Molecular Reproduction

An early step in mammalian fertilization is species‐restricted binding of sperm to the egg's zona pellucida (ZP), a thick extracellular coat that surrounds eggs. Sperm bind to the ZP of unfertilized eggs, but not to the ZP of fertilized eggs. Shortly after binding to the unfertilized egg ZP, sperm undergo the acrosome reaction, a form of cellular exocytosis that enables sperm to penetrate the ZP. Three glycoproteins, mZP1‐3, constitute the mouse egg's ZP and participate in the process of fertilization. For example, sperm exposed to unfertilized egg mZP3 at nanomolar concentrations are inhibited from binding to eggs and undergo the acrosome reaction. Neither mZP1 nor mZP2 has an effect on sperm binding or the acrosome reaction. Furthermore, mZP3 from fertilized eggs has no effect on sperm binding and is unable to induce the acrosome reaction. These and other properties of mZP3 suggest that it is a receptor for sperm and inducer of the acrosome reaction. Mapping of the mZP3 co...

Background: Some traditional herbs disrupt endocrine-endometrial synchrony, affecting embryo-endometrium communication during fertility. Hormonal imbalances cause non-receptive conditions, leading to anti-implantation or abortion. Historically, Mentha pulegium has served for contraceptive and abortive purposes. Its effects on the postcoital contraceptive and hormonal activities were evaluated. Methods: Thirty-six Sprague-Dawley female rats were selected based on the presence of copulation plugs and received 200, 500, and 1000 mg/kg of Hydroethanolic leaf extract of Mentha pulegium L. (MPE) for seven days. Reproductive organ weights and serum estrogen and progesterone levels were measured. The resorption index, antiimplantation activity, and pre-implantation loss were also calculated using the number of implantation sites and resorptions in all treatments. Data were presented as mean±standard error mean (SEM), and significance was defined as p<0.05 using one-way ANOVA. Results: Post-coital administration of MPE resulted in resorptive, pre-implantation loss, and anti-implantation activity. A dose of 200 mg/kg reduced the number of implantations and exhibited a high resorption index, percentage pre-implantation loss, and anti-implantation activity. A marked decline in serum progesterone levels and a significant reduction in serum estrogen and progesterone ratio was observed at 200 mg/kg MPE. A significant increase in uterine weight was observed in the 500 mg/kg treatment. Doses of 500 and 1000 mg/kg resulted in a significant reduction in anti-implantation activity. Conclusions: The observed anti-implantation activity and pre-implantation loss suggest the abortifacient properties of MPE. However, its effects were seen to be dose-dependent.

Previous studies from our laboratory have identified MPS, a 100‐kDa protein, as the major phosphoprotein substrate of caprine sperm ecto‐cyclic AMP independent protein kinase. In this study the isolated 32P‐labelled MPS has been incorporated into mature caprine (Capra indicus) cauda‐epididymal spermatozoa with the help of cell electroporation technique to investigate the effect of MPS on sperm flagellar motility. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2 × 108/ml) to external electric field of intensity 1.5 kV/cm and capacitation of 25 µF at 4°C and post‐pulse incubation at 37°C for 1 hr. when nearly 50% of the cells lost motility. Scanning electron micrographs (SEM) demonstrate the formation of micro‐pores and local osmotic swelling in the electroporated spermatozoa. MPS incorporation was maximal when its concentration was 30 µg/ml (300 pmol) in the medium and when the post‐pulse incubation time was 60 min. At maximu...

In mammalian eggs, activation by sperm that leads to resumption of meiosis is characterized by an explosive transient increase in intracellular calcium ion concentration ([Ca2+]i), followed by [Ca2+]i oscillations. In addition to the spermatozoon, various treatments can induce parthenogenetic activation, accompanied by an elevation of [Ca2+]i. It has been reported that cooling can induce egg activation, yet the mechanism of this phenomenon has not been elucidated. In the present study we followed changes in egg [Ca2+]i (measured by Fura‐2 fluorescence ratio imaging) during activation by cooling, using conditions that ensure a low rate of spontaneous activation.Our present findings demonstrate that cooling induces egg activation as manifested by [Ca2+]i transient(s) and second polar body extrusion. Seventy‐eight of 104 eggs responded to cooling with increased [Ca2+]i. Thirty‐five percent of the responding eggs displayed a single [Ca2+]i transient, while 65% exhibited at least two [Ca...

Under the influence of gonadotropins or growth factors, a close cooperation develops between cumulus cells and the oocyte that is implicated in transmitting signals involved in maintaining or releasing the meiotic arrest in the oocyte. While cyclic adenosine 5′‐monophosphate (cAMP) is a key molecule in maintaining the meiotic arrest, calcium (Ca2+) may play a role in controlling either spontaneous or gonadotropin‐induced oocyte maturation, possibly by modulating intracytoplasmic cAMP concentrations via Ca2+‐sensitive adenylate cyclases. This review focuses on the mechanisms related to the origin of the Ca2+ wave that travels from the cumulus cells to the oocyte, and discusses the source of variations affecting the dynamics of this wave. Mol. Reprod. Dev. 78:744–756, 2011. © 2011 Wiley Periodicals, Inc.

Atlantic salmon males may mature already as small parr in freshwater. Sexual maturation in teleosts as in vertebrates is characterized by the activation of the brain-pituitary-gonad axis. The endocrine regulation of early puberty is still not well understood. In the present study, one-summer-old male Atlantic salmon parr were sampled regularly from December several months prior to the beginning of spermatogenesis until spawning in October. Pituitary expression levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) b subunit genes were analyzed in parallel with testis expression of FSH receptor (FSHR) and LH receptor (LHR) genes by RT-PCR and plasma 11-ketostestosterone (11-KT) was measured. Expression levels of FSHb, low during winter and spring started to increase prior to the onset of gonadal growth at the end of May while LHb mRNA levels were hardly detectable. Both gonadotropin receptor genes were expressed in immature testis with FSHR transcripts being more abundant (8-fold). FSHR transcript levels increased in parallel to FSHb levels from early spermatogenesis onwards, while LHR mRNA started to increase prior to any large changes in LHb expression. Both transcript levels of LHb and LHR were highest during spermiation. Plasma 11-KT increased at the beginning of spermatogenesis reaching highest levels at spermiogenesis suggesting a possible role of FSH in inducing 11-KT production during early spermatogenesis while LH stimulates via its specific receptor 11-KT production at spermiogenesis. The commitment into sexual maturation appears to be dependant on both the presence of FSHR in immature testis and the increase of FSH expression.

The present studies were carried out to characterize the CAMP-phosphodiesterase enzyme (PDE) in luteal cells recovered from pseudopregnant rats with streptozotocin-induced diabetes. A significant increase in the specific activity of the enzyme was detected in luteal cells from diabetic rats (Group D) with respect to control rats (Group C). This increase could not be prevented by insulin therapy (Group I). Luteal cells from Groups C and D rats responded in vitro to insulin by increasing their PDE activity (% of stimulus of specific activity: C = 75%, D = 110%). However, in cells isolated from Group I, the hormone caused an inhibition of PDE activity (% of inhibition of specific activity: 48%). When cytosolic fractions from Groups C, D, and I were submitted to ion exchange chromatography, two PDE activity peaks could be observed and the activity of the different fractions was increased in the presence of Ca2' and calmodulin. Nevertheless, the CaZtcalmodulin effect was much lower in the extracts from Groups D and I than for controls. Kinetic studies of luteal PDE showed nonlinear Lineweaver-Burk graphs with two apparent ATP hydrolysis sites. Similar K, values were found for PDE from groups C, D, and I, whereas the Vmax2 for the enzyme was higher in Groups D and I. The endogenous concentration of CAMP, measured by RIA, showed no significant differences among Groups C, D, and I. On the basis of these results, we conclude that the specific activity of PDE is significantly increased in luteal cells from streptozotocin-induced diabetic animals, which could explain the previously described reduction in LH-stimulated progesterone production by luteal cells in diabetic rats.

The control of protein synthesis during maturation in oocytes is mainly exerted through cytoplasmic polyadenylation of stored mRNAs. We first analyzed the polyadenylation status of cyclins A2 and B1 during in vitro maturation (IVM) of bovine oocytes, using Rapid Amplification of cDNA Ends-Polyadenylation Technique (RACE-PAT). An inconstant elongation of the poly(A) tail was observed for cyclin A2 transcripts after maturation, while a constant lengthening was observed for cyclin B1, occurring during the first 12 hr of incubation. We then evaluated the effects of the polyadenylation inhibitor 3 0-deoxyadenosine (3 0-dA), on polyadenylation and nuclear maturation. The presence of 0.02 mM 3 0-dA during the whole incubation period or from 6 hr after its beginning completely prevented meiosis progression in 100% of the oocytes. Polyadenylation of cyclin B1 was also completely prevented when 3 0-dA was added at 0 hr, and greatly reduced when added at 6 hr. When 3 0-dA was added at 12 hr, around metaphase I (MI), 46.9% of the oocytes have reached metaphase II (MII, vs. 78.8% in the control group) at 24 hr. The use of the same concentration of 3 0-deoxyguanosine (3 0-dG), that impairs transcription but not polyadenylation, did not affect cyclins polyadenylation, nor nuclear maturation, whatever was the timing of addition. These results suggest that the polyadenylation of cyclin B1 could be related to the first peak of activity of MPF, occurring around MI (10-12 hr after the onset of the maturation period). They also show that, in our culture conditions, inhibition of polyadenylation prevents meiosis progression, especially up to the MI stage, while inhibition of transcription does not.

Preimplantation embryos utilize mitogen‐activated protein kinase signaling (MAPK) pathways to relay signals from the external environment to prepare appropriate responses and adaptations to a changing milieu. It is therefore important to investigate how MAPK pathways are regulated during preimplantation development. This study was conducted to investigate whether PP2Cδ (Ppm1d, WIP1) is expressed during mouse preimplantation development and to determine the influences of p38 MAPK inhibition on expression of Trp53 (p53), Ppm1d, (WIP1), and Cdkn2a (p16) during mouse preimplantation development. Our results indicate that Trp53, Ppm1d, and Cdkn2a mRNAs and TRP53 and PP2Cδ proteins are expressed throughout mouse preimplantation development. Treatment of 2‐cell embryos with SB220025 (potent inhibitor of p38 MAPK α/β/MAPK 14/11) significantly increased Trp53, Ppm1d and Cdkn2a and Mapk14 mRNA levels at 12 and 24 hr. Treatment of 8‐cell embryos with SB220025 for 12 hr increased Trp53, Ppm1d, ...

The macromolecular composition of zona pellucida (ZP) isolated from human oocytes and embryos was characterized by one-and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (I-D-and 2-D-SDS-PAGE) under reducing conditions after silver staining. ZP specimens obtained after in vitro fertilization were removed from pools of heavily fragmented embryos and inseminated oocytes that failed to fertilize. For unfertilized oocytes, two major bands with an apparent molecular weight of, respectively, 96 and 76-54 kDa were observed after 1-D-SDS-PAGE and silver staining. When ZP were isolated from fragmented embryos, the electrophoretic pattern showed a marked attenuation of the 96-kDa band. Silver-stained 2-D-SDS-PAGE analysis of ZP components from unfertilized oocytes revealed the presence of four protein trains: ZP1 (Mr = 92-80 kDa, pl = 4.9-5.9), ZP2 (Mr = 66-58 kDa, pl = 5.0-6.0), Z P~H (high) (Mr = 72-58 kDa, pl = 35-5.1), and Z P~L (low) (Mr = 62-54 kDa, pl = 3.5-5.1). The human ZP3 family (Z P~H and Z P~L) showed marked heterogeneity. Fertilization-associated changes were apparent in the electrophoretic pattern. ZP1 (Mr = 92-86 kDa, pl = 5.0-5.8) displayed a dramatic decrease in intensity, and a new component had migrated to a position similar to that of ZP2. This modification may have been responsible for one aspect of the zona reaction, and could have contributed to a zona block to polysperma. Q 1995 Wiley-Liss, Inc.

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8-cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF-derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF-derived blastomeres with parthenogenetic blsatomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVFderived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or eight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were constructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zonafree) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demiembryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypicallychimeric viable male calf was born. We conclude that the IVF-derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentally competent.

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8-cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF-derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF-derived blastomeres with parthenogenetic blsatomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVFderived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or eight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were constructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zonafree) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demiembryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypicallychimeric viable male calf was born. We conclude that the IVF-derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentally competent.

Amphibians evolved in the Devonian period about 400 Mya and represent a transition step in tetrapod evolution. Among amphibians, high-throughput sequencing data are very limited for Caudata, due to their largest genome sizes among terrestrial vertebrates. In this paper we present the transcriptome from the fire bellied newt Cynops orientalis. Data here presented display a high level of completeness, comparable to the fully sequenced genomes available from other amphibians. Moreover, this work focused on genes involved in gametogenesis and sexual development. Surprisingly, the gsdf gene was identified for the first time in a tetrapod species, so far known only from bony fish and basal sarcopterygians. Our analysis failed to isolate fgf24 and foxl3, supporting the possible loss of both genes in the common ancestor of Rhipidistians. In Cynops, the expression analysis of genes described to be sex-related in vertebrates singled out an expected functional role for some genes, while others...

In mammals, fertilization initiates Ca2+ oscillations in metaphase II oocytes that are required for the activation of embryo development. Germinal vesicle (GV) oocytes also display Ca2+ oscillations, although they unfold spontaneously in the absence of any known agonist(s) and their function remain unclear. We found that the main intracellular store of Ca2+ in GV oocytes, the endoplasmic reticulum ([Ca2+]ER), constitutively “leaks” Ca2+ through the type 1 inositol 1,4,5-trisphosphate receptor. The [Ca2+]ER leak ceases around the resumption of meiosis, the GVBD stage, which coincides with the first noticeable accumulation of Ca2+ in the stores. It also concurs with downregulation of the Ca2+ influx and termination of the oscillations, which seemed underpinned by the inactivation of the putative plasma membrane Ca2+ channels. Lastly, we demonstrate that mitochondria uptake Ca2+ during the Ca2+ oscillations mounting their own oscillations, which stimulate the mitochondrial redox state ...

Attempts were made to identify species in commercial frozen shrimp meat samples collected in Taiwan. First, approximately 20 fresh specimens of each of the 10 common commercial shrimp species were identified by morphological methods. A 401 to 407-bp fragment of the cytochrome b gene was amplified from the shrimp meat by polymerase chain reaction (PCR) using the primer pair UCYTB151F/270R. By comparing the gene sequences and PCR-restriction fragment length polymorphism (RFLP) maps obtained using the endonucleases Alu I, Ssp I and Hae III, the 10 species of shrimp could be successfully differentiated. The PCR-RFLP method was proved applicable for the species identification of 10 marketed shrimp samples.

An early step in mammalian fertilization is species‐restricted binding of sperm to the egg&#39;s zona pellucida (ZP), a thick extracellular coat that surrounds eggs. Sperm bind to the ZP of unfertilized eggs, but not to the ZP of fertilized eggs. Shortly after binding to the unfertilized egg ZP, sperm undergo the acrosome reaction, a form of cellular exocytosis that enables sperm to penetrate the ZP. Three glycoproteins, mZP1‐3, constitute the mouse egg&#39;s ZP and participate in the process of fertilization. For example, sperm exposed to unfertilized egg mZP3 at nanomolar concentrations are inhibited from binding to eggs and undergo the acrosome reaction. Neither mZP1 nor mZP2 has an effect on sperm binding or the acrosome reaction. Furthermore, mZP3 from fertilized eggs has no effect on sperm binding and is unable to induce the acrosome reaction. These and other properties of mZP3 suggest that it is a receptor for sperm and inducer of the acrosome reaction. Mapping of the mZP3 co...

The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol‐17β. As males of both species synthesize these proteins after treatment with estradiol‐17β, the origin is not restricted to the ovaries. Vitellogenin of both Eurpoean sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa. © 1995 Wiley‐Liss, Inc.

Renaturation kinetics of DNA obtained from chicken testis cell nuclei separated by sedimentation at unit gravity showed that the undermethylation, previously observed in meiotic and postmeiotic cells, is not a peculiarity of repetitive sequences, but is also a feature of unique sequences. The large proportion of slowly renaturing, intermediately renaturing and rapidly renaturing DNAs contain 27, 32 and 31% less methylcytosines in meiotic and postmeiotic cells than the corresponding fractions of premeiotic cells. DNA methyltransferase activity is lower in meiotic and postmeiotic cells containing undermethylated DNA than in immature testis, enriched in spermatogonia, with higher levels of DNA methylation.

Exposure of male rats to cyclophosphamide, a commonly used anticancer and immunosuppressive drug, has been shown to alter fertility and progeny outcome in a male germ cell phase‐specific manner. The effect of toxicant exposure on male germ cells depends in part on the stress response mechanisms present during the different stages of spermatogenesis. To assess how acute cyclophosphamide exposure affects the expression of stress response genes, we examined the expression of 216 genes, using gene expression arrays, in isolated rat spermatogenic cell types (pachytene spermatocytes, round spermatids, and elongating spermatids). Cyclophosphamide exposure affected gene expression in all cell types but most dramatically in round spermatids. Increased transcript levels were observed for 30 genes in round spermatids compared to seven genes in pachytene spermatocytes and two in elongating spermatids. The expression of genes involved in apoptosis, DNA‐damage recognition and repair, transcriptio...

We have identified a bone cell adhesion molecule, osteopontin, in the rat testis and epididymis by Northern analysis, RT-PCR, Western immunoblot analysis, and immunocytochernistry. A polyclonal antibody raised against rat epididymal fluid proteins was used to detect fusion proteins produced by a testis Xgtll cDNA library. Sequence analysis of one of four positive cDNA clones, designated as pREP5, revealed identity with the rat osteopontin (OPN) cDNA. The partial cDNA clone pREP5 encompasses 64% of the 1,457 residues reported by Oldberg et al. (1986; Proc Natl Acad Sci USA 83:8819-8823). lmrnunoblot analysis with a monoclonal antibody against OPN detects the presence of imrnunoreactive polypeptides in rat testis homogenates as well as in epididymal fluid and sperm extracts. lrnmunocytochemical localization to the basal and adluminal region of the seminiferous tubule suggests that OPN could be a Sertoli cell product. Indeed, Northern blot analysis of testicular cell preparations demonstated positive hybridization to Sertoli cellenriched RNA, but not to RNA isolated from interstitial cell preparations or to isolated germ cell RNA preparations. OPN is also detected in the rat epididymis and on epididy-ma1 spermatozoa. This is the first report on the presence of OPN mRNA and protein in rat testis and epididymis and on the presence of OPN on the surface of epididymal spermatozoa. The characterization of this protein in other tissue suggests that OPN could play a role in testicular cell adhesion during spermatogenesis and/or epididymal maturation, although other potential functions in the male reproductive tract are discussed. o 1995 Wiley-Liss, tnc.

Only competent oocytes are able to undergo complete maturation and normal embryonic development. Therefore, the identification of genes that are differentially expressed in competent oocytes would contribute to our understanding of the factors controlling competency. It is well known that time of cleavage after insemination in vitro is highly correlated with embryonic developmental potential and this can be used to distinguish between oocytes of different quality. The main objective of this study was to identify genes associated with competency and rapid cleavage. We examined the expression of 16 candidate genes (IDH,

The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52,48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol-17P. As males of both species synthesize these proteins after treatment with estradiol-17p, the origin is not restricted to the ovaries. Vitellogenin of both Eurpoean sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa.

Hemocyanin (HC) was purified from hemolymph of Fenneropenaeus merguiensis (de Man, 1888). Purified HC showed one band in nondenaturing PAGE with a molecular mass of 457 kDa, determined by gel filtration. Using an ELISA, HC content was 85.6% of total hemolymph protein. HC's hexamer form contained no phenoloxidase activity under trypsin or SDS activation. FmHC, cloned from F. merguiensis, had a full-length cDNA comprised of 2128 bp with an open reading frame encoding a peptide of 661 amino acids with two Cu-binding sites. Expression of FmHC was found only in the hepatopancreas where it was up-regulated after exposure to Vibrio harveyi injection into shrimp and with tissue incubations. HC protein levels in the hemolymph of bacterial injected shrimp also increased. FmHC expression was also up-regulated after shrimp were challenged with a lipopolysaccharide, which is a surface component of gram-negative bacteria. These results indicate that HC is inducible and up-regulated in response to pathogenic challenge, confirming that HC participates in the innate immune response in F. merguiensis.

A complete cDNA sequence of vitellogenin (Vg-H) was cloned from the hepatopancreas of Fenneropenaeus merguiensis (De Man, 1888). The full-length Vg-H gene consists of 7958 bp and contains an ORF of 7761 bp. It encodes a polypeptide of 2587 amino acids, comprising a predicted molecular mass of 283 kD and the theoretical pI is 6.5. The amino acid sequences of the mature vitellogenin (Vg) composed of a signal peptide, three possible O-glycosylation sites, three phosphorylation sites and putative possessing sites (R-X-K/R-R) recognized by subtilisin-like endoproteases. Cleavage at residues 725 to 728 will produce two Vg subunits (78 and 200 kD), which the N-terminal amino acid sequence of 78 kD subunit is identical to that of vitellin purified from the ovary. These properties suggest that Vg protein encoding by Vg-H undergoes processing at its synthetic sites prior to being transported to developing oöcytes. The deduced primary structure of Vg-H showed the highest identity (98.8%) to the Vg-O previously cloned from the ovary of the same species. It is more related to the penaeid Vg sequences than that of nonpenaeids. In situ hybridization revealed that mRNA encoding Vg was expressed both in follicle cells in the ovary and parenchyma cells in the hepatopancreas. In the developing ovary, highest levels were detected during the early vitellogenic stage 2, declining thereafter. In the hepatopancreas, Vg mRNA levels reached a maximum at stage 3 of ovarian development. Profiles of Vg mRNA expression in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenesis is inversely related during gonad maturation. Thus, Vg gene sequences expressed in the ovary and hepatopancreas are most likely identical and both tissues are responded for yolk protein synthesis in F. merguiensis.

In order to explore the significance of DNA methylation in proliferation and differentiation of germ cells in testis, 5-aza,2'-deoxyCytidine (5-azaCdR), a hypomethylating agent, was administered in vivo to neonatal mice having only spermatogonial (premeiotic) cells. End-labeling of the Mspl, Hpall, and Hhal digested DNA revealed considerable loss of methylation following the treatment. Cellular and histological preparations of the testis showed complete inhibition of differentiation into spermatocytic stage. Analysis of protein synthesis in the treated and control testis by growing the cells in 35S-Methionine medium and resolving the lysate by SDS-PAGE revealed that the programme of expression of at least 5 polypeptides (35.0, 31.5, 27.0, 22.5, and 18.0 KD) was altered as a result of 5-azaCdR incorporation. It appears that DNA methylation plays a critical role in the differentiation of gonia into primary spermatocytes.

A complete cDNA sequence of vitellogenin (Vg-H) was cloned from the hepatopancreas of Fenneropenaeus merguiensis (De Man, 1888). The full-length Vg-H gene consists of 7958 bp and contains an ORF of 7761 bp. It encodes a polypeptide of 2587 amino acids, comprising a predicted molecular mass of 283 kD and the theoretical pI is 6.5. The amino acid sequences of the mature vitellogenin (Vg) composed of a signal peptide, three possible O-glycosylation sites, three phosphorylation sites and putative possessing sites (R-X-K/R-R) recognized by subtilisin-like endoproteases. Cleavage at residues 725 to 728 will produce two Vg subunits (78 and 200 kD), which the N-terminal amino acid sequence of 78 kD subunit is identical to that of vitellin purified from the ovary. These properties suggest that Vg protein encoding by Vg-H undergoes processing at its synthetic sites prior to being transported to developing oöcytes. The deduced primary structure of Vg-H showed the highest identity (98.8%) to the Vg-O previously cloned from the ovary of the same species. It is more related to the penaeid Vg sequences than that of nonpenaeids. In situ hybridization revealed that mRNA encoding Vg was expressed both in follicle cells in the ovary and parenchyma cells in the hepatopancreas. In the developing ovary, highest levels were detected during the early vitellogenic stage 2, declining thereafter. In the hepatopancreas, Vg mRNA levels reached a maximum at stage 3 of ovarian development. Profiles of Vg mRNA expression in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenesis is inversely related during gonad maturation. Thus, Vg gene sequences expressed in the ovary and hepatopancreas are most likely identical and both tissues are responded for yolk protein synthesis in F. merguiensis.

Brahman preimplantation embryos are less affected by exposure to heat shock than Holstein embryos. Two experiments were conducted to test whether the ability of Brahman embryos to resist the deleterious effects of heat shock was a result of the genetic and cellular contributions from the oocyte, spermatozoa, or a combination of both. In the first experiment, Brahman and Holstein oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from an Angus bull. A different bull was used for each replicate to eliminate bull effects. On day 4 after fertilization, embryos ! 9 cells were collected and randomly assigned to control (38.58C) or heat shock (418C for 6 hr) treatments. The proportion of embryos developing to the blastocyst (BL) and advanced blastocyst (ABL; expanded and hatched) stages was recorded on day 8. Heat shock reduced the number of embryos produced from Holstein oocytes that developed to BL (P < 0.001, 55.6 AE 4.2% vs. 29.8 AE 4.2%) and ABL (P < 0.01, 37.7 AE 3.6% vs. 12.2 AE 3.6%) on day 8 as compared to controls. In contrast, heat shock did not reduce development of embryos produced from Brahman oocytes (BL ¼ 42.1 AE 4.8% vs. 55.6 AE 4.8% for 38.5 and 418C, respectively; ABL ¼ 17.6 AE 4.2% vs. 32.4 AE 4.2%). In the second experiment, oocytes from Holstein cows were fertilized with semen from bulls of either Brahman or Angus breeds. Heat shock of embryos ! 9 cells reduced development to BL (P < 0.002) and ABL (P < 0.005) for embryos sired by both Brahman (BL ¼ 54.3 AE 7.7% vs. 23.4 AE 7.7%; ABL ¼ 43. AE 7.4% vs. 7.9 AE 7.4%, for 38.5 and 418C, respectively) and Angus bulls (BL ¼ 57.9 AE 7.7% vs. 31.0 AE 7.7%; ABL ¼ 33.6 AE 7.4% vs. 18.4 AE 7.4%, for 38.5 and 418C, respectively). There were no breed  temperature interactions. Results suggest that the oocyte plays a more significant role in the resistance of Brahman embryos to the deleterious effects of heat shock than the spermatozoa.

This study is intended to characterize the ovarian development and hemolymph vertebrate-type steroids concentration during the reproductive cycle of the freshwater prawn, Macrobrachium rosenbergii. A five-stage classification based on the external observation of the ovary's size and color as seen through the tegument was used. The results showed the existence of a direct correspondence between the ovarian stages and the gonadosomatic index, the oöcyte diameter, and the characteristics of ovarian histology. In each stage total bleeding of the prawns was conducted and the hemolymph concentrations of 17b-estradiol (E 2), testosterone (T) and 17a-hydroxyprogesterone (17-OHP; conjugated and unconjugated) were determined by solid-phase radioimmunoassay (RIA). High levels of unconjugated 17-OHP, relatively constant concentrations of unconjugated T, and null concentration of unconjugated E 2 were found throughout the five stages considered. Nonetheless, low levels of E 2 were determined in all stages, in conjugated (glucoronide) form. The highest levels were obtained in stage II and III (18.14 6 14.52 pg/ml hemolymph) and progressively declined in the other stages (7.53 6 6.76 pg/ml hemolymph). These results point out the possible involvement of vertebrate-type steroids in the endocrine regulation of the Macrobrachium rosenbergii's ovarian cycle. To our knowledge this is the first study concerning the vertebrate-like steroid levels throughout the ovarian cycle and is a step needed to characterize the hemolymph profile of these steroids in order to elucidate the possible role in the regulation of the reproductive crustacean cycle.

We studied apoptotic fragmentation in ovulated mouse oocytes. Some cells were fixed immediately after isolation, while others were cultured for 3 hours with or without prostaglandin F2 alpha. Membrane organelles, fibrillar actin and DNA were stained with DiOC6, TRITC-phalloidin and Hoechst 33342, respectively. While fragmented cells were generally rare, most of them were in samples treated with prostaglandin F2α, revealing it as a potential inducer of apoptosis. The chromatin had interphase appearance, indicating exit from meiosis or arrest at germinal vesicle stage. Fragmentation tended to be more pronounced in the vicinity of chromatin, which could be explained with the concentration of actin in the cap region. These preliminary data confirm the active participation of cytoplasm in oocyte apoptosis, and suggest that this process could be induced by mediators of inflammation.

Preantral follicles (PAF) and early antral follicles (EAF) were isolated from bovine ovaries and classified under a stereomicroscope as atretic or healthy. The atretic follicles were all considered as group I (in vivo atresia), whereas healthy follicles were assigned to five groups (group II, in vivo normal control; group 111, in vitro normal control; group IV, in vitro induced atresia; groups V and VI, Lipopolysaccharide (LPSI-induced atresia in vitro). Group I and II follicles were immediately snap-frozen (-70°C) until DNA extraction, whereas group Ill-VI follicles were incubated (39"C, 5% C02, 95% air) for periods up to 72 hr under various conditions. Group 111 follicles were maintained in complete medium (M199, bovine calf serum, sodium pyruvate, epidermal growth factor, insulin, transferrin, sodium selenite, penicillin, streptomycin, and amphotericin), whereas group IV follicles were incubated in the same medium, but without serum. Group V and VI follicles were maintained in complete medium, but in the presence of LPS (10 or 50 pg/ml, respectively). Results showed that follicles incubated in the absence of serum and those exposed to both doses of LPS became atretic. DNA isolated from all atretic follicles showed fragmentation typical of that described for apoptosis; this was also confirmed by in situ DNA labeling and histology. Atretic follicles did not produce estradiol (P < 0.001), but progesterone values increased with follicle size (P < 0.001) and time of incubation (P < 0.001). We concluded that in the absence of serum or in the presence of LPS, follicles undergo atresia via apoptosis. o 1996 WiIey-Liss, Inc.

The purposes of this study were to determine how early in time endotoxin can trigger apoptosis of bovine ovarian follicles in vitro, and to further characterize if these inductions are mediated via adenine nucleotides and the P2 purinergic receptors. Healthy preantral and early antral follicles (400 and 700 p,m) isolated from bovine ovaries were sandwiched between two layers of collagen gel and incubated (39°C 5% C02, 95% air) for various time periods up to 72 hr, floating in complete medium with either 2-MethylThioATP or with 2-ChloroATP, or with or without LPS (10 or 50 p,g/rnl), or with combinations of LPS with 2-MethylThioATP or 2-ChloroATP. Data from histological examination, and in situ detection of apoptotic DNA cleavage, showed that by 2 hr from start of incubation, both doses of LPS had triggered apoptosis of granulosa cells (P < 0.001), and simultaneously decreased estradiol concentrations to nondetectable levels (P < 0.001), but progesterone values increased (P < 0.001) with time of incubation. Both 2-MethylThioATP and 2-ChloroATP inhibited (P < 0.001) LPS (10 and 50 p,g/ ml)-induced apoptosis by 30% to 100%. We concluded that adenine nucleotides play a fundamental role in endotoxininduced apoptosis/atresia of bovine follicles, probably via the P2 purinergic receptors. It is possible that during the first 2 hr of incubation, the apoptotic events associated with LPS-induced follicular atresia might not be detectable with the procedures used in this study.

A precise regulation of the uterine fluid volume and pH is essential for a successful embryo implantation. Progesterone has been reported to participate in uterine fluid volume regulation during this period, however its effect on the uterine fluid pH is unknown. As endometrial fluid absorption has been proposed to occur secondary to sodium (Na +) absorption under progesterone mediated effect, we therefore hypothesize that there may be a concomitant changes in fluid volume and pH if sodium-hydrogen exchanger (NHE), a protein responsible for both luminal Na + absorption and H + extrusion is involved. In view of these, our study aimed to investigate the possibility that progesterone affect the uterine fluid pH and endometrial NHE expression. Ovariectomised female Sprague-Dawley (SD) rats were treated with peanut oil (vehicle), oestradiol-3-benzoate and progesterone for three consecutive days. On the fourth day, in-vivo uterine perfusions were performed on anaesthetized rats. The collected perfusate were analyzed for the changes in pH. The effect of amiloride, a non-specific Na +-channel blocker on the pH was investigated. The expression of uterine NHE-1 protein was detected by Western blotting and immunohistochemistry. Our findings indicate that the fluid pH is the lowest in progesterone-treated group and amiloride administration significantly increased the pH in the same treatment group (p < 0.05). NHE-1 proteins were significantly expressed in the progesterone-treated group. In conclusion, progesterone induces a reduction of the uterine fluid pH and is amiloride-sensitive. The up-regulation of NHE-1 under a common progesterone effect may explain the role of this exchanger in regulating the uterine fluid pH.

During cryopreservation, the immature oocyte is subjected to anisosmotic conditions potentially impairing subsequent nuclear and cytoplasmic maturation in vitro. In preparation for cryopreservation protocols and to characterize osmotic tolerance, cat cumulus-oocyte complexes (COC) at the germinal vesicle (GV) stage were exposed for 15 min to sucrose solutions ranging from 100 to 2,000 mOsm and then examined for structural integrity and developmental competence in vitro. Osmolarities ≥200 and ≤750 mOsm had no effect on incidence of oocyte nuclear maturation, fertilization success, and blastocyst formation compared to control COC (exposed to 290 mOsm). This relatively high osmotic tolerance of the immature cat oocyte appeared to arise from a remarkable stability of the GV chromatin structure as well as plasticity in mitochondrial distribution, membrane integrity, and ability to maintain cumulus-oocyte communications. Osmolarities < 200 mOsm only damaged cumulus cell membrane integrity, which contributed to poor nuclear maturation but ultimately had no adverse effect on blastocyst formation in vitro. Osmolarities >750 mOsm compromised nuclear maturation and blastocyst formation in vitro via disruption of cumulus-oocyte communications, an effect that could be mitigated through 1,500 mOsm by adding cytochalasin B to the hyperosmotic solutions. These results (1) demonstrate, for the first time, the expansive osmotic tolerance of the immature cat oocyte, (2) characterize the fundamental role of cumulus-oocyte communications when tolerance limits are exceeded, and (3) reveal an interesting hyperosmotic tolerance of the immature oocyte that can be increased twofold by supplementation with cytochalasin B.

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P Ͻ 0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P Ͻ 0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P Ͻ 0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P Ͻ 0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P Ͼ 0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.

According to the World Health Organization, infertility affects up to 14% of couples under reproductive age, leading to an exponential rise in the use of assisted reproduction as a route for conceiving a baby. In the same way, thousands of embryos are produced in cattle and other farm animals annually, leading to increased numbers of individuals born. All reproductive manipulations entail deviations of natural phenotypes and genotypes, with in vitro embryo technologies perhaps showing the biggest effects, although these alterations are still emerging. Most of these indications have been provided by animal models, in particular the bovine species, due to its similarities to human early embryo development. Oocytes and embryos are highly sensitive to environmental stress in vivo and in vitro. Thus, during in vitro culture, a number of stressful conditions affect embryonic quality and viability, inducing subfertility and/or long-term consequences that may reach the offspring. A high pro...

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k, = 0.003 hr-'; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k, = 0.145 and 0.162 hr-', respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37°C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A, may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation. o 1995 Wiley-Liss, Inc.

Myostatin is a dominant inhibitor of skeletal muscle development and growth. As transgenic over-expression of myostatin propeptide dramatically enhanced muscle mass, we hypothesized that administration of myostatin propeptide will increase muscle growth. In this study, the wild-type form of porcine myostatin propeptide and its mutated form at the cleavage site of metalloproteinases of BMP-1/TLD family were produced from insect cells. In vitro A204 cells reporter assays showed that both wildtype and the mutated propeptides depressed myostatin activity. The recombinant propeptides at four-fold myostatin concentration can effectively block myostatin function during co-incubation with A204 cells. In particular, the mutated propeptide appeared much more effective than wild-type propeptide over a long period during the in vitro co-incubation. Administration of the mutated propeptide to neonatal mice at the age of 11 and 18 days was tested and showed significant increase in growth performance by 11-15% from the age of 25 to 57 days (P < 0.05). The major skeletal muscles of mice that were injected with mutated propeptide were 13.5-24.8% heavier than the control group (P < 0.05) as a result of muscle fiber hypertrophy. In conclusion, administration of the mutated myostatin propeptide during the neonatal period is an effective way for promoting muscle growth.

To investigate whether GABA/progesterone (P(4)) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa(2+) for 5.5 h and then labeled with [(32)P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5x10(7) cells/mL and exposed to 2 mmol/L Ca(2+), 5 micromol/L GABA, 10 micromol/L P(4) and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermatozoa were treated with GABA,(32)P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP(2)), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic acid (PA). The loss of label from PPI was almost completed by 10 min. The time-course of the AR was much slower than PPI when spermatozoa reached a maximal response by 15 min; (ii) the pattern of PPI hydrolysis and stimulation...

Intrarea în Uniunea Europeanã conferã enorme posibilitãţi tinerilor studioşi. Adaptarea la sistemul european de credite transferabile face ca orice student al universitãţii noastre sã-şi poatã continua studiile sau sã facã un semestru (modul) la orice universitate de profil din ...

A complete cDNA sequence of vitellogenin (Vg-H) was cloned from the hepatopancreas of Fenneropenaeus merguiensis (De Man, 1888). The full-length Vg-H gene consists of 7958 bp and contains an ORF of 7761 bp. It encodes a polypeptide of 2587 amino acids, comprising a predicted molecular mass of 283 kD and the theoretical pI is 6.5. The amino acid sequences of the mature vitellogenin (Vg) composed of a signal peptide, three possible O-glycosylation sites, three phosphorylation sites and putative possessing sites (R-X-K/R-R) recognized by subtilisin-like endoproteases. Cleavage at residues 725 to 728 will produce two Vg subunits (78 and 200 kD), which the N-terminal amino acid sequence of 78 kD subunit is identical to that of vitellin purified from the ovary. These properties suggest that Vg protein encoding by Vg-H undergoes processing at its synthetic sites prior to being transported to developing oöcytes. The deduced primary structure of Vg-H showed the highest identity (98.8%) to the Vg-O previously cloned from the ovary of the same species. It is more related to the penaeid Vg sequences than that of nonpenaeids. In situ hybridization revealed that mRNA encoding Vg was expressed both in follicle cells in the ovary and parenchyma cells in the hepatopancreas. In the developing ovary, highest levels were detected during the early vitellogenic stage 2, declining thereafter. In the hepatopancreas, Vg mRNA levels reached a maximum at stage 3 of ovarian development. Profiles of Vg mRNA expression in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenesis is inversely related during gonad maturation. Thus, Vg gene sequences expressed in the ovary and hepatopancreas are most likely identical and both tissues are responded for yolk protein synthesis in F. merguiensis.

In brief Post-ovulatory ageing of oocytes leads to poor oocyte and embryo quality as well as abnormalities in offspring. This review provides an update on the contributions of oxidative stress to this process and discusses the current literature surrounding the use of antioxidant media to delay post-ovulatory oocyte ageing. Following ovulation, the metaphase II stage oocyte has a limited functional lifespan before succumbing to a process known as post-ovulatory oocyte ageing. This progressive demise occurs both in vivo and in vitro and is accompanied by a deterioration in oocyte quality, leading to a well-defined sequelae of reduced fertilisation rates, poor embryo quality, post-implantation errors, and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been characterised, less is known regarding the molecular mechanisms that drive this process. This review presents an update on the established relationships between t...

Under the influence of gonadotropins or growth factors, a close cooperation develops between cumulus cells and the oocyte that is implicated in transmitting signals involved in maintaining or releasing the meiotic arrest in the oocyte. While cyclic adenosine 5 0-monophosphate (cAMP) is a key molecule in maintaining the meiotic arrest, calcium (Ca 2þ) may play a role in controlling either spontaneous or gonadotropin-induced oocyte maturation, possibly by modulating intracytoplasmic cAMP concentrations via Ca 2þ-sensitive adenylate cyclases. This review focuses on the mechanisms related to the origin of the Ca 2þ wave that travels from the cumulus cells to the oocyte, and discusses the source of variations affecting the dynamics of this wave.

We have identified a bone cell adhesion molecule, osteopontin, in the rat testis and epididymis by Northern analysis, RT-PCR, Western immunoblot analysis, and immunocytochernistry. A polyclonal antibody raised against rat epididymal fluid proteins was used to detect fusion proteins produced by a testis Xgtll cDNA library. Sequence analysis of one of four positive cDNA clones, designated as pREP5, revealed identity with the rat osteopontin (OPN) cDNA. The partial cDNA clone pREP5 encompasses 64% of the 1,457 residues reported by Oldberg et al. (1986; Proc Natl Acad Sci USA 83:8819-8823). lmrnunoblot analysis with a monoclonal antibody against OPN detects the presence of imrnunoreactive polypeptides in rat testis homogenates as well as in epididymal fluid and sperm extracts. lrnmunocytochemical localization to the basal and adluminal region of the seminiferous tubule suggests that OPN could be a Sertoli cell product. Indeed, Northern blot analysis of testicular cell preparations demonstated positive hybridization to Sertoli cellenriched RNA, but not to RNA isolated from interstitial cell preparations or to isolated germ cell RNA preparations. OPN is also detected in the rat epididymis and on epididy-ma1 spermatozoa. This is the first report on the presence of OPN mRNA and protein in rat testis and epididymis and on the presence of OPN on the surface of epididymal spermatozoa. The characterization of this protein in other tissue suggests that OPN could play a role in testicular cell adhesion during spermatogenesis and/or epididymal maturation, although other potential functions in the male reproductive tract are discussed. o 1995 Wiley-Liss, tnc.

We studied apoptotic fragmentation in ovulated mouse oocytes. Some cells were fixed immediately after isolation, while others were cultured for 3 hours with or without prostaglandin F2 alpha. Membrane organelles, fibrillar actin and DNA were stained with DiOC6, TRITC-phalloidin and Hoechst 33342, respectively. While fragmented cells were generally rare, most of them were in samples treated with prostaglandin F2α, revealing it as a potential inducer of apoptosis. The chromatin had interphase appearance, indicating exit from meiosis or arrest at germinal vesicle stage. Fragmentation tended to be more pronounced in the vicinity of chromatin, which could be explained with the concentration of actin in the cap region. These preliminary data confirm the active participation of cytoplasm in oocyte apoptosis, and suggest that this process could be induced by mediators of inflammation.

Arginine methylation is a post-translational modification that regulates protein function. RNAbinding proteins are an important class of cell function mediators, some of which are methylated on arginine. Early studies of RNA-binding proteins and arginine methylation are briefly introduced, and the enzymes that mediate this post-translational modification are described. We review the most common RNA-binding domains and briefly discuss how they associate with RNAs. We address the following groups of RNA-binding proteins: hnRNP, Sm, Piwi, Vasa, FMRP, and HuD. hnRNPs were the first RNA-binding proteins found to be methylated on arginine. The Sm proteins function in RNA processing and germ cell specification. The Piwi proteins are largely germ cell specific that are also required for germ cell production, as is Vasa. FMRP participates in germ cell formation in Drosophila but is more widely known for its neuronal function. Similarly, HuD plays a role in nervous system development and function. We review the effects of arginine methylation on the function of each protein, then conclude by addressing remaining questions and future directions of arginine methylation as an important and emerging area of regulation.

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface.