Beneficial influence of Vero cells on in vitro maturation and fertilization of bovine oocytes

The aim of this study was to examine the effects of Vero cells and other somatic cells on in vitro maturation of bovine oocytes. Both denuded oocytes and oocytes with intact cumuli (COCs) were cultured on monolayer of Vero cells, cumulus cells and granulosa cells. The effect of gonadotropins was investigated after the addition of gonadotropins to the culture medium. The evaluation using analysis of variance revealed that removal of cumulus cells generally reduced the percentage of oocytes completing their maturation in vitro and that this effect could not be overcome by the addition of gonadotropins to the culture medium. However, in individual experiments, when oocytes were co-cultured with different monolayers of somatic cells, Vero cells were able significantly support the maturation of denuded oocytes, and their beneficial effect was further enhanced by the addition of gonadotropins (76 vs 80.9%). We did not observe a similar effect after the co-culture of oocytes with a monolayer of cumulus cells (65.3 and 53%, respectively). Granulosa cell monolayer delayed maturation in the both COCs and denuded oocytes (10.5 and 16.5%, respectively). In vitro fertilization was successful in most of the experimental groups. However, when denuded oocytes were cultured without any somatic cell support, they did not decondense the penetrated sperm head after in vitro fertilization. This study demonstrates that 1) Vero cells beneficially affect the in vitro maturation of bovine oocytes; 2) cumulus cells in the form of monolayer lose their beneficial influence on in vitro maturation of bovine oocytes; and 3) granulosa cells and FSH and LH alone (without somatic cells) do not show positive effects on in vitro maturation of bovine oocytes.     

PTGS2-related PGE2 affects oocyte MAPK phosphorylation and meiosis progression in cattle: late effects on early embryonic development

During the periovulatory period, the induction of prostaglandin G/H synthase-2 (PTGS2) expression in cumulus cells and associated prostaglandin E2 (PGE2) production are implicated in the terminal differentiation of the cumulus-oocyte complex. During the present study, the effects of the PTGS2/PGE2 pathway on the developmental competence of bovine oocytes were investigated using an in vitro model of maturation, fertilization, and early embryonic development. The specific inhibition of PTGS2 activity with NS-398 during in vitro maturation (IVM) significantly restricted mitogen-activated protein kinase (MAPK) activation in oocytes at the germinal vesicle breakdown stage and reduced both cumulus expansion and the maturation rate after 22 h of culture. In addition, significantly higher rates of abnormal meiotic spindle organization were observed after 26 h of culture. Periconceptional PTGS2 inhibition did not affect fertilization but significantly reduced the speed of embryo development. Embryo output rates were significantly decreased on Day 6 postfertilization but not on Day 7. However, total blastomere number was significantly lower in embryos obtained after PTGS2 inhibition. The addition of PGE2 to IVM and in vitro fertilization cultures containing NS-398 overrode oocyte maturation and early embryonic developmental defects. Protein and mRNA expression for the prostaglandin E receptor PTGER2 were found in oocytes, whereas the PTGER2, PTGER3, and PTGER4 subtypes were expressed in cumulus cells. This study is the first to report the involvement of PGE2 in oocyte MAPK activation during the maturation process. Taken together, these results indicate that PGE2-mediated interactions between somatic and germ cells during the periconceptional period promote both in vitro oocyte maturation and preimplantation embryonic development in cattle.     

Positive feedback loop between prostaglandin E2 and EGF-like factors is essential for sustainable activation of MAPK3/1 in cumulus cells during in vitro maturation of porcine cumulus oocyte complexes

During in vitro maturation of porcine cumulus-oocyte complexes (COCs), follicle-stimulating hormone (FSH) increases both prostaglandin E2 (PGE2) production and the expression levels of EGF-like factors. The ligands act on cumulus cells by the autocrine system due to their specific receptors, EP2, EP4, or EGF receptor. When each pathway is suppressed by inhibitors, complete cumulus expansion and oocyte maturation do not occur. In this study, we examined the relationship between both of these pathways in cumulus cells of porcine COCs. When COCs were cultured with FSH, Fshr mRNA expression was immediately decreased within 5 h, whereas Ptger2, Ptger4, and Ptgs2 expression levels were significantly increased in cumulus cells in the culture containing FSH for 5 or 10 h. The PTGS2 inhibitor NS398 significantly suppressed not only PGE2 secretion at any culture time point but also Areg, Ereg, and Tace/Adam17 expression in cumulus cells at 10 and 20 h but not at 1 or 5 h. During the early culture period, phosphorylation of MAPK3 and MAPK1 (MAPK3/1) was not affected by NS398; however, at 10 and 20 h, phosphorylation was suppressed by the drug. Furthermore, down-regulations of MAPK3/1 phosphorylation and expression of the target genes by NS398 was overcome by the addition of either PGE2 or EGF. FSH-induced cumulus expansion and meiotic progression to the MII stage were also suppressed by NS398, whereas these effects were also overcome by addition of either PGE2 or EGF. These results indicated that PGE2 is involved in the sustainable activation of MAPK3/1 in cumulus cells via the induction of EGF-like factor, which is required for cumulus expansion and meiotic maturation of porcine COCs.     

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.     

Effect of gonadotropins and prostaglandin on cumulus mucification in cultures of intact follicles

Follicles of 28 day-old pregnant mare serum gonadotropin (PMSG)-primed rats, were cultured for up to 24 hours in the presence or absence of ovine gonadotropins, highly purified rat gonadotropins, dibutyryl cyclic AMP (dbcAMP), methylisobutylxanthine (MIX), choleratoxin (CT), or prostaglandin E2 (PGE2). The morphology of the cumulus-oocyte complexes isolated from these follicles was subsequently examined with the light microscope. Cumulus mucification was studied under the different culture conditions using scanning electron microscopy (SEM) and the hyaluronidase sensitivity test. The features of the cumulus-oocyte complexes in the control cultures did not change throughout the incubation period, while complexes from follicles incubated with LH, FSH, dbcAMP, MIX, CT, or PGE2 changed their appearance and accumulated extracellular mucoid material. Treatment of these cumuli with hyaluronidase resulted in lysis of the extracellular mucus and dispersal of the cumulus masses. The results of this study agree with our earlier observation that the maturation of the cumulus-oophorus, which occurs in vivo following the LH surge, can be induced in vitro by either gonadotropins or cAMP. Prostaglandin E2 did not affect cumulus cells, unless incubated enclosed by their follicles. This suggests that this hormone may influence the cumulus cells indirectly, probably via other components.     

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/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 μg/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 stages 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. Mol. Reprod. Dev. 49:141–149, 1998. © 1998 Wiley-Liss, Inc.     

Metabolic coupling, cumulus expansion and meiotic resumption in mouse cumuli oophori cultured in vitro in the presence of FSH or dcAMP, or stimulated in vivo by hCG

The temporal changes of metabolic coupling between the mouse oocyte and the cumulus cells which follow hCG injection in vivo and FSH treatment in vitro were studied by measuring what fraction of [3H]uridine taken up by cumulus cells was transferred to the oocyte. Meiotic resumption and a partial coupling loss (to 35% of the initial value) spontaneously occurred in cumuli cultured in control medium. The addition of 1 microgram FSH/ml in vitro, or the injection of hCG in vivo caused a delay of about 3 h in both phenomena and a near total uncoupling, together with cumulus expansion. FSH caused uncoupling even if cumulus expansion was prevented by the addition of heparin. The presence of 2 mM-dcAMP prevented meiotic resumption in cumulus-enclosed oocytes and maintained a high level of co-operation for at least 6 h. The slow uncoupling observed at later times was due to cumulus expansion, because it was totally prevented by heparin. We suggest that metabolic co-operation with the cumulus oophorus and meiotic resumption are both regulated by FSH through variations of intracellular levels of cAMP.     

Cumulus cell dispersion induced by estradiol in mouse oviduct in vitro

Dispersion of cumulus cells in nonmated mice is completed in the oviduct 15–20 h after ovulation. Oviducts, isolated 1 h after ovulation (13 h post-human chorionic gonaditropin), were cultured in vitro for 40 h. In these oviducts, denuded oocytes were first seen at 30 h of culture, indicating that cumulus dispersion proceeded at a slower rate in vitro. Oocyte denudation was accelerated in a dose-dependent manner by the addition of estiadiol to the culture medium in which oviducts were incubated. The addition of progesterone or cycloheximide to the culture medium strongly inhibited oocyte denudation even in the presence of estradiol. When isolated cumuli were incubated in the absence of oviductal tissue, the rate of cell dispersion was slower than that of cumuli incubated inside the oviduct and the addition of estradiol to the culture failed to accelerate this process. On the basis of these data, we propose that cumulus cell dispersion is accelerated by an estrogen-dependent protein produced by the oviduct and that this effect of estrogen is antagonized by progesterone.     

Evidence of new antigens in the mouse cumulus oophorus during preovulatory cumulus expansion

The effects of an antiserum (anti-COC) against ovulated mouse cumulus-oocyte complexes (COC) on in vitro fertilization of mouse oocytes were studied. Preincubation of ovulated COC with various concentrations of anti-COC led to dose-dependent impairment of fertilization rates as well as to a decrease in the number of spermatozoa attached to the zona pellucida. Anti-COC was used to probe Western blots of cumulus proteins. These cumuli were obtained from 2 experimental groups of mice corresponding to 2 different maturational stages (preovulatory immature COC or preovulatory mature COC). Two antigens (70 and 80 kDa) present in cumulus intercellular matrix from mature COC were only found as traces in matrix from immature COC. In addition, the protein pattern of the cumulus intercellular matrix was different from that of cumulus cells, whatever the COC maturational stage. These results indicate the appearance of new proteins in the cumulus oophorus during preovulatory expansion and are consistent with the contraceptive action of anti-COC. © 1993 Wiley-Liss, Inc.     

Sulfated glycosaminoglycans inhibit hyaluronic acid synthesizing activity in mouse cumuli oophori

Prior to ovulation, the cumulus cells that surround the oocyte become embedded in a matrix containing hyaluronic acid (HA). Sulfated glycosaminoglycans (GAGs) prevent the hormonally stimulated deposition of this matrix in vitro. The goal of this project was to determine the effect of sulfated GAGs on the HA-synthesizing activity of the cumuli oophori. This activity was measured in lysates of mouse cumuli oophori after stimulation of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) in the presence or absence of sulfated GAGs. FSH treatment resulted in a 5-fold stimulation of HA-synthesizing activity by 3 h in vitro. This induction was inhibited in a dose-dependent manner by heparin and chondroitin sulfate B. However, addition of heparin or chondroitin sulfate B to the assay mixtures containing lysates of FSH-stimulated cumuli oophori had no effect on the HA-synthesizing activity. Heparin also suppressed HA-synthesizing activity stimulated by dibutyryl cyclic adenosine monophosphate. Heparin inhibited the continued increase in hyaluronic acid synthesizing activity when added to cultures after 3 h of FSH stimulation. Also, addition of heparin to cultures of cumuli oophori after 3 or 6 h of incubation in medium containing FSH resulted in only partial cumulus expansion. These results indicate that sulfated GAGs, which are found in ovarian follicular fluid and are a component of extracellular matrix, inhibit some cellular process(es) that results in increased HA-synthesizing activity. The sulfated GAGs also have the ability to suppress HA-synthesizing activity after it has been induced to levels that result in partial cumulus expansion. However, the sulfated GAGs are not direct enzyme inhibitors.     

Hyaluronic acid synthesis by mural granulosa cells and cumulus cells in vitro is selectively stimulated by a factor produced by oocytes and by transforming growth factor-beta

In ovarian antral follicles cumulus cells (approximately 1,000/follicle) closely surround the oocyte, and mural granulosa cells (approximately 50,000/follicle) are distributed at the periphery. Previous work (Salustri, A., Yanagishita, M., and Hascall, V. C. (1990) Dev. Biol. 138, 26-32) showed that oocytes produce a factor(s) which stimulates hyaluronic acid (HA) synthesis by cumulus cells during expansion of the cumulus cell-oocyte complex. We now show that mural granulosa cells also respond in vitro to the oocyte factor(s) with greatly increased HA synthesis. As with cumulus cells, a factor(s) present in fetal calf serum is required to retain newly synthesized HA in the extracellular matrix. Unlike cumulus cells, follicle-stimulating hormone (FSH) is not required for maximal stimulation, in part because mural granulosa cells synthesize prostaglandin E2 which can substitute for FSH in promoting cumulus cell-oocyte complex expansion. Of several growth factors studied, only transforming growth factor-beta 1 (TGF-beta 1) stimulated HA synthesis in both cell types. However, the stimulation of HA synthesis by TGF-beta 1 was additive with that for the oocyte factor(s), and neutralizing antibodies to TGF-beta did not inhibit the response to the oocyte factor(s). The results indicate that the oocyte factor(s) and TGF-beta 1 are not the same and that they operate through different receptors in stimulating HA synthesis. Epidermal growth factor was able to replace FSH in amplifying the response of cumulus cells to the oocyte factor(s) and in stimulating synthesis of dermatan sulfate proteoglycans.     

PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/micrograms cell protein) in response to an acute FSH stimulus (5 micrograms/ml NIH-FSH-S11, 2 H). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/micrograms cell protein). Cells cultured with prostaglandin E2 (PGE2, 1 microgram/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/microgram cell protein, respectively) than that of control cultures. Therefore the presence of PGE2 or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH. Paradoxically, granulosa cells cultured with PGE2 produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 vs. 250.3 fm/micrograms cell protein). This may be due to a suppressive effect of prior exposure to PGE2 on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.     

Prostaglandin production by phagocytic cells of the mouse thymic reticulum in culture and its modulation by indomethacin and corticosteroids

The production of prostaglandins by phagocytic cells of the thymic reticulum in culture (P-TR) was studied by using high pressure liquid chromatography and radioimmunoassay. Radioimmunologic determinations showed that thromboxane B2 (TXB2), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1 alpha (6 keto-PGF1 alpha) were the major compounds released into the culture medium, whereas prostaglandin F2 alpha (PGF2 alpha) was only a minor component. Indomethacin and dexamethasone exerted a similar pattern of differential inhibition of the secretion of prostanoids. PGE2 and 6-keto PGF1 alpha productions were markedly decreased by these anti-inflammatory drugs, whereas those of TXB2 and PGF2 alpha were not or were only slightly affected. Experiments performed with an antiglucocorticoid compound (RU 38486) showed that the steroid-induced inhibition of prostanoid secretion is a classical receptor-mediated action. These results demonstrated that phagocytic cells of the thymic reticulum, which resemble the thymic interdigitating cells, produce several types of prostaglandins. Because it has been described that P-TR regulate thymocyte proliferation in vitro via the secretion of both interleukin 1 and PGE2, these results suggest that anti-inflammatory agents may be able to modulate the thymic microenvironment and, consequently, thymocyte proliferation.     

FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte

Although it has been shown that granulosa cells regulate the growth and meiotic maturation of mammalian oocytes, there is little evidence of a role for the oocyte in the differentiation or function of granulosa cells. To test the hypothesis that the oocyte participates in the regulation of granulosa cell function, oocytes were removed from isolated oocyte-cumulus cell complexes by a microsurgical procedure and oocytectomized complexes were tested for their ability to undergo expansion in response to follicle-stimulating hormone (FSH). FSH increased the levels of intracellular cAMP, the activity of the hyaluronic acid-synthesizing enzyme system, and induced cumulus expansion in intact complexes. In contrast, FSH did not induce increased hyaluronic acid-synthesizing enzyme activity or cumulus expansion in oocytectomized complexes. Therefore, the participation of the oocyte is necessary for the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in vitro in response to stimulation with FSH. FSH induced the elevation of intracellular cAMP to the same extent in both intact and oocytectomized complexes and the cAMP analog 8-bromo cyclic adenosine monophosphate (8Br-cAMP) did not stimulate expansion in oocytectomized complexes. Therefore, the influence of the oocyte on cumulus expansion occurs downstream from the elevation of cAMP levels in the cumulus cells. Epidermal growth factor (EGF), a potent stimulator of cumulus expansion in intact complexes, which probably acts by a mechanism at least initially different from FSH, failed to stimulate cumulus expansion after oocytectomy. Next, oocytectomized complexes were either cocultured with germinal vesicle stage denuded oocytes or cultured in medium conditioned by denuded oocytes. In both cases, FSH or EGF stimulated expansion by oocytectomized complexes. The degree of expansion was directly correlated to the number of oocytes used to condition the medium. Contact between the oocyte and the cumulus cells is not necessary for cumulus expansion. Rather, a factor(s) secreted by the oocyte is necessary for the cumulus cells to undergo expansion in response to either FSH or EGF. FSH did not induce expansion of oocytectomized complexes in media conditioned by various somatic cells such as granulosa cells, fibroblasts, and Sertoli cells; by a mixed population of male germ cells; or by spermatozoa. This suggests that the expansion enabling activity is specific to the oocyte. These results demonstrate that the oocyte participates in the regulation of cumulus cell function.     

The effect of sera, bovine serum albumin and follicular cells on in vitro maturation and fertilization of porcine oocytes

The effects of fetal calf serum (FCS), estrus gilt serum (EGS) BSA, dispersed granulosa cells, hemi-sections of follicular wall, and replacement of medium after 24 hours on in vitro maturation and fertilization of porcine oocytes were studied. The results indicate that the use of BSA for 24 or 48 hours inhibited the expansion of cumulus cells and the maturation of oocytes. An incubation of 24 hours culture in FCS followed by a second 24 hours in BSA containing medium did not decrease the rate of maturation but significantly decreased the polyspermy and mean number of spermatozoa penetrated/oocyte. Renewing the medium with or without removal of cumulus cells during the second incubation increased the maturation rate. Removal of cumulus cells decreased the penetrability, the polyspermy rates of the oocyte and the mean number of spermatozoa/oocyte penetrated. The EGS-supplemented medium, dispersed granulosa cells or hemi-sections of follicular wall did not affect the maturation or fertilization rates. In conclusion, BSA, a protein supplement in maturation medium, inhibited cumulus cell expansion and maturation of porcine oocytes. After resumption of meiosis triggered by FCS, BSA did not influence maturation. The FCS-BSA treatment reduced the incidence of polyspermy and the mean number of spermatozoa penetrated/oocyte without decreasing the rate of maturation and fertilization.     

Maintenance of meiotic arrest and the induction of oocyte maturation in mouse oocyte-granulosa cell complexes developed in vitro from preantral follicles.

During the development of oocyte-granulosa cell complexes from preantral follicles in vitro, oocytes grow and acquire competence to undergo germinal vesicle breakdown (GVB). In the culture system used here, GVB-competent oocytes were maintained in meiotic arrest solely by endogenous physiological mechanisms of the granulosa cells without supplementation with meiosis-arresting substances. Addition of mycophenolic acid, an inhibitor of inosine monophosphate (IMP) dehydrogenase, induced GVB in about 70% of the GVB-competent oocytes grown in vitro. The mechanism for meiotic arrest in this system is, therefore, similar to that for arrest in vivo insofar as it requires the participation of the IMP dehydrogenase pathway. Rp-cyclic adenosine monophosphothioate, a membrane-permeable antagonist to cAMP, induced GVB by about 30% of the competent oocytes. Cyclic AMP-dependent pathways, therefore, participate in the physiological mechanism by which mouse granulosa cells maintain meiotic arrest. Complexes were grown for 10 days in medium containing 0, 1, 5, or 10 ng/ml FSH, were stimulated with either 1 microgram/ml FSH or LH, and were assessed for GVB and cumulus expansion. GVB was stimulated by FSH whether or not the complexes were grown in medium containing FSH, but LH or hCG induced GVB only when the complexes were grown in medium containing FSH. Cumulus expansion occurred in response to either FSH or LH only when complexes were grown in medium containing FSH. FSH, therefore, promotes the differentiation of granulosa cells from preantral follicles in vitro so that LH can stimulate GVB and cumulus expansion.     

Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa

Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 μm vs. 240 μm in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group nor on cumulus expansion (246 μm vs. 240 μm in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles. © 1996 Wiley-Liss, Inc.