studies on the effect of the cell cycle on nuclear transfer. A signficant effect was found. When nuclei from 8-cell embryos in G1 or S-phase were used as nuclei donors, development to blastocyst was respectively 27% and none. ©Wiley-Liss, Inc.
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  • 46
    ISSN: 1040-452X
    Keywords: Chick embryo ; Germline chimerism ; Transplantation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Germline chimeric chickens were produced by transfer of primordial germ cells from White Leghorn to Barred Plymouth Rock, and vice versa. Blood was collected from stage 13-15 embryos and primordial germ cells were concentrated by Ficoll density gradient centrifugation. Approximately 200 primordial germ cells were injected into the bloodstream through the dorsal aorta of stage 14-15 recipient embryos from which blood had been drawn via the dorsal aorta prior to the injection. Intact embryos were also prepared as recipients for White Leghorns only. The manipulated embryos were cultured in recipient eggshells until hatching. Germline chimerism of the chickens reaching maturity was examined by mating them with Barred Plymouth Rocks and donor-derived offspring were identified based on their feather color. The efficiency of production of germline chimeras was 95% (19/20). When primordial germ cells were transferred from White Leghorn to Barred Plymouth Rock, the average frequency of donor-derived offspring was 81% for three male chimeras (96% for one female chimera), and it was ∼3.5 times higher for transfer in the opposite direction (23% for 6 male chimeras). Removing blood from recipient embryos prior to primordial germ cell injection enhanced the frequency of donor-derived offspring by 10% in resulting male chimeras. Male chimeras produced donor-derived offspring more frequently (∼3.8 times) than female chimeras. Increases, decreases, or no changes were observed in the frequency of donor-derived offspring from the germline chimeras with increasing age. Male to female ratio of the offspring derived from the donor primordial germ cells did not deviate significantly in male and female chimeras, suggesting that primordial germ cells that have different sex from recipient embryos could not differentiate into functional gametes. The technique for primordial germ cell transfer employed in this experiment is simple to perform and resulted in the efficient production of germline chimeras with high transmission rates of donor-derived gametes. This system provides a powerful tool for avian embryo manipulation. © 1994 Wiley-Liss, Inc.
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  • 47
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    Molecular Reproduction and Development 39 (1994), S. 182-183 
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 48
    ISSN: 1040-452X
    Keywords: Fish ; Oogenesis ; Chorion ; Zona radiata ; Estradiol-17β ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to study the initial formation of the vitelline envelope and the appearance of vitellogenin in oocytes of rainbow trout, females were sampled monthly from 19 to 5 mo before ovulation. lmmunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 μm. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol-17β increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 μm) present just inside the oolemma, when the oocytes reached a diameter of 600 μm. This was interpreted as an active uptake of vitellogenin. Oocytes of this size were first found in females sampled 9 mo before ovulation at the time when estradiol 17β levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 μm had an immunoreactive vitelline envelope with a thickness of about 3 μm. It is apparent that the initial formation of the vitelline envelope starts before the active uptake of vitellogenin and that the low previtellogenic plasma levels of estradiol-17β observed in females are of physiological significance. © 1994 Wiley-Liss, Inc.
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  • 49
    ISSN: 1040-452X
    Keywords: Heterogeneity of human sperm ; Condensation ; Chromatin structure ; Laser scanning cytometry ; Flow cytometry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation.We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion.We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley-Liss, Inc.
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  • 50
    ISSN: 1040-452X
    Keywords: Demembranated Sperm ; Dynein ; Calcium ; Oxygen consumption ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The action of carnitine in regulating fowl sperm motility was investigated. As the concentration of L-carnitine was increased (0-20 mM), the motility of intact and demembranated fowl spermatozoa was reduced at 30°C. Even the presence of 1 mM CaCl2 before the addition of 10 mM carnitine could not prevent the inhibition of motility at 30°C and 40°C. However, motility was restored by reducing the concentrations of carnitine. Carnitine also inhibited the oxygen consumption and ATP concentrations of intact spermatozoa, and caused a reduction in intracellular free Ca2+ concentrations. Phosphorylation of a 50 kDa protein and dephosphorylation of 24 kDa and 30 kDa proteins of demembranated spermatozoa were observed after the addition of carnitine. In contrast, the flagellar ATPase activity of crude dynein extract was not affected by the addition of carnitine. These results suggest that inhibitory effect of carnitine for motility may be directly on the axonemal phosphoproteins, but not directly on the dynein ATPase activity. The physiological role of carnitine for fowl spermatozoa in the ductus deferens is discussed. © 1994 Wiley-Liss, Inc.
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  • 51
    ISSN: 1040-452X
    Keywords: Protease ; Oocyte ; Germinal vesicle breakdown ; Amphibian ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The inhibition of progesterone-induced oocyte maturation by diisopropylfluorophosphate (DFP), a typical serine protease inhibitor, was investigated in oocytes of the Japanese toad Bufo japonicus for the first time. Oocytes to which DFP was externally applied did not undergo germinal vesicle breakdown (GVBD), which is an early signal of oocyte maturation, in response to progesterone. The more inhibitory period was found to be 0-0.5 GVBD50 on a relative time scale [when the time at which 50% of the oocytes had completed GVBD (GVBD50) was set at 1.0], namely, before the beginning of GVBD. DFP-sensitive proteases, which seem to be multifunctional nonlysosomal protease complexes (proteasomes), may already be present in the cytosol of premature oocytes. Peptide hydrolyzing activity, as reflected by proteasome activity, was found to be regulated before and after GVBD. In addition, immunoblotting regarding the native electrophoretic protein profile of the proteasomes throughout the maturational process demonstrated that they undergo alterations in mobility dependent upon the maturational process.These findings raise the possibility that the activities of some endogenous DFP-sensitive proteasomes play distinct, essential roles in oocyte maturation triggered by progesterone in Bufo. © 1994 Wiley-Liss, Inc.
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  • 52
    ISSN: 1040-452X
    Keywords: Cumulus cells ; Meiosis ; GVBD ; Cow ; mRNA ; Adenosine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) is an analog of the nucleoside adenosine that has been used to inhibit transcription in a variety of cell types. The present studies were designed to evaluate the ability of DRB to block germinal vesicle breakdown (GVBD) in bovine oocytes matured in vitro and to characterize culture conditions required for DRB-mediated arrest of meiosis. Administration of DRB (60-90 μM) at 2-3 h intervals during culture of COC blocked GVBD in approximately 70 percent of oocytes. The inhibitory effect of DRB was reversible and required the presence of cumulus cells. Treatment with DRB was associated with a 57% decrease in 3H-uridine incorporation into total COC RNA and a 90.8% decrease into mRNA but did not affect the incorporation of 3H-leucine into COC proteins. The ability of DRB to arrest meiosis was significantly compromised if supplemental gonadotropin preparations were absent from the maturation media. Gonadotropin-induced GVBD as well as cumulus cell expansion was blocked by treatment with DRB but no with adenosine. GVBD in cultured bovine COC was initially inhibited and then stimulated when supplemental gonadotropin preparations were included in the culture media. DRB treatment in the presence of gonadotropin supplementation blocked the stimulatory effect of gonadotropins on GVBD. In conclusion, DRB can be used to arrest GVBD in bovine COC in a specific and reversible manner. The data support the hypothesis that gene transcription is required for the stimulatory phase of gonadotropin-mediated GVBD in cultured bovine COC. © 1994 Wiley-Liss, Inc.
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  • 53
    ISSN: 1040-452X
    Keywords: Sperm antigen ; Acrosome reaction ; Zona solution ; Ionophore A23187 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have previously prepared an anti-mouse sperm monoclonal antibody (A-1) which inhibited sperm penetration into the egg zona pellucida. By indirect immunofluorescence (IIF), the A-1 antibody was shown to recognize an antigen localized in the acrosomal area of sperm. This antibody bound negligibly to fresh sperm, while binding to methanol-fixed sperm was almost complete. After methanol fixation, no sperm that penetrated into the zona were immunoreactive for this antibody. In the present study we examined the localization and fate of A-1 antigen during the acrosome reaction by IIF and flow cytometry (FCM). Cauda epididymal sperm were treated with either calcium ionophore A23187 or zona solution, immunostained indirectly, and subjected to FCM. Treatment with A23187 reduced the percentage of immunoreactive sperm to 59% from the 80% obtained in the untreated sperm. The treatment also reduced the average fluorescence intensity per fluorescence-positive spermatocyte to 65 channels, while this intensity was 89 channels in the untreated sperm. A similar result was obtained from treatment with zona solution. The proportion of sperm that was immunoreactive with A-1 antibody was reduced to 55% by incubation in zona-containing media from the 80% obtained in zona-free media. On the other hand, neither A23187 nor the zona solution affected the immunoreactivity or the fluorescence intensity of caput epididymal sperm, while the A-1 antigen was present in both the immature sperm from the caput epididymis of adult mice and in the mature sperm from the cauda epididymis of the same mice. These findings suggest that the intramembrane antigen recognized by the A-1 monoclonal antibody is released from sperm as a result of the acrosome reaction. © 1994 Wiley-Liss, Inc.
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  • 54
    ISSN: 1040-452X
    Keywords: Macaca fasicularis ; Semen ; Capacitation ; Zona binding ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Capacitation of macaque sperm with caffeine and dbcAMP is required for fertilization in vitro. This study determined the separate effects of caffeine and dbcAMP on sperm-zona pellucida binding and the acrosome reaction of zona bound sperm. Semen from 6 cynomolgus macaques was washed through 60% Percoll, resuspended, and washed with BWW media and incubated for 2.5 hr. Caffeine, dbcAMP (2 mM each), or both (1 mM each) were added to aliquots of the sperm suspensions. Immature macaque oocytes were placed into drops of sperm suspensions, coincubated with sperm for 30 sec, and either fixed immediately or removed to sperm-free media and incubated 1 hr before fixation. There were no significant diffences between groups in the percentage of live, acrosome-reacted sperm in suspension. Treatment with caffeine and dbcAMP or with caffeine alone, significantly increased the number of sperm bound to each zona pellucida (96 ± 16 and 81 ± 17, respectively) compared to control and dbcAMP treatment (15 ± 4 and 28 ± 13). However, treatment with dbcAMP, alone and with caffeine, resulted in a higher percentage of acrosome-reacted sperm on the zona (15.2 ± 2.1 and 9.0 ± 0.6) than control or caffeine treatment (3.0 ± 1.4 and 2.4 ± 0.5). Effects on sperm motility consistent with hyperactivation were detected only when both caffeine and dbcAMP were present. Although both caffeine and dbcAMP are presumed to increase or to produce the same effects as increased intracellular cAMP levels, these compounds have different effects on the ability of sperm to bind to the zona and to undergo the acrosome reaction. © 1994 Wiley-Liss, Inc.
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  • 55
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    Molecular Reproduction and Development 37 (1994), S. 305-317 
    ISSN: 1040-452X
    Keywords: HeLa extracts ; Chromatin decondensation ; Remodelling ; Recondensation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: HeLa cell extracts induced decondensation of lysolecithin permeabilized Xenopus, pig, and human sperm chromatin; decondensation began almost immediately on incubation in the extract and was completed within 10-20 min. The average enlargements of human and pig sperm nuclei were 15-fold and 3-fold, respectively. The structural organization of pig and human sperm chromatin was significantly differnt. Decondensation was differentially inhibited by Mg++ and polyamines; inhibition was least for Xenopus and most for pig sperm nuclei. The nuclear membrane was disintegrated on chromatin dispersion, whereas the nuclei which failed to decondense exhibited distinct nuclear envelopes. The decondensing factors were stable at 65°C for 15 min. The dispersed chromatin was remodelled to somatic nucleosomal structures within 60 min. The remodelled chromatin could be recondensed to chromosome-like structures, when incubated further in extracts from mitosis arrested HeLa cells. © 1994 Wiley-Liss, Inc.
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  • 56
    ISSN: 1040-452X
    Keywords: Aging ; Male infertility ; Mitochondrial DNA ; Ischemia ; Free radicals ; Spermatogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
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  • 57
    ISSN: 1040-452X
    Keywords: Sperm acrosome reaction ; Sperm motility ; A23187 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A method to generate a population of motile, acrosome-reacted mouse sperm is described. Sperm retrieved from the cauda epididymis and vas deferens were first capacited in a 3% bovine serum albumin (BSA) containing medium. Sperm were then resuspended in medium with low BSA content (0.01%) and treated with 30 nM of the calcium ionophore, A23187, which was added as a singel dose of 30 nM for 15 min at 37°C; or three sequential 10 nM doses over three 5 min intervals. Approximately 55-60% of the treated sperm population became acrosome reacted. The motility of the treated sperm sample was 40-65%, slightly lower than that of the control sperm, following addition of medium containing 3% BSA, This is in contrast to the 〈10% motility observed for capacitated mouse sperm treated with 10 μM A23187, a concentration that had been used by other investigators to induce the acrosome reaction. The ultrastructure of the 30 nM A23187-induced acrosome-reacted sperm ws similar to that of the acrosome-reacted sperm induced by solubilized zonae pellucidae. These motile, acrosome-reacted sperm were able to penetrate zone-free mouse eggs at a higher rate than the control sperm. Thus this method of treatment will be useful for further physiological experimentation with acrosome-reacted sperm. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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