Roles of cAMP in regulation of both MAP kinase and p34(cdc2) kinase activity during meiotic progression, especially beyond the MI stage

Time-dependent changes in the level of adenosine cyclic AMP (cAMP) in porcine oocytes during meiotic progression from the germinal vesicle stage (GV stage) to the metaphase II stage (MII stage) were examined using reversed-phase HPLC with UV detection. The concentration of cAMP in oocytes reached a peak at 8 hr of cultivation of cumulus-oocyte complexes (COCs), but it was dramatically decreased after 12-hr cultivation. After a 28-hr cultivation period, the level of cAMP in the oocytes had significantly reduced further, and the basal level of cAMP was observed in oocytes cultured at 32 hr and for up to 48 hr. When phosphatidylinositol 3-kinase (PI 3-kinase) or protein kinase C (PKC) in cumulus cells [which were required for meiotic progression to the MII stage in porcine oocytes (Shimada and Terada, 2001: Biol Reprod 64:1106-1114)] was suppressed by each specific inhibitor following initial 24-hr cultivation of COCs, cAMP level in the oocytes was significantly increased. After 24-hr cultivation in the maturation medium, COCs, which were cultured for an additional 24 hr in the presence of either forskolin or 3-isobutyl-1-methylxanthine (IBMX), exhibited a significant increase in the oocyte cAMP level to the similar level of that in oocytes cultured with PI 3-kinase inhibitor or PKC inhibitor, and the addition of each agent significantly suppressed meiotic progression from the MI to the MII stage and the activity of mitogen-activated protein kinase (MAPK) and p34(cdc2) kinase. These results demonstrated that when transported into oocytes from the cumulus cells via gap junctions, cAMP plays an important role not only in meiotic resumption, but also in the regulation of meiotic progression beyond the MI stage in porcine oocytes.     

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.     

Fundamental significance of specific phosphodiesterases in the control of spontaneous meiotic resumption in porcine oocytes

The meiosis of mammalian oocytes begins during the fetal life and stops at the dictyate stage. This study has assessed the role of specific phosphodiesterase (PDE) inhibitors on the control of meiotic resumption in porcine oocytes investigating the influence of PMSG-hCG and cAMP stimulation. Cumulus-oocytes complexes (COCs) and denuded oocytes (DOs) were collected from gilt ovaries obtained at a local slaughterhouse. Oocytes were cultured in NCSU23 with different PDE inhibitors. The EC(50) for oocytes maintained in germinal vesicle (GV) stage was evaluated using different doses of both cilostamide (CIL), PDE3 inhibitor and 3-isobutyl-1-methylxanthine (IBMX), a nonspecific PDE inhibitor. In presence of PMSG-hCG, meiotic resumption is observed after 24 hr of culture. Both CIL and IBMX reversibly blocked meiotic resumption. In absence of PMSG-hCG, meiotic resumption is reduced after 24 hr of culture. After 48 hr of culture, only CIL significantly blocked meiotic resumption. Still in absence of PMSG-hCG, significant effect of treatment was only observed in COCs using the combination of CIL and rolipram (PDE3 and PDE4 inhibitor, respectively) compared to the use of IBMX. To assess the contribution of cAMP synthesis, a low dose of an adenylyl cyclase (AC) stimulator, forskolin, has been used in combination with CIL showing a significant effect of this combination. In CIL-treated COCs and DOs, significant higher percentages of oocytes were maintained in GV stage when cultured in combination with forskolin instead of PMSG-hCG. In conclusion, these results demonstrate that the control of meiotic resumption in porcine oocytes is highly regulated by cAMP. Both the degradation by specific PDE3 enzyme and the synthesis by an active AC are highly involved.     

Possible involvement of Class III phosphatidylinositol-3-kinase in meiotic progression of porcine oocytes beyond germinal vesicle stage

Phosphatidylinositol-3-kinases (PI3Ks) play pivotal roles in meiotic progression of oocytes from metaphase I to metaphase II stage. Using a Class III-specific inhibitor of PI3K, 3-methyladenine (3MA), this study shows that Class III PI3K may be essential for meiotic progression of porcine oocytes beyond germinal vesicle (GV) stage. Treatment of immature porcine oocytes with 3MA for 22-42 h arrested them at the GV stage, irrespective of the presence or absence of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). The GV-arresting effect of 3MA was, however, completely reversible upon their further culture in the absence of 3MA for 22 h. When cumulus-oophorus-complexes (COCs), arrested at the GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached the MII stage at 42 h of IVM and did not differ from non-treated control oocytes with respect to their ability to fertilize, cleave and form blastocyst (P > 0.05) upon in vitro fertilization (IVF) or parthenogenetic activation (PA). These data suggest that 3MA efficiently blocks and synchronizes the meiotic progression of porcine oocytes at the GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocyte beyond the GV stage.     

Cumulus cells suppress meiotic progression in pig oocytes cultured in vitro

The objective of this study was to examine the effect of cumulus cell removal from cumulusoocyte complexes (COCs) on meiotic progression. In Experiments 1, 2 and 3, pig COCs were cultured for 16, 20 and 24 h, respectively. The cumulus cells were then removed, and the denuded oocytes were incubated in fresh medium for another 32 h in Experiment 1, for 28 h in Experiment 2 and for 24 h in Experiment 3. In Experiment 4, the denuded oocytes and COCs were co-cultured in a drop of fresh medium from 24 h of cultivation to the end of the culture period (48 h). Removal of the cumulus cells after 16 h of cultivation had no effect on the proportions of oocytes both undergoing germinal vesicle breakdown (GVBD) and reaching MII. When the denuded oocytes were further cultured for 24 h, following the removal of their cumulus cells after 24 h of cultivation, the proportion of oocytes undergoing GVBD was significantly higher (90%, P < 0.05) than that of oocytes that were continuously cultured for 48 h without removing the cumulus cells (80%). Removal of the cumulus cells after 20 and 24 h of incubation produced a significant increase in the proportion of oocytes reaching the MII stage (84%, P < 0.05 and 76%, P < 0.01, respectively) as compared with COCs cultured continuously for 48 h without removing cumulus cells (71% and 55%, respectively). The maturation rate of denuded oocytes co-cultured with COCs for the second 24 h of cultivation was comparable to that of denuded oocytes cultured without COCs (77 and 74%, respectively). From these results, it was concluded that cumulus cells surrounding oocytes suppressed meiosis of both the GVBD process and progression from GVBD to MII in pig oocytes cultured in vitro, and that the suppressive factor in meiotic progression produced by the cumulus cells might be transferred to the oocytes through gap junctions rather than through the medium.     

A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles

Growing porcine oocytes from early antral follicles (1.2-1.5 mm in diameter) do not mature to metaphase II (MII, 4%) under culture conditions which supported maturation (MII, 95%) of fully grown oocytes from large (4-6 mm) antral follicles. We hypothesized that FSH and dbcAMP supported growth and acquisition of meiotic competence. Growing oocytes (113.0 ± 0.4 μm, mean ± SEM) were cultured for 5 d in medium supplemented with 1 mM dbcAMP, 0.01 IU/mL FSH or both; in these media, oocytes reached, 120.5 ± 0.4, 123.5 ± 0.4 and 125.7 ± 0.2 μm, respectively, after 5 d, and then were matured in vitro for 48 h. Oocytes remained enclosed by cumulus cells when cultured with FSH (82%) or both FSH and dbcAMP (80%), but not with dbcAMP alone (0%). Furthermore, oocytes cultured with FSH maintained trans-zonal projections of cumulus cells. Oocytes remained at the GV stage at higher rates when cultured with dbcAMP and FSH (99%), or dbcAMP (97%), than with FSH (64%), or without either (75%). Following in vitro maturation, oocytes reached MII after in vitro growth with dbcAMP (19%), FSH (11%), or both (68%). When oocytes were cultured with both FSH and dbcAMP, activation of Cdc2 and MAP kinases in growing oocytes was similar to fully grown oocytes. In conclusion, growing porcine oocytes grew and acquired meiotic competence in medium supplemented with dbcAMP and FSH; the former maintained oocytes in meiotic arrest, whereas the latter maintained trans-zonal projections of cumulus cells to oocytes during in vitro growth culture.     

Possible role of 5'AMP-activated protein kinase in the metformin-mediated arrest of bovine oocytes at the germinal vesicle stage during in vitro maturation

The 5'AMP-activated protein kinase (AMPK) activation is involved in the meiotic maturation of oocytes in the ovaries of mice and pigs. However, its effects on the oocyte appear to be species-specific. We investigated the patterns of AMPK and mitogen-activated protein kinases (MAPK3/1) phosphorylation during bovine in vitro maturation (IVM) and the effects of metformin, an AMPK activator, on oocyte maturation in cumulus-oocyte complexes (COCs) and denuded bovine oocytes (DOs). In bovine COCs, PRKAA Thr172 phosphorylation decreased, whereas MAPK3/1 phosphorylation increased in both oocytes and cumulus cells during IVM. Metformin (5 and 10 mM) arrested oocytes at the GV stage in COCs but not in DOs. In COCs, this arrest was associated with the inhibition of cumulus cell expansion, an increase in PRKAA Thr172 phosphorylation, and a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. However, the addition of compound C (10 muM), an inhibitor of AMPK, accelerated the initiation of the GV breakdown (GVBD) process without any alteration of MAPK3/1 phosphorylation in oocytes from bovine COCs. Metformin decreased AURKA and CCNB1 protein levels in oocytes. Moreover, after 1 h of IVM, metformin decreased RPS6 phosphorylation and increased EEF2 phosphorylation, suggesting that protein synthesis rates were lower in oocytes from metformin-treated COCs. Most oocytes were arrested after the GVBD stage following the treatment of COCs with the MEK inhibitor, U0126 (100 micromoles). Thus, in bovine COCs, metformin blocks meiotic progression at the GV stage, activates PRKAA, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during IVM. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK3/1 phosphorylation was not.     

Development of functional LH Receptors on pig cumulus–oocyte complexes cultured in vitro by a novel two‐step culture system

We show in the present study that freshly isolated pig cumulus–oocyte complexes (COCs) display a limited response to LH, as assessed by the expression of hyaluronan synthase 2 (Has2) mRNA, activation of protein kinase A (PKA), production of hyaluronic acid (HA) and progesterone, cumulus cell expansion and resumption of meiosis. These data indicate that freshly isolated COCs do not possess a sufficient number of functional LH receptors (LHR). However, the expression of Lhr significantly increased during the culture of COCs in vitro in a medium supplemented with FSH. Assuming that the effect of FSH on LHR induction is mediated via cAMP signaling pathways, we developed a new culture system, in which the COCs were pre‐cultured for 72 hr in a medium supplemented with dbcAMP. The pre‐cultured COCs remained in the germinal vesicle stage, their cumulus investment underwent a dramatic increase in size and gap junctions between the cumulus cells were preserved. The stimulation of such COCs with either FSH or LH led to the resumption and completion of meiosis, activation of PKA, expression of Has2, synthesis of large amounts of HA and progesterone, and extensive expansion of cumulus cells. We conclude that the formation of functional LHR is stimulated in cumulus cells during the culture in vitro in a cAMP‐dependent pathway. The dbcAMP‐treated COCs thus represent a new model in which the resumption of meiosis and cumulus expansion can be induced exclusively by the action of recombinant LH. Mol. Reprod. Dev. 76: 751–761, 2009. © 2009 Wiley‐Liss, Inc.     

Comparison between effects of 3-isobutyl-1-methylxanthine and FSH on gap junctional communication, LH-receptor expression, and meiotic maturation of cumulus-oocyte complexes in pigs

We investigated cAMP content, gap junctional communications (GJCs) status, and LH-receptor (LH-R) expression in porcine cumulus-oocyte complexes (COCs) during in vitro maturation treated with the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) or with FSH. COCs were cultured for 20 hr (1st culture) in M199 containing 10% FBS (basic medium, BM group) or BM supplemented with FSH (FSH group) or IBMX (IBMX group). Each COC was then transferred into BM containing both FSH and LH and cultured for an additional 24 hr (2nd culture). The proportions of metaphase-II (M-II) oocytes at the end of the 2nd culture did not differ between the FSH (75.7%) and IBMX (68.2%) groups, whereas only 10.1% of oocytes in the BM group reached the M-II stage. During the 1st culture, the cAMP content of COCs and oocytes became significantly higher in the FSH and IBMX groups than in the BM group; the FSH group had a far greater increment than did the IBMX group. GJCs in the FSH and BM groups gradually closed with increasing duration of the 1st culture, whereas a significantly higher proportion of COCs in the IBMX group still had open GJCs than in the other two groups. Furthermore, LH-R mRNA expression significantly increased in both the FSH and IBMX groups compared with the BM group. These results suggest that inhibition of PDEs in porcine COCs make the oocyte ready for release from meiotic arrest, and that maintenance of a moderate cAMP content may prolong GJCs and stimulate LH-R expression.     

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

Inhibition of phosphatidylinositol 3-kinase or mitogen-activated protein kinase kinase leads to suppression of p34(cdc2) kinase activity and meiotic progression beyond the meiosis I stage in porcine oocytes surrounded with cumulus cells

In this study, the effects of U0126 that inhibits the activity of mitogen-activated protein (MAP) kinase kinase (MEK), and LY294002, which is a phosphatidylinositol (PI) 3-kinase inhibitor, on meiotic progression beyond the metaphase I (MI) stage in porcine oocytes were examined. Cumulus-oocyte complexes (COCs) were cultured for 22 h with 50 microM LY294002 or 10 microM U0126 following cultivation for the initial 22 h. MAP kinase activity in oocytes cultured with LY294002 or U0126 was significantly lower than that in control oocytes cultured for up to 44 h. U0126 and LY294002 significantly decreased p34(cdc2) kinase activity and the proportion of oocytes reaching the MII stage compared to those in control oocytes. Oocytes denuded after COCs had been cultured for 22 h were cultured further for 22 h with U0126 or LY294002. In the denuded oocytes, U0126 suppressed MAP kinase activity, p34(cdc2) kinase activity, and meiotic progression to the MII stage; however, LY294002 did not significantly affect the activity of these kinases and meiotic progression. These results suggest that increasing MAP kinase activity in oocytes via the PI 3-kinase signaling pathway in cumulus cells is involved in the stimulation of maturation promoting factor, leading to meiotic progression beyond the MI to MII stage in porcine oocytes.     

Phosphatidylinositol 3-kinase in cumulus cells and oocytes is responsible for activation of oocyte mitogen-activated protein kinase during meiotic progression beyond the meiosis I stage in pigs

The roles of phosphatidylinositol 3-kinase (PI 3-kinase) during meiotic progression beyond the meiosis I (MI) stage in porcine oocytes were investigated. PI 3-kinase exists in cumulus cells and oocytes, and the PI 3-kinase inhibitor, LY294002, suppressed the activation of mitogen-activated protein (MAP) kinase in denuded oocytes during the beginning of the treatment. However, in denuded oocytes cultured with LY294002, the MAP kinase activity steadily increased, and at 48 h of cultivation MAP kinase activity, p34(cdc2) kinase activity, and proportion of oocytes that had reached the meiosis II (MII) stage were at a similar level to those of oocytes cultured without LY294002. In contrast, LY294002 almost completely inhibited the activation of MAP kinase, p34(cdc2) kinase activity, and meiotic progression to the MII stage in oocytes surrounded with cumulus cells throughout the treatment. Treating cumulus oocyte complexes (COCs) with LY294002 produced a significant decrease in the phosphorylation of connexin-43, a gap junctional protein, in cumulus cells compared with that in COCs cultured without LY294002. These results indicate that PI 3-kinase activity in cumulus cells contributes to the activation of MAP kinase and p34(cdc2) kinase, and to meiotic progression beyond the MI stage. Moreover, gap junctional communications between cumulus cells and oocytes may be closed by phosphorylation of connexin-43 through PI 3-kinase activation in cumulus cells, leading to the activation of MAP kinase in porcine oocytes.     

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.     

Meiotic state of bovine oocytes is regulated by interactions between cAMP,cumulus, and granulosa

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.     

Effects of LH and FSH on the maturation of pig oocytes in vitro

This research was designed to investigate the effects of LH and FSH (50 ng/ml) on pig oocyte maturation in vitro. The following parameters were studied: a) the degree of heterologous coupling between cumulus cells and oocytes, evaluated by measuring the (3)H-uridine and (3)H-choline uptake in cumulus enclosed oocytes; b) meiotic maturation; c) cytoplasmatic maturation, evaluated by analyzing the ability of the oocytes to promote male pronucleus formation after in vitro fertilization. Despite the marked cumuli expansion induced by gonadotropins, uridine uptake was not influenced by LH or FSH. By contrast, choline uptake in LH-treated oocytes was significantly higher than in FSH-treated or control oocytes (3199 cpm +/- 251 vs 1686 cpm +/- 142, P<0.01). Gonadotropins accelerated meiotic progression, and after 30 hours of culture the percentage of oocytes at the germinal vesicle stage was significantly lower (P<0.01) in LH-(24%, 24 102 ) and FSH-(20%, 18 90 ) treated oocytes than in control oocytes (76%, 64 84 ). After 44 hours of culture, the percentage of oocytes reaching the MII stage was significantly higher (P<0.01) in the presence of LH (76%, 92 120 ) and FSH (86%, 92 108 ) than in the controls (35%, 40 116 ). The percentage of oocytes capable of sustaining male pronucleus formation was similar in the control (48.4%, 63 132 ) and FSH-treated oocytes (44.3%, 51 116 ), while it was markedly increased (P<0.01) by the addition of LH (72.7%, 143 197 ). The data reported indicate that in vitro pig oocytes tend to undergo meiotic maturation even in the absence of hormones. However, in our in vitro system, LH and FSH accelerated and facilitated meiotic progression, and LH selectively improved cytoplasmic maturation which is required to promote the formation of a male pronucleus.     

The releasing action of calcium upon cyclic AMP-dependent meiotic arrest in hamster oocytes

The effect of increasing cytoplasmic calcium on cyclic adenosine monophosphate (cAMP)-dependent meiotic arrest (%GV where GV is germinal vesicle) in hamster oocytes was investigated. The hypotheses tested were that calcium is required for the spontaneous maturation of hamster oocytes, elevation of calcium in the oocyte-cumulus complex can antagonize cAMP-dependent meiotic arrest, and the intraoocyte level of cAMP remains unchanged, but heterologous metabolic coupling decreases, concomitant with calcium-stimulation of germinal vesicle breakdown (GVBD). Levels of cAMP were elevated by culturing cells in the presence of dibutyryl cAMP (dbcAMP), isobutylmethylxanthine (IBMX) or forskolin and intracellular levels of calcium were manipulated by altering the CaCl2 concentration in the medium and/or by utilizing EGTA or A23187. Intracellular cAMP was determined by RIA, functional metabolic coupling was assessed by determination of the fraction of radiolabeled uridine marker transferred from the cumulus mass to the oocyte, and meiotic stage was determined cytogenetically. Compared with the proportion of oocytes that underwent meiotic maturation in control medium containing 1.53 mM CaCl2, that of cumulus-free (denuded) oocytes was unaffected by culture in the absence of added CaCl2, while that of cumulus-enclosed (intact) oocytes was significantly decreased (%GV = 59.5 +/- 4.8 and 4.2 +/- 0.9 in 0 and 1.53 mM CaCl2, respectively, P less than 0.001, where GV is germinal vesicle). EGTA prevented, in a dose-dependent manner, the spontaneous maturation of denuded oocytes that occurred in 0 mM CaCl2 (ID50 = 0.05 mM, where ID50 is the dose of EGTA that inhibited GVBD in 50% cultured oocytes). In contrast, compared with the control, less than 1 mM EGTA failed to increase the %GV of intact oocytes, although 5 mM EGTA significantly increased meiotic arrest. The %GVBD of oocytes cultured in medium containing 0 mM CaCl2 was dose-dependent on A23187 for both intact oocytes (ID50 = 3.0 microM) and for denuded oocytes cultured in the presence of 0.5 mM EGTA (ID50 = 2.7 microM). Elevated extracellular calcium significantly antagonized dbcAMP-maintained meiotic arrest in both types of oocyte and the %GV was significantly correlated with the pH of the medium [(r) = -0.78 and -0.60 for intact and denuded oocytes, respectively, P less than 0.001 in both cases]. Both CaCl2 and A23187 induced dose-dependent antagonistic effects on forskolin-maintained meiotic arrest in intact oocytes but neither antagonism was accompanied by significant dose-dependent decreases in either the intraoocyte content of cAMP or the extent of heterologous metabolic coupling.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative inhibitory influence of porcine cumulus cells upon the maturation of pig and cattle oocytes in vitro

Porcine cumulus oocyte complexes (COCs) were cultured together in 10-microliters droplets of culture medium. When 10 COCs were cultured for 24 h, germinal vesicle breakdown (GVBD) occurred in 81% of them. When more COCs (20 or 40) were put into the same volume of medium the frequency of GVBD gradually decreased. This inhibition was not observed in denuded oocytes. The process of GVBD was adversely influenced when 10 COCs were cultured in cumulus-preconditioned medium. It is concluded that porcine cumulus cells produced a factor inhibiting GVBD. After removing the inhibitory block and extensive washing, GVBD of arrested oocytes was significantly accelerated. The addition of LH or heparin only partially overcame the inhibitory action. This factor produced by porcine cumulus cells negatively influenced maturation of bovine oocytes; however, a similar effect was not demonstrated in the mouse. Our results suggest that a high concentration of porcine cumulus cells exerts a quantitative inhibitory effect upon GVBD of porcine and cattle oocytes cultured in vitro.     

Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin

The efficacy of follicle-stimulating hormone (FSH), epidermal growth factor (EGF), and dibutyryl cGMP (dbcGMP) as inducers of germinal vesicle breakdown (GVBD) in cumulus cell-enclosed mouse oocytes was examined when meiotic arrest was maintained in vitro with purines, dibutyryl cAMP (dbcAMP), or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). When FSH was added to hypoxanthine (HX)-containing medium, the effect on oocyte maturation was at first inhibitory and later stimulatory. EGF stimulated GVBD at all time points tested. FSH and EGF also induced GVBD when oocytes were arrested with dbcAMP, IBMX, or guanosine. Dibutyryl cGMP stimulated GVBD when meiotic arrest was maintained with HX, but not when oocytes were meiotically arrested with guanosine, and was inhibitory in dbcAMP-supplemented medium. FSH and dbcGMP produced a transient delay of oocyte maturation in control medium, but the FSH effect was much more pronounced. EGF had no effect on maturation kinetics. The actions of FSH and EGF required the presence of cumulus cells. Both agents significantly stimulated cAMP production in oocyte-cumulus cell complexes. A brief exposure of complexes to a high concentration of dbcAMP induced GVBD, suggesting that FSH and EGF may act via a cAMP-dependent process. The frequency of FSH- and EGF-induced GVBD in cumulus cell-enclosed oocytes was significantly higher than the frequency of GVBD when oocytes were cultured while denuded of cumulus cells. of maturation is apparently not mediated solely by oocyte-cumulus cell uncoupling and termination of the transfer of an inhibitory meiotic signal from cumulus cells to the oocyte. The data suggest the generation of a positive signal within cumulus cells in response to hormone treatment that acts upon the oocyte to stimulate GVBD in the continued presence of inhibitory factors.