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PubMed1988

Effects of cyclic AMP on the spontaneous meiotic maturation of cumulus-free bovine oocytes cultured in chemically defined medium.

The rate of spontaneous meiotic maturation and the period of commitment to this process were determined in bovine oocytes devoid of surrounding cumulus cells, cultured in chemically defined medium with bovine serum albumin in the absence of serum. The effects of compounds that are known to elevate levels of intracellular cyclic adenosine monophosphate (cAMP) on the resumption and progression of meiosis were investigated. Bovine oocytes were mass-harvested, denuded of cumulus cells, and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin. Intracellular cAMP levels were indirectly modified using 8-bromo-cAMP, dibutyryl cAMP (dbcAMP), forskolin, or 3-isobutyl-1-methyl xanthine (IBMX). Meiotic maturation was scored cytogenetically. Ninety percent of denuded bovine oocytes mature after 24 h, with 65% progressing beyond anaphase I. These oocytes remain at the germinal vesicle (GV) stage for up to 8 h in culture. GV breakdown (GVBD) occurs in 40.5% of oocytes at 9 h. The peak times for the different meiotic stages were 12 h for diakinesis, 15 h for late diakinesis to metaphase I, 20 h for metaphase I, and 24 h for telophase I. By 48 h, most had reached metaphase II. There is a 2-h lag period between the time at which they become irreversibly committed to mature (at 7 h) and when they demonstrate GVBD (at 9 h). Incubation for 12 h with high concentrations of 8-bromo-cAMP and forskolin significantly inhibited GVBD, while the effect of dbcAMP was similar but less pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Histochemical characterization of anionic constituents in oocyte-cumulus complex of rats.

Fertilization is a process that involves the recognition and adhesion of negatively charged spermatozoa to the oocyte investments. It is not known, however, if charge properties of the interacting gametes play a role in fertilization. The present study evaluates the content and distribution of anionic constituents in the oocyte-cumulus complex of rats. Polycationic colloidal gold (PCG), ruthenium red (RR) and wheat germ agglutinin (WGA) were used as cytochemical markers of anionic sites at the light (LM) and electron microscopical (EM) levels. Isolated oocyte-cumulus complexes were fixed with glutaraldehyde (GA) and OsO4 containing RR, or with GA without RR, and embedded in araldite or LR-gold. For LM, deresined, semi-thin, araldite-embedded sections were labelled with PCG intensified by silver, or with biotinylated lectins visualized by avidin-peroxidase. For EM, thin LR-gold sections were labelled with PCG, whereas RR labelling was examined in araldite sections. The zona pellucida (ZP) failed to bind any of the polycationic markers used, but intensely bound neutralized WGA. In contrast, cumulus cell membranes bound PCG but not RR, whereas the oolemma bound RR but not PCG. The results indicate that the ZP is practically devoid of negatively charged constituents, and tends to repel positively charged ligands possibly due to the presence of cationic determinants. The binding of PCG to cumulus cells probably reflects a high content of membrane-bound heparan sulphate, whereas the binding of RR to the oolema indicates the presence of membrane sialoglycoconjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development.

Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6-12 mm) were retrieved 34-36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

Growth and maturation of follicles and oocytes following xenotransplantation of porcine ovarian tissues and in vitro maturation.

This is the first report to show morphological evidence of in vitro maturation of oocytes recovered from xenotransplanted antral follicles. To develop a suitable tool for studing the growth and maturation of follicles and oocytes, we xenotransplanted small pieces of ovarian cortical tissue from sows, which contained small preantral follicles (primordial, primary, and secondary follicles; less than 0.05, 0.1 and 0.3 mm in diameter, respectively), under the capsules of kidneys of adult female severe combined immunodeficient (SCID) mice for 2 and 8 weeks, and then recovered cumulus-oocyte complexes from the growing tertiary follicles in xenografted tissues. The distribution of processes from cumulus cells to oocytes and the follicular growth, development, and maturation during xenotransplantation were histochemically analyzed. Tertiary follicles, 0.5 to 3.0 mm in diameter, were obtained from grafted tissues 2 (85%: 52 follicles/61 grafted tissues) and 8 (50%: 15/30) weeks after xenotransplantation, and then oocytes, which were tightly attached to cumulus cells, were collected from each tertiary follicle and cultured to assess their quality. At 2 weeks after grafting, 17.6% of the oocytes had matured to the metaphase II stage, but no such maturation was observed 8 weeks after grafting. Thus, in the 2 weeks group, preantral follicles rapidly grew in xenotransplanted porcine ovarian tissues to the tertiary stage, and oocytes could be recovered and matured from them by in vitro culture.

Actin Cytoskeleton↗
PubMed1998

Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation.

The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n = 30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-(35S). Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significant by 20 h, with 89% of cow oocytes (P < 0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation.

Aging↗
PubMed1995

In vitro maturation and fertilization of prepubertal goat oocytes.

The aim of this work was to study the IVM-IVF of prepubertal goat oocytes collected from a slaughterhouse as an alternative source of oocytes to those of FSH-primed adult goats. In Experiment 1, IVM of prepubertal goat oocytes in co-culture with granulosa cells were compared with IVM in 50 microl microdrops of medium. There was no significant difference in the percentage of maturation (72.0 vs 76.9%) between the 2 groups. In Experiment 2, a low percentage of normal fertilization (24.4%) was observed for prepubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro-matured (48.7%) oocytes from adult goats. There were no significant differences with respect to the oocytes from adult goats matured in vitro when prepubertal goat oocytes were cultured with adult goat granulosa cells (33.3%) or in microdrops (29.7%). No differences were observed among the treatments in the percentage of oocytes showing evidence of fertilization (normal fertilization + abnormal fertilization + polyspermy). In Experiment 3, it was shown that there were no differences in the percentage of normally fertilized oocytes after in vitro maturation in microdrops containing oocytes with 1 to 2 and 3 or more complete layers of cumulus cells (32.1 and 33.3% respectively). In conclusion, the ovaries of prepubertal slaughterhouse goats were found to be an economical alternative for an abundant source of oocytes for IVM-IVF research. In vitro maturation of oocytes in microdrops yielded maturation and fertilization rates comparable to those obtained with oocytes from FSH-primed adult goats. Moreover, similar maturation and fertilization rates were obtained using oocytes with 1 to 2 layers or 3 or more layers of cumulus cells.

Journal Article↗
PubMed2005

Nutrient concentrations in murine follicular fluid and the female reproductive tract.

The culture of murine oocytes and preimplantation embryos in vitro has been used successfully for many years. However, this practice can result in cellular stress and reduced viability. Since this phenomenon is partly attributable to differences in nutrient composition between culture media and maternal tract fluids, we determined the concentrations of glucose, pyruvate, lactate and 19 amino acids in murine preovulatory follicles and oestrous oviductal and uterine fluids. Follicular fluids were aspirated from hyperstimulated ovaries, whereas oviductal fluids (with/without oocyte-cumulus complexes) and uterine fluids were collected from naturally cycling animals. Glucose, pyruvate and lactate concentrations were analysed using ultramicrofluorometric methods, whilst amino acid profiles were determined by reverse-phase high performance liquid chromatography. Mean glucose concentrations in follicular, oviduct (with/without cumulus cells) and uterine fluids were 0.46, 1.09/1.65 and 0.61 mmol l(-1), respectively. Pyruvate concentrations were 0.38, 0.37/0.17 and 0.25 mmol l(-1), respectively, and lactate concentrations were 17.34, 10.92/11.68 and 9.41 mmol l(-1), respectively. Oviductal pyruvate concentration was significantly higher, and glucose significantly lower, in the presence of cumulus cells. Taurine, glycine, alanine, glutamine and glutamate were the major amino acids detected. Concentrations of amino acids differed among fluids, with highest levels being found in the oviduct. The follicular fluid and tract nutrient profiles differed from those of murine maturation, fertilisation and embryo culture media. These data extend our understanding of cellular metabolism and of nutritional environments of the oocyte and early embryo as they progress along the reproductive tract in vivo. These results may also contribute to the formulation of nutritionally more physiological media for mouse oocyte maturation and embryo culture.

Amino Acids↗
PubMed2006

Differential effects of culture and nuclear transfer on relative transcript levels of genes with key roles during preimplantation.

It is well known that the preimplantation culture environment to which embryos are exposed influences the expression of developmentally important genes. Recently, it has been reported that MEMalpha, a culture medium commonly used for somatic cells, allows high rates of preimplantation development and development to term of mouse somatic cell nuclear transfer (SCNT) embryos. The objective of this study was to compare the differential effects of this medium and of the nuclear transfer procedure on the relative mRNA abundance of several genes with key roles during preimplantation. The relative mRNA levels of nine genes (Glut 1, Glut 5, G6PDH, Bax, Survivin, Gpx 1, Oct4, mTert and IGF2bp1) were quantified at blastocyst stage on cumulus cell cloned embryos cultured in MEMalpha, as well as on in vivo cultured and MEMalpha cultured controls. Only three of the nine transcripts analysed (Glut 5, Gpx 1 and Igf2bp1) were significantly down-regulated at blastocyst stage in in vitro produced controls. However, most genes analysed in our MEMalpha cultured cloned embryos showed altered transcription levels. Interestingly, between cloned and in vitro produced controls only the transcription levels measured for Glut 1 were significantly different. This result suggests that Glut 1 may be a good marker for embryo quality after cumulus cell nuclear transfer.

Animals↗
PubMed1994

Inhibition of germinal vesicle breakdown in bovine oocytes by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

5,6-Dichloro-1-beta-D-ribofuranosyl-benzimidazole (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 microM) 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 not 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.

Animals↗
PubMed2006

Meiosis activating sterol (MAS) regulate FSH-induced meiotic resumption of cumulus cell-enclosed porcine oocytes via PKC pathway.

Meiosis activating sterol (MAS) have been found to be able to promote oocytes meiotic maturation of small animals in vitro, such as mouse, rat and rabbit. But in large animals, whether MAS play the same function, especially the physiological mechanisms of MAS on oocytes maturation are not clear. To our knowledge, this is the first time to investigate the role and signal pathway of MAS on FSH-induced porcine oocytes meiotic resumption. Porcine cumulus-enclosed oocytes (CEOs) isolated from 3 to 5mm follicles were cultured in the FSH-medium for 24h supplemented with 0-50 microM RS21745 or 0-100 microM RS21607 (two specific inhibitors of lanosterol 14alpha-demethylase that converts lanosterol to FF-MAS), or cultured in FSH-medium with 25 microM RS21745 for 0-24h firstly, then transferred into a new FSH-medium (the total culture time is 24h). The results revealed that RS21745 or RS21607 could inhibit FSH-induced porcine CEOs meiotic resumption in a dose and time-dependent manner. Meanwhile, FSH-induced cumulus expansion could also be inhibited dose-dependently by RS21745 or RS21607. Otherwise, AY9944-A-7, an inhibitor of Delta14-reductase which promotes cholesterol accumulation from FF-MAS, had no effect on both denuded oocytes (DOs) cultured for 24 or 44 h and CEOs cultured for 24h meiotic resumption, but it could promote CEOs meiotic resumption after 44 h culture. In addition, we got that 10(-8) to 10(-6)M PMA, an activator of PKC pathway, could reverse the inhibiting effect of RS21745 on FSH-induced CEOs meiotic resumption and enhance the rate of germinal vesicle breakdown (GVBD) of CEOs cultured in medium with hypoxanthine (HX). Moreover, 5-10 microM chelerythrine chloride, an inhibitor of PKC, could enhance the inhibitory effect of RS21745 on FSH-induced porcine oocytes resumption of meiosis. All the data of this study support that endogenous FF-MAS takes part in the FSH-induced porcine oocytes meiotic resumption and might play an active role via PKC signal pathway.

Aniline Compounds↗
PubMed1995

Effects of supplementation of the maturation media with insulin on in vitro maturation and in vitro fertilization of bovine oocytes.

This study was carried out to determine the effects of supplementation of the maturation media with insulin on in vitro maturation and fertilization of bovine oocytes. In Experiment 1, cumulus-intact bovine oocytes were cultured in a maturation medium (TCM-199 containing 10% fetal calf serum, 0.02 U/ml follicular stimulating hormone and 1 microgram/ml estradiol-17 beta) with or without insulin supplementation (10 micrograms/ml). The maturation and fertilization rates of oocytes and subsequent embryonic development to the blastocyst stage were not affected by the treatment with insulin in the presence of serum and the hormones during the maturation period. In Experiment 2, to avoid the effects of serum and the hormones, a serum- and hormone-free maturation medium (TCM-199 containing 1 mg/ml polyvinyl alcohol) was used. In the absence of serum and hormones during the maturation period, the maturation rate was not affected by treatment with insulin, but the fertilization rate was improved. In Experiment 3, when denuded oocytes were inseminated together with cumulus cells cultured in serum- and hormone-free maturation medium supplemented with insulin, the fertilization rate was increased. These results demonstrate that the addition of insulin to the serum- and hormone-free maturation medium improves the fertilization rate of bovine oocytes in vitro, and suggest that insulin may stimulate the secretion of sperm capacitating agent (s) from cumulus cells.

Animals↗
PubMed2006

Cumulus oophorus-associated glycodelin-C displaces sperm-bound glycodelin-A and -F and stimulates spermatozoa-zona pellucida binding.

Spermatozoa have to swim through the oviduct and the cumulus oophorus before fertilization in vivo. In the oviduct, spermatozoa are exposed to glycodelin-A and -F that inhibit spermatozoa-zona pellucida binding. In this study, we determined whether these glycodelins would inhibit fertilization. The data showed that the spermatozoa without previous exposure to glycodelin-A and -F acquired glycodelin immunoreactivity during their passage through the cumulus oophorus. On the other hand, when glycodelin-A or -F-pretreated spermatozoa were exposed to the cumulus oophorus, the zona pellucida binding inhibitory activity of glycodelin-A and -F was not only removed, but the spermatozoa acquired enhanced zona pellucida binding ability. These actions of the cumulus oophorus were due to the presence of a cumulus isoform of glycodelin, designated as glycodelin-C. The cumulus cells could convert exogenous glycodelin-A and -F to glycodelin-C, which was then released into the surrounding medium. The protein core of glycodelin-C was identical to that in other glycodelin isoforms, as demonstrated by mass spectrum, peptide mapping, and affinity to anti-glycodelin antibody recognizing the protein core of glycodelin. In addition to having a smaller size and a higher isoelectric point, glycodelin-C also had lectin binding properties different from other isoforms. Glycodelin-C stimulated spermatozoazona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound glycodelin-A and -F. In conclusion, the cumulus cells transform glycodelin-A and -F to glycodelin-C, which in turn removes the spermatozoazona binding inhibitory glycodelin isoforms and enhances the zona binding capacity of spermatozoa passing through the cumulus oophorus.

Female↗
PubMed1997

The expression of leptin and its receptors in pre-ovulatory human follicles.

The expression of leptin and its receptors was examined by reverse transcriptase-polymerase chain reaction and immunofluorescence in granulosa and cumulus cells of pre-ovulatory follicles and in meiotically mature oocytes obtained from women undergoing in-vitro fertilization. Leptin concentrations were measured in newly aspirated follicular fluids and in maternal serum before and after the administration of an ovulatory dose of human chorionic gonadotrophin. The findings demonstrate leptin expression at the mRNA and protein levels by granulosa and cumulus cells, and the presence of leptin in mature human oocytes. While an association between follicular leptin concentration and embryo development was not observed, a post-ovulatory increase in serum leptin concentration was associated with implantation potential. The results are discussed with respect to possible roles of leptin in early human development.

Adult↗
PubMed1995

Partial deletion of the Y chromosome removes the effect of paternal genome imprinting on periovum sensitivity to hyaluronidase in mice.

Cumulus-oocyte complexes from the two consomic strains of mice, B10.BR and B10.BR-Ydel (with a partial deletion of the Y chromosome) and the BALB/c strain, as well as reciprocal F1 and F2 hybrids, were tested for their susceptibility to attack by hyaluronidase. The cumulus cell dispersal was significantly more rapid in the eggs of B10.BR females and of F1 and F2 hybrid females sired by the B10.BR males than in those of BALB/c females and hybrids sired by BALB/c males. These results confirm the earlier data of Bander et al. (1989) which were interpreted as evidence of paternal imprinting of the C57BL genome. In contrast, cumulus cell dispersal in the eggs of females sired by B10.BR-Ydel males was significantly slower than in females sired by B10.BR males, and did not differ from that in the BALB/c strain. The results suggest that the partial deletion of the Y chromosome abolished the effect of paternal genome imprinting which is observed in the B10.BR strain.

Animals↗
PubMed1996

In vitro maturation and transmission electron microscopic observation of horse oocytes after vitrification.

The study was designed to examine the suitability of immature horse oocytes for vitrification. Immature oocytes derived from slaughtered horse ovaries were transferred to a vitrification solution (EFS; 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in modified phosphate-buffered saline) directly (Groups 1 and 4) or were first exposed to 20% ethylene glycol solution for 10 min (Groups 2 and 5) or 20 min (Groups 3 and 6). Oocytes were handled at 20 degrees C (Groups 1, 2, and 3) or 30 degrees C (Groups 4, 5, and 6). After vitrification and warming, their viability was assessed by maturation culture for 32 h. The percentages of oocytes reaching the metaphase II stage after the in vitro maturation in Groups 2, 3, 5, and 6 (16.0, 16.7, 10.0. and 8.2%, respectively) were higher than those in Groups 1 and 4 (2.2 and 1.9%, respectively). In untreated control oocytes, 55.6% completed meiosis in vitro. Transmission electron microscopy was used to compare the fine structure of vitrified oocytes (treated as Group 2) with those of untreated control oocytes and EFS-exposed, nonvitrified oocytes (n = 10 each). The viability of EFS-exposed oocytes, assessed by in vitro maturation, was 27.7%. Vitrification induced some ultrastructural changes, such as the swelling of mitochondria together with reduced matrix density, the destruction of communication between oocytes and their surrounding cumulus cells, and the presence of vacuoles located in the periphery of the ooplasm. However, these changes were not always observed. Exposure of the oocytes to EFS solution induced similar ultrastructural changes in mitochondria and cell-cell communication but to a lesser extent. However, the exposure to EFS induced vacuoles in the periphery of the ooplasm to the same extent as did the vitrification. Thus, immature horse oocytes can be cryopreserved by vitrification with EFS solution. Reduced viability of EFS-exposed and/or vitrified horse oocytes may relate to morphological changes such as destruction of the intercellular communications between cumulus cells and oocytes.

Animals↗
PubMed1994

Embryo development capacity of oocytes fertilized by immature sperm and sperm treated with motility stimulants.

The fertilizing ability of mouse spermatozoa develops during maturation and coincides with the acquisition of motility. The lack of progressive motility of spermatozoa from the testis and precaudal segments of the epididymis interferes with their ability to fertilize oocytes after insemination in vitro. The removal of cumulus cells for insemination in vitro and the use of subzonal injection of a single spermatozoon resulted in a higher number of oocytes fertilized by immature caput and corpus spermatozoa. High rates of embryonic arrest and retarded development were observed in oocytes fertilized by caput and corpus spermatozoa when compared with oocytes fertilized by cauda spermatozoa. However, when the oocytes were enclosed in their cumulus cells or microinjected with a single spermatozoon, these effects were reduced. A block in embryonic development was also observed after human and mouse oocytes were exposed to the sperm motility stimulants pentoxifylline (PTF) and 2-deoxyadenosine (DOA). These observations suggest that exposure of oocytes to PTF and DOA should be avoided.

Animals↗
PubMed1987

Cellular origin, hormonal regulation and biochemical characteristics of polypeptides secreted by graafian follicles of sheep.

The pattern, chemical nature and biological role of polypeptides secreted by ovine follicles exposed to gonadotrophins in vivo were studied. Follicles were removed at intervals before ovulation (in-vivo groups), separated into granulosa and cumulus compartments, and incubated for 3 h with radiolabelled amino acids. No differences were detected in the polypeptides (Mr 46,000-60,000) secreted by granulosa and cumulus cells before exposure to the preovulatory LH surge. Each class of polypeptides was characterized by different degrees of phosphorylation, sulphation and glycosylation. Within 15 h of the LH surge the secretion of the Mr 46,000-60,000 polypeptides had ceased and was replaced by a non-sulphated Mr 30,000 secretory product. Significant differences in the secretory pattern of granulosa and cumulus cells were detected after exposure to LH. Intact follicles and granulosa cells were cultured for 24 h (in-vitro groups) and then incubated with radiolabelled amino acids. The profile of polypeptides secreted by intact follicles cultured in the presence or absence of LH corresponded closely with the profile observed in vivo. By contrast, granulosa cells grown as monolayers switched spontaneously to the secretion of Mr 30,000 polypeptides in medium devoid of gonadotrophin. This aberrant secretory switch did not occur in granulosa cells maintained in suspension culture. Inhibition of transcription in follicles exposed to LH prevented both the appearance of the Mr 30,000 polypeptide and the disappearance of the Mr 46,000-60,000 polypeptides. Although the inhibition of steroidogenesis by a variety of steroid enzyme inhibitors was without effect on secretion, evidence was obtained to suggest that one of the secreted polypeptides binds oestradiol-17 beta.

Animals↗
PubMed2005

Plasminogen detection in oocytes and plasminogen activator activities in the porcine oviduct during the estrous cycle.

Plasminogen activators (PAs) are highly specific serine proteases that convert the extracellular zymogen plasminogen into the active proteinase plasmin. Plasminogen-dependent proteolytic activity was detected by zymography both in the tissue membrane fraction of oviducts and in the oviductal flushing obtained at the preovulatory (Pre-Ov), postovulatory (Post-Ov) and mid-luteal (Mid-L) stages of the estrous cycle. A main proteolytic band, with a relative mobility similar to a human melanoma cell tissue-type plasminogen activator (t-PA), was found in all samples. Two additional components were observed in Pre-Ov and Post-Ov oviductal flushing but not in the tissue membrane fraction. In the oviductal flushing the PA activity was significantly higher in the Post-Ov stage than in the Pre-Ov one. Both urokinase-type plasminogen activator (u-PA, 50 kDa) and t-PA (72 kDa) were detected by Western blot; they showed differences in their relative concentration between Post-Ov and Pre-Ov oviductal flushing. The main PA substrate, plasminogen, was detected by indirect immunofluorescence in the cumulus cell extracellular matrix (ECM) and oocyte zona pellucida (ZP). In denuded oocytes, plasminogen was also detected on the surface of the plasma membrane. It is possible that oviductal PAs may act on the plasminogen present in the cumulus cell ECM and ZP; consequently, the generated plasmin could be involved in the rebuilding or degradation of these oocyte structures during fertilization or early development.

Animals↗