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PubMed1997

Glutathione S-transferases in human ovarian follicular fluid.

OBJECTIVE: To study the levels of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 in human preovulatory ovarian follicular fluid (FF) and pooled granulosa and cumulus cells. DESIGN: The relation of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 with P and 17 beta-E2 concentrations were studied. SETTING: The Department of Obstetrics and Gynecology, the Department of Gastroenterology, and the Laboratory of Endocrinology and Reproduction of the University Hospital Nijmegen in Nijmegen, the Netherlands. PATIENT(S): Infertile women participating in an IVF program. RESULT(S): Detectable amounts of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 were found in ovarian FF and pooled cumulus and granulosa cells. Concentrations of glutathione S-transferase Alpha 1-1 were always much higher than those of glutathione S-transferase Pi 1-1. Both ovarian FF concentrations of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 did not correlate with ovarian FF concentrations of 17 beta-E2 and P. CONCLUSION(S): The high FF concentrations of glutathione S-transferase Pi 1-1 and especially of glutathione S-transferase Alpha 1-1 suggest that these enzymes may play an important role in the detoxification processes in the follicles. The lack of correlation between follicular P and 17 beta-E2 and glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 indicates that both enzymes presumably are not present as a result of the high steroid levels.

Estradiol↗
PubMed2001

TSG-6 is concentrated in the extracellular matrix of mouse cumulus oocyte complexes through hyaluronan and inter-alpha-inhibitor binding.

During development of ovarian follicles in mammals, cumulus cells and the oocyte form a mucoelastic mass that detaches itself from peripheral granulosa cell layers upon an ovulatory surge. The integrity of this cumulus-oocyte complex (COC) relies on the cohesiveness of a hyaluronan (HA)-enriched extracellular matrix (ECM). We previously identified a serum glycoprotein, inter-alpha-inhibitor (IalphaI), that is critical in organizing and stabilizing this matrix. Following an ovulatory stimulus, IalphaI diffuses into the follicular fluid and becomes integrated in the ECM through its association with HA. TSG-6 (the secreted product of the tumor necrosis factor-stimulated gene 6), another HA binding protein, forms a complex with IalphaI in synovial fluid. The purpose of this study was to investigate whether TSG-6 is involved in the ECM organization of COCs. Immunolocalization of TSG-6 and IalphaI in mouse COCs at different ovulatory stages was analyzed by immunofluorescence and laser confocal microscopy. IalphaI, TSG-6, and HA colocolized in the cumulus ECM. Western blot analyses were consistent with the presence of both TSG-6 and TSG-6/IalphaI complexes in ovulated COCs. These results suggest that TSG-6 has a structural role in COC matrix formation possibly mediating cross-linking of separate HA molecules through its binding to IalphaI.

Alpha-Globulins↗
PubMed1998

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles.

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/microliter), cumulus cells from oocytectomized complexes (1 OOX/microliter), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/microliter), or oviductal cells (1000 cells/microliter) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 microgram/ml) to microdrops of the conditioned medium. After 16-18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all states of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo.

Animals↗
PubMed1984

Chemical signals that regulate mammalian oocyte maturation.

The nature and functions of the chemical signals involved in the acquisition of competence to resume meiosis, and the maintenance of meiotic arrest in antral follicles are the subjects of this paper. Evidence indicating that gonadotropins are not required for the development of competence to undergo spontaneous maturation is discussed. However, gonadotropins may promote optimal conditions for oocyte development via an estrogen-dependent action on follicular cells. Evidence for the participation of cyclic AMP (cAMP), steroids and a putative maturation-inhibiting factor in the maintenance of meiotic arrest in mammalian oocytes is discussed. Cyclic AMP seems to play a critical role in the maintenance of meiotic arrest by actions in both granulosa/cumulus cells and oocytes. However, cAMP does not appear to equilibrate between cumulus cells and oocytes. In the granulosa/cumulus cells, cAMP may promote the generation/activation of a maturation-inhibiting factor which is transferred to the oocyte. Oocyte cAMP appears to be produced in the oocyte itself. The putative maturation-inhibiting factor may be maintained in an active form by a cAMP-dependent process in the oocyte. Alternatively, the putative maturation-inhibiting factor may play a role in maintaining oocyte cAMP levels. Some steroid hormones act synergistically with a cAMP-dependent process in the oocyte to maintain meiotic arrest. However, the physiological significance of this observation remains in question.

Animals↗
PubMed1994

Viability of one-cell bovine embryos cultured in vitro: comparison of cell-free culture with co-culture.

In this study, the quality (number of cells) and pregnancy rates of bovine blastocysts produced by in vitro maturation/in vitro fertilization (IVM/IVF) following cultivation in either cell-free culture or co-culture were compared. Bovine one-cell IVM/IVF embryos obtained 6 h after insemination were stripped of cumulus cells and assigned to either cell-free culture or co-culture with granulosa cell monolayers for 9 days (Expt 1) or 10 days (Expts 2 and 3). In Expt 3, day-7 (day 0 = day of insemination) blastocysts, day-8 expanded blastocysts and day-9 hatched blastocysts were air-dried, fixed and stained to determine the number of cells. Expanded blastocysts obtained in Expt 1 were cryopreserved using propylene glycol as a cryoprotectant and were used later for embryo transfer. There were no significant differences between cell-free culture and co-culture in the percentage of one-cell embryos that developed to 2- to 16-cells (66.7% versus 72.4% for Expt 1, 71.0% versus 78.2% for Expt 2). However, significantly more (P < 0.05) of the one-cell embryos co-cultured with granulosa cell monolayers developed to morula, blastocyst and expanded blastocyst stages compared with those in cell-free culture (35.0 versus 27.1%, 25.1 versus 14.2%, 15.6 versus 5.4% for Expt 1; 37.6 versus 24.0%, 25.7 versus 11.0%, 16.8 versus 3.0% for Expt 2). Only embryos co-cultured with granulosa cell monolayers hatched (Expt 2). Embryos co-cultured with granulosa cell monolayers also had higher (P < 0.01) numbers of cells (92 +/- 42 versus 56 +/- 21 for blastocysts, 149 +/- 53 versus 81 +/- 29 for expanded blastocysts).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

In vitro growth and maturation of pig oocytes.

Preantral follicles containing oocytes of 70-89.5 microns in diameter were isolated from pig ovaries and cultured in collagen gel for up to 16 days, in the presence of serum, FSH and oestradiol. Formation of follicular antra occurred as the culture proceeded. The oocytes had been enclosed by granulosa cells and contacts between the oocytes and processes of the enclosing cumulus cells were maintained over the culture period. After 16 days of culture, 30-40% of the oocytes were of normal appearance, and the diameters of about half of these oocytes were larger than 100 microns. When the oocytes grown in vitro were liberated from the follicles and cultured for a further 48 h in modified Krebs-Ringer bicarbonate solution, 6, 30 and 60% of the oocytes larger than 90, 100 and 110 microns underwent germinal vesicle breakdown, respectively. Progression to metaphase II was observed in 40% of oocytes that were over 110 microns in diameter, whereas no oocyte less than 90 microns in diameter resumed meiosis. The relationship between the size and meiotic competence of oocytes was similar for oocytes grown in vitro or in vivo. Oocytes grown and matured in vitro were penetrated by spermatozoa and formed a female pronucleus, but decondensation of the sperm head was incomplete. The results demonstrate for the first time that pig oocytes from preantral follicles can grow up to their final size, acquire meiotic competence, and be penetrated by spermatozoa in vitro.

Animals↗
PubMed2001

Direct exposure of chromosomes to nonactivated ovum cytoplasm is effective for bovine somatic cell nucleus reprogramming.

We examined the in vitro developmental potential of nonactivated and activated enucleated ova receiving cumulus cells at various stages of the cell cycle. Eleven to 29% of activated ova receiving donor cells stopped developing at the 8-cell stage but 21% to 50% of nonactivated ova receiving donor cells at either the G(0), G(1), G(2), or M phase, or cycling cells developed into blastocysts. One normal calf was born after transferring five blastocysts that had developed from ova receiving donor cells at the M phase. The present study demonstrated that direct exposure of donor chromosomes to nonactivated ovum cytoplasm is effective for somatic cell nucleus reprogramming, and activated ovum cytoplasm does not reprogram the nucleus.

Animals↗
PubMed1982

Preovulatory changes in cumulus-oocyte complex of the hamster.

An orderly series of associations was found between the cumulus and the stages of nuclear development in the hamster oocyte as the oocyte-cumulus complex underwent preovulatory maturation. Ten hours prior to ovulation meiotic resumption was first seen in the oocyte. The onset of cumulus dissociation with corresponding morphological changes in cumulus cells was observed eight hours prior to ovulation. Completion of the first meiotic division in the oocyte and full cumulus dissociation occurred 2 hours prior to ovulation. Cumulus dissociation was found to occur only in vesicular follicles destined to ovulate and not in follicles undergoing atresia. Cells with large vacuolar inclusions with distorted, hyperchromatic nuclei were found in the maturing cumulus.

Animals↗
PubMed2001

Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse.

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.

Animals↗
PubMed2002

Mitochondrial activity in response to serum starvation in bovine (Bos taurus) cell culture.

In nuclear transfer procedures, in addition to nuclei, donor cell mitochondria are routinely transferred into recipient oocytes, and mitochondrial heteroplasmy has been reported. However, various protocols have resulted in either homoplasmy for recipient oocyte mitochondria or varying heteroplasmic levels in cloned animals. In nuclear transfer protocols, donor cells are subjected to serum-starvation prior to electroporation. Therefore, the relationship between culture conditions and mitochondrial activity was explored. Fibroblast cell lines were propagated from bovine ear epithelium, skin, skeletal muscle, or cumulus cells. In vitro mitochondrial viability was assessed in proliferative and confluent cells, cultured under serum-starvation or supplemented conditions. Cells were stained with MitoTracker Red CMXRos and comparative fluorescence intensities were assessed. The mitochondrial activity per cell was highest under proliferation, significantly lower at confluency (p < 0.001), and remained depressed after serum starvation for within a week (p < 0.001). Serum starvation induced an increase in mitochondrial viability in confluent cells. These results demonstrate that mitochondrial viability is dramatically affected by cell culture conditions. Consequently, specific cell culture parameters provide one explanation for the varying incidence of heteroplasmy identified in cloned animals. Future research should reveal whether specific cell culture parameters represent one of the factors for the varying incidence of heteroplasmy identified in cloned animals.

Animals↗
PubMed2003

Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals.

Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases that are widely distributed in eukaryotic cells. Studies in the last decade revealed that MAPK cascade plays pivotal roles in regulating the meiotic cell cycle progression of oocytes. In mammalian species, activation of MAPK in cumulus cells is necessary for gonadotropin-induced meiotic resumption of oocytes, while MAPK activation is not required for spontaneous meiotic resumption. After germinal vesicle breakdown (GVBD), MAPK is involved in the regulation of microtubule organization and meiotic spindle assembly. The activation of this kinase is essential for the maintenance of metaphase II arrest, while its inactivation is a prerequisite for pronuclear formation after fertilization or parthenogenetic activation. MAPK cascade interacts extensively with other protein kinases such as maturation-promoting factor, protein kinase A, protein kinase C, and calmodulin-dependent protein kinase II, as well as with protein phosphatases in oocyte meiotic cell cycle regulation. The cross talk between MAPK cascade and other protein kinases is discussed. The review also addresses unsolved problems and discusses future directions.

Animals↗
PubMed2004

Effects of retinol on the in vitro development of Bos indicus embryos to blastocysts in two different culture systems.

The objective of this study was to evaluate the effect of retinol on the in vitro development of early embryos of cultured Bos indicus (Expt 1) to the blastocyst stage in medium simplex of optimization (KSOM) or sintetic fluid of oviduct (SOF) or co-cultured (Expt 2) with an oviduct cell monolayer (OCM) in KSOM or SOF. A total of 3149 cumulus-oocyte complexes obtained by aspirating follicles (2-5 mm diameter) from ovaries of slaughtered animals were selected for IVM and incubated in TCM 199 supplemented with 25 mM HEPES at 39 degrees C in air with 5% CO(2) and maximum humidity for 24 h. In vitro fertilization (IVF) was performed in modified defined medium (mDM) medium. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes were randomly allocated to the experimental groups. Zygotes cultured (Expt 1) in KSOM + retinol, KSOM, SOF + retinol and SOF were incubated in maximum humidity at 39 degrees C, 5% CO(2), 5% O(2) and 90% N(2). Zygotes co-cultured (Expt 2) in KSOM + retinol + OCM, KSOM + OCM, SOF + retinol + OCM and SOF + OCM were incubated at 39 degrees C, 5% CO(2). In both experiments media were partially changed 48 h after IVF and unfertilized ova were removed. Afterwards embryos were kept in culture or co-culture for further 9 days. In Expt 1, blastocyst rates (day 7) were 14.6% (KSOM + retinol), 15.8% (KSOM), 16.4% (SOF + retinol) and 15.9% (SOF). In Expt 2, the blastocyst rates (day 7) were 25.4% (KSOM + retinol + OCM) 14.2% (KSOM + OCM), 24.3% (SOF + retinol + OCM) and 15.9% (SOF + OCM). The same influence profile of retinol was observed in the formation of the expanded (day 9) and hatched (day 11) blastocysts. The results obtained in Expt 2 demonstrated that the addition of 0.28 microg/ml retinol to the embryo culture media used in this study had a significant (p < 0.05) positive effect on bovine early embryonic development, under the conditions tested, and can be used to enhance in vitro embryo production.

Animals↗
PubMed1996

Differential expression of four connexin genes, Cx-26, Cx-30.3, Cx-32, and Cx-43, in the porcine ovarian follicle.

Connexin genes expressed in porcine ovaries were isolated by reverse transcription-PCR, and the expression of four connexin genes (Cx-43, -32, -30.3, and -26) was detected by in situ hybridization in internal and surrounding compartments of large antral follicles. Cx-43 and Cx-30.3 were expressed in the theca interna as well as in the granulosa cell compartment. However, two zones were observed in the theca interna by probing with Cx-43; about a quarter of the region close to the lamina basalis appeared devoid of Cx-43 messenger RNA (mRNA) and protein as well. Cx-30.3, in contrast, seemed to be expressed ubiquitously in these two compartments. Cx-26 and Cx-32 were expressed only in the thecal cells. Both Cx-43 and -30.3 also gave positive signals in the cumulus cells to the same extent as obtained in the granulosa cells with the respective probes, whereas the expression of Cx-32 and -26 was undetectable. Cx-43 mRNA drastically decreased in the functioning stage of the corpus luteum, whereas Cx-30.3 mRNA increased significantly in the early stage of luteinization in the estrus cycles. These results suggest that Cx-43 is not the only gap junction protein to be expressed in the ovarian follicle; other proteins, such as Cx-32, -30.3, and -26 seem to be expressed, and their expressions are regulated differently.

Aging↗
PubMed1988

G-proteins in mammalian gametes: an immunocytochemical study.

The presence of transductory GTP(G)-regulatory proteins in mammalian gametes has been examined by indirect fluorescence immunocytochemistry. Using rabbit antisera to bovine rod beta gamma-transducin (RA beta gamma T), bovine rod holotransducin (AS-1), bovine rod alpha-transducin (RA alpha T), synthetic bovine rod alpha-transducin C-terminal decapeptide (AS-6), bovine brain alpha 39Go (RA alpha 39), and two mouse monoclonal antibodies raised against frog retinal transducin (4A), and rat brain beta-tubulin, we demonstrated the presence of corresponding immunoreactive material in both rat oocytes and bovine ejaculated sperm. Immunostaining in the oocyte was evenly distributed on the oolemma, excluding the cell cytoplasm and zona pellucida. Immunoreactive material was also present in the cumulus cells that encapsulate the oocyte. In contrast, the immunofluorescence corresponding to transductory G-proteins was confined in sperm to functionally defined regions in the head and tail, in a manner specific for each antibody. While RA beta gamma T, AS-1 and RA alpha 39 all stained the entire acrosome, AS-6 and RA alpha T stained only the acrosomal tip. Monoclonal antibody 4A stained the midpiece exclusively and anti-rat betaq-tubulin (a structural G-protein) stained the full length of the sperm tail. The existence of several G-protein types in mammalian gametes suggests their possible involvement in the regulation of various effector systems, in a manner reminiscent of somatic cells. The unique situation in sperm, where different G-proteins show distinct and specific patterns of distribution, further suggests their association with various effector systems in discrete functional domains.

Animals↗
PubMed2003

Effects of cycloheximide treatment on in-vitro development of porcine parthenotes and somatic cell nuclear transfer embryos.

This study aimed to verify the beneficial effect of cycloheximide (CHX) treatment on the development of porcine somatic cell nuclear transfer (NT) embryos, which were constructed with enucleated oocytes and cumulus cells by using a single direct current (DC) pulse. In the first experiment, a single DC pulse applied to the induction of fusion and activation of NT embryos gave a high fusion rate. However, cleavage and subsequent development of fused couplets (NT embryos) to the blastocyst stage were poor. Experiment 2 was conducted to determine whether CHX treatment could enhance metaphase II (M II) oocyte activation and improve the subsequent parthenogenetic development. After giving the DC pulse and incubation with or without CHX, M II oocytes incubated with CHX showed higher cleavage and development to blastocysts compared with those incubated without CHX (P < 0. 05). Experiment 3 was carried out to verify the beneficial effect of CHX on the development of NT embryos. The NT embryos treated with the DC pulse and CHX treatment showed higher cleavage and subsequent development compared with those treated with the DC pulse alone (P < 0.05) . The present study demonstrates that CHX treatment enhances the electrical stimulus-induced activation of oocytes and NT embryos, and improves the subsequent development of parthenotes and NT embryos. The results indicate that protein synthesis inhibition treatment required for the induction of oocyte activation promotes the development of NT embryos.

Animals↗
PubMed1989

Orchestrated expression of steroidogenic side-chain cleavage cytochrome P-450 during follicular development in the rat ovary.

Antibody to the mitochondrial cholesterol side-chain cleavage cytochrome P-450 (P-450scc) was used to study the expression of this key steroidogenic enzyme during follicular development. Immunofluorescence staining of tissue cryosections and isolated cells revealed unique and cell-specific patterns of P-450scc expressed in four cell types of the prepubertal rat ovary. Without hormonal treatment, the interstitial gland expressed ample amounts of P-450scc. Within 24 h after PMSG administration, theca interna acquired the cytochrome, and after an additional 24 h, the enzyme appeared also in granulosa cells. However, expression of P-450scc in granulosa cells was observed only in preovulatory follicles. This was in contrast to the theca interna cells which differentiated in various follicles unrelated to their stages of development. Cumulus cells were the last cell type to express P-450scc, occurring as late as 2-3 h before ovulation. These observations indicate that the concerted expression of P-450scc in the different ovarian cell types is a well-timed phenomenon which is precisely co-ordinated during follicular development.

Animals↗
PubMed1990

Maturation of pig oocytes: observations on membrane potential.

The membrane-potential changes of pig oocytes during maturation are described. Cumulus-enclosed oocytes have a resting potential of -41.81 +/- 0.60 mV; the removal of cumulus cells caused this potential to drop to -30.95 +/- 0.43 mV. Adding LH to the culture medium did not influence the potential of denuded oocytes but depolarized the potential of cumulus-enclosed oocytes to -32.90 +/- 0.43 mV. FSH did not affect the membrane potential of denuded or cumulus-enclosed oocytes, but significantly reduced the amplitude of the depolarization induced by LH. The effect of gonadotropins on cultured granulosa cells was also investigated. Plated granulosa cells have a resting potential of -45.21 +/- 0.72 mV, similar to that of cumulus-enclosed oocytes. As recorded in cumulus-enclosed oocytes, LH depolarized granulosa cell membrane potential (-30.33 +/- 0.69 mV) and FSH reduced this effect. To evaluate if oocyte maturation in vivo is accompanied by membrane-potential depolarization, follicular growth and oocyte maturation were induced in 6 prepubertal gilts by using an eCG-hCG treatment. Twenty hours after the beginning of oocyte maturation in vivo (induced by hCG), the membrane potential of the oocyte was depolarized to -28.84 +/- 1.01 mV, a value similar to that observed in vitro. These data indicate that both LH and FSH can influence the membrane potential of follicular somatic cells and, consequently, that of the oocyte. The electrical coupling between somatic cell and oocyte may represent a means by which the gonadotropin message is passed to the germinal cell by the somatic compartment.

Animals↗
PubMed1991

Lack of effect of oocytectomy on expansion of the porcine cumulus.

Oocyte-cumulus cell complexes (OCC) and complexes with an attached piece of membrana granulosa (C + P), isolated from prepubertal or cyclic gilts stimulated with pregnant mares' serum gonadotrophin, were cultured in media supplemented with follicle-stimulating hormone (FSH; 0.01-1.0 micrograms/ml) or forskolin (50-100 mumol/l) for 24 and 32 h. FSH and forskolin each induced dose-dependent cumulus and membrana granulosa expansion. After 2 h of culture, FSH (0.1 microgram/ml) or forskolin (100 mumol/l) increased the contents of intracellular adenosine 3',5'-phosphate (cAMP) in OCC from prepubertal gilts to almost 10 times that in unstimulated complexes. After 24 h of culture in media supplemented with FSH (0.1 microgram/ml) or forskolin (100 mumol/l), the oocytectomized OCC and C + P showed similar expansion to that of the control groups. The intracellular cAMP contents in intact and oocytectomized OCCs were similar in all groups except those treated with FSH, in which the intact OCCs had significantly higher contents than their oocytectomized counterparts (P less than 0.01). After hyaluronidase treatment, cumulus and membrana granulosa cells of intact and oocytectomized OCC and C + P were suspended, except for those of the innermost layers of the corona radiata. The results suggest that increases in cAMP contents and synthesis of an extracellular, hyaluronidase-sensitive mucus by pig OCC and C + P induced by FSH or forskolin are not dependent on the oocyte.

Animals↗