Cumulus cells secrete a meiosi-inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed. The GVBD of unstimulated DO also increased significantly when cocultured with cumulus cells from primed CEO. The percentage of GVBD in unprimed DO and in DO isolated from primed CEO was the same. We suggest that within 1–2 hr, forskolin and cAMP stimulate cumulus cells to produce a diffusible meiosis-inducing substance which overcomes HX-inhibition and induces oocyte maturation, including both GVBD and PB formation. The CEO must be primed for more than 2 hr before the resumption of meiosis in DO isolated from such CEO is induced. Oocyte-cumulus connections are crucial as far as initiating the production of a meiosis-inducing substance is concerned. Oocyte-cumulus connections are not needed for transferring this substance to the oocyte. © 1994 Wiley-Liss, Inc.     

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.     

Induction of meiotic maturation of follicle-enclosed oocytes of rabbits by a transient increase followed by an abrupt decrease in cyclic AMP concentration.

The involvement of cyclic adenosine monophosphate (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without forskolin at 10(-4), 10(-5) or 10(-6) mol l-1 for 3-6 h. At 3 or 4 h spontaneous meiotic maturation was significantly (P < 0.05) inhibited by forskolin at 10(-4) mol l-1. With prolonged incubation, spontaneous maturation progressed despite exposure to forskolin. In the second experiment ovaries were perfused for 12 h with forskolin (10(-4), 10(-5) or 10(-6) mol l-1) or medium alone. Neither ovulation nor degeneration of follicular oocytes occurred in any perfused ovary. The percentage of follicular oocytes achieving germinal vesicle breakdown was significantly (P < 0.001) increased in response to forskolin in a dose-related manner. In an additional experiment, ovaries were perfused with forskolin at 10(-4) mol l-1. A significant increase in the cAMP content in the follicle was observed within 30 min, but the ability to produce cAMP in response to forskolin decreased as the duration of perfusion was increased. Intraoocyte cAMP increased significantly within 30 min and reached its maximum 2 h after exposure to forskolin. Thereafter, cAMP levels in the oocytes decreased abruptly. This drop in intraoocyte cAMP concentration was followed by the resumption of meiosis. The alterations of intraoocyte cAMP contents following exposure to hCG in vivo paralleled those observed in the ovaries perfused with forskolin. These data suggest that a transient, but not continuous, increase in cAMP concentration after the gonadotrophin surge may be required to initiate oocyte maturation.     

Induction and Inhibition of Meiotic Maturation in Follicle-enclosed Mouse Oocytes by Forskolin

A continuous exposure of follicle-enclosed mouse oocytes to ovine luteinizing hormone (LH, 10 μg/ml) in vitro resulted in a 3-fold elevation of CAMP levels in the follicle cells, but not the oocytes, with subsequent oocyte maturation. When follicle-enclosed oocytes were exposed to forskolin (0.01–10 μM) for 2 hr and then incubated in forskolin-free medium (transient exposure group), oocytes underwent germinal vesicle breakdown in a dose-dependent manner. In contrast, a continuous exposure of the follicles to forskolin (10 μM) for up to 10 hr failed to induce resumption of meiosis. Follicle cell cAMP levels increased within 2 hr after the initial exposure to forskolin, and thereafter decreased rapidly regardless of whether forskolin treatment was transient or continuous. A similar transient increase in oocyte cAMP levels was observed after transient or continuous treatment with forskolin. It was evident, however, that at any time examined oocyte cAMP levels were consistently higher in the continuous exposure group than in the transient exposure group. Furthermore, a continuous exposure to forskolin also blocked LH-induced meiotic maturation. These findings suggest that elevated levels of cAMP in the oocyte block meiotic maturation in mouse oocytes. The present results further suggest that an increase in follicle cell cAMP levels is essential to the LH-induced meiotic maturation.     

Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes

Phosphodiesterase (PDE)‐mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3‐isobutyl‐1‐methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV‐stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double‐strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 μM dbcAMP and 10.0 μM IBMX efficiently inhibited meiotic resumption in GV‐stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.     

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

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

Effect of testosterone and dibutyryl c-AMP on the meiotic competence in pig oocytes of various size categories

Spontaneous meiosis resumption was investigated in small pig oocytes of various size categories. Two parameters were studied: 1) the possibility for inhibiting spontaneous meiosis resumption in small oocytes of various size categories and 2) the level of meiotic competence of small oocytes in which meiosis resumption had been temporarily blocked. It was observed that 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) combined with 0.5 μM testosterone were able to prevent spontaneous maturation in pig oocytes of various size categories cultured in vitro for up to 10 d. Moreover, a culture of these oocytes with dbcAMP and testosterone has a beneficial effect on the integrity of the oocyte-granulosa cell complex. When oocytes were allowed to mature after long-term inhibition of maturation, the percentage of oocytes able to resume maturation was significantly increased. However, the portion of oocytes which completed maturation, reaching the metaphase II (M II) stage, did not increase. It was found that dbcAMP, in combination with testosterone, also efficiently inhibited spontaneous maturation in fully grown pig oocytes. When the inhibitory effect of dbcAMP and testosterone was reversed in fully grown oocytes, maturation was significantly accelerated.     

Interaction of signal cascades induced by cAMP and prolactin in bovine oocyte-cumulus complexes

Prolactin (PRL) is one of the pituitary hormones participating in the control of mammalian folliculo- and oogenesis. In the present study, the joint effect of PRL (50 ng/ml) and dibutyryl cAMP (dbcAMP, 1 mM) on oocyte maturation and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. It has been shown that PRL totally suppresses the braking impact of dbcAMP on meiosis reinitiation and the completion of oocyte nuclear maturation. Furthermore, PRL partly inhibited cumulus expansion induced by dbcAMP, although it exerted the opposite effect in the control medium. In the presence of PRL, the inhibitory impact of dbcAMP on the proliferative activity of cumulus cells and on the PRL-elicited braking of destructive processes in the cells has been found. In cumulus cells, mRNA expression of PRL receptor long isoform was revealed by the RT-PCR method. The data obtained suggest an interaction of signal cascades induced by PRL and cAMP in bovine oocyte-cumulus complexes, with the coupling site of these cascades in oocytes being apparently different from that in cumulus cells.     

Roles of protein kinase A and C in spontaneous maturation and in forskolin or 3-isobutyl-1-methylxanthine maintained meiotic arrest of bovine oocytes

Four hypotheses were tested using isolated bovine oocytes. (1) Cumulus oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with the protein kinase A (PKA) inhibitor, H-89, to test if meiotic arrest induced by forskolin or IBMX was due to cAMP-stimulated PKA activity or nonspecific effects of these cAMP elevators. (2) COCs were cultured with a protein kinase C (PKC) stimulator (PDDβ) or inhibitor (GF109203x) to test if PKC modulation altered oocyte maturation. (3) COCs were prestimulated for 15 min with (a) PDDβ followed by cotreatment with forskolin, or (b) with H-89 or H-7 followed by cotreatment with GF109203x, to test for interaction between the PKA and PKC signal transduction pathways. (4) H-89 was added to spontaneously maturing COCs at intervals 0–18 hr to test if H-89 interfered with the transition between meiosis I and II. The results were as follows: H-89 interfered with forskolin or IBMX arrested oocytes in a dose-response manner (IBMX ED₅₀ = 41 μM for COCs; forskolin ED₅₀ = 9 μM for denuded oocytes). Prestimulation with PKC induced meiotic resumption in COCs in spite of the presence of forskolin [PDDβ followed by PDDβ + forskolin: 41–47% germinal vesicle (GV) oocytes; forskolin alone: 90–95% GV], while PKC inhibition induced meiotic arrest to a similar extent as forskolin (GF109230x, 85% GV; forskolin, 67–80% GV). Additionally, pretreatment of COCs with H-89 interfered with GF109203x induced arrest (41% vs. 90% GV, respectively). Finally, H-89 interfered with the timely progression of COCs from meiosis I and II. These results indicate that the PKA and PKC pathways can modulate the maturation of bovine oocytes in vitro. © 1996 Wiley-Liss, Inc.     

Forskolin and mouse oocyte maturation in vitro

Oocytes isolated from mature follicles undergo spontaneous maturation when cultured in vitro. Forskolin, an adenylate cyclase stimulator, inhibited resumption of meiosis of cumulus-free mouse oocytes in vitro. Germinal vesicle breakdown (GVBD) was prevented in more than 85% of the oocytes treated by forskolin at concentrations of 20 micrograms/ml and higher. The inhibiting effect of forskolin was dose-dependent and reversible. FSH, LH, FSH plus LH, estrogen, progesterone, and estrogen plus progesterone did not reverse the block induced by forskolin in cumulus-free and cumulus-enclosed oocytes. The present results suggest that intracellular cAMP may play a role in the regulation of oocyte maturation.     

绵羊卵母细胞体外核成熟抑制及其对胚胎发育潜力的影响

本研究旨在探讨次黄嘌呤 +dbcAMP或IBMX +dbcAMP对绵羊卵母细胞体外成熟的可逆性抑制作用 ,以及这种抑制作用对卵母细胞胚胎发育潜力的影响。自绵羊卵巢分离卵丘 -卵母细胞复合物进行体外培养 ,培养基中分别加入或不加入上述抑制剂。培养 6h后各组取部分卵母细胞固定染色检查卵母细胞核成熟情况 ;将其余的卵母细胞分别移入无抑制剂的成熟培养基中继续培养 18h后 ,再次检查各组卵母细胞核成熟情况 ,并进行体外受精和胚胎培养。结果表明 :次黄嘌呤 +dbcAMP或IBMX +dbcAM都分别能使 6 0 %以上的绵羊卵母细胞抑制在GV期。这种抑制是可逆性的 ,去除抑制剂后卵母细胞能恢复减数分裂 ,并加快由GVBD到MⅡ的成熟过程。各处理组受精率、卵裂率和囊胚发育率与对照组相比无显著性差异 ,表明卵母细胞的胚胎发育潜力没有受损。上述物质对卵母细胞成熟的可逆性抑制可用于研究卵母细胞成熟及其胚胎发育潜力的调节机制。     

Interaction of signal cascades induced by cAMP and prolactin in bovine oocyte-cumulus complexes

Prolactin (PRL) is one of the pituitary hormones that participate in controlling mammalian folliculo-and oogenesis. In the present study, the combined action of PRL (50 ng/ml) and dibutyryl cAMP (dbcAMP, 1 mM) on oocyte maturation and the morphologic-functional state of the surrounding cumulus cells was investigated in vitro. It has been shown that PRL completely suppresses the inhibitory effect of dbcAMP on meiosis reinitiation and completion of the oocyte nuclear maturation. Moreover, PRL partly inhibited the dbcAMP-induced cumulus expansion, although it produced an opposite effect in the control medium. In the presence of PRL, the inhibitory effect of dbcAMP on the proliferative activity of cumulus cells, as well as on the PRL-induced suppression of destructive processes in these cells, was revealed. In cumulus cells, the mRNA expression of the long PRL receptor isoform was established by the RT-PCR method. The obtained data indicate an interaction of signal cascades induced by PRL and cAMP in the bovine oocyte-cumulus complexes, with the coupling site of these cascades in oocytes seeming to differ from that in cumulus cells.     

Effect of type 3 and type 4 phosphodiesterase inhibitors on the maintenance of bovine oocytes in meiotic arrest.

The use of broad-spectrum inhibitors first suggested that phosphodiesterases (PDEs) are involved in the maturation of bovine oocytes. Modulation of individual PDE families is now possible with the use of newly developed type-specific PDE inhibitors. This study evaluated the role of type 3- and type 4-specific PDE inhibitors on the meiotic arrest of bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DOs). It also evaluated the role of these specific inhibitors on meiotic arrest when COCs are incubated in the presence or absence of theca cell monolayers. Bovine COCs were aspirated from ovaries collected at the abattoir. Denuded oocytes and COCs were incubated for 12 h in culture medium alone or culture medium containing the type 3 PDE inhibitors cilostamide (10 and 20 microM) or milrinone (10 and 50 microM) or the type 4 PDE inhibitor rolipram (10 and 50 microM). Oocytes were then fixed and classified according to the status of nuclear maturation. Cumulus-oocyte complexes were coincubated with untreated theca cell monolayers or theca cell monolayers treated with the different specific PDE inhibitors. Bovine COCs or DOs incubated in culture medium resumed meiosis, but supplementation of the culture medium with the PDE3 inhibitors cilostamide or milrinone resulted in meiotic arrest. On the other hand, supplementation of the culture medium with rolipram did not prevent oocyte maturation. Furthermore, PDE3 inhibitors, but not PDE 4 inhibitors, had an additive effect on the inhibitory action of theca cell monolayers on oocyte maturation. These data support the hypothesis that inhibition of PDE3 prevents the meiotic resumption of bovine oocytes, whereas inhibition of PDE4 does not block oocyte maturation even under normally inhibitory conditions. The additive effect of PDE3 inhibitors on the ability of theca cells to maintain bovine oocytes in meiotic arrest suggests that type 3 PDE has an important role in meiotic resumption of bovine oocytes.     

Regulation of murine oocyte meiosis: evidence for a gonadotropin- induced, cAMP-dependent reduction in a maturation inhibitor

We have developed an assay that can detect relative changes in the amount of a non-cAMP inhibitor of maturation present in cumulus cells (Eppig et al., 1983, Dev. Biol., 100:39-49). Using this assay in which accelerated maturation of a group of treated cumulus cell-oocyte complexes relative to untreated complexes indicates a decrease in the amount of inhibitor, results of the experiments described here suggest a possible relationship between elevation of cAMP levels and subsequent decreased amounts of a non-cAMP inhibitor. Mouse oocytes obtained from cumulus cell-oocyte complexes treated with luteinizing hormone (LH) resumed meiosis prior to oocytes obtained from untreated complexes; the degree of acceleration of maturation was dependent on LH concentration. A similar result was obtained with follicle-stimulating hormone (FSH). Correlated with LH- or FSH-acceleration of maturation was an LH- or FSH-induced elevation of cumulus cell cAMP levels. Inhibiting LH-induced elevation of cumulus cell cAMP levels inhibited LH-induced acceleration of maturation. An initial incubation of complexes in medium containing dibutyryl cAMP (dbcAMP) also promoted acceleration of maturation. In contrast, maturation of denuded oocytes was not altered by treatment with either LH, FSH, or dbcAMP. Complexes initially incubated in dbcAMP-containing medium still demonstrated acceleration of maturation after a subsequent 2 h incubation in dbcAMP-free medium. Relative to untreated complexes, none of these treatments disrupted intercellular communication between cumulus cells and the oocyte. Elevating follicle cAMP levels with cholera toxin induced maturation of follicle-enclosed oocytes when cumulus cell-oocyte coupling was still fully maintained. These results are interpreted to indicate that gonadotropin-mediated acceleration of maturation is via a cAMP-dependent reduction in the level of a maturation inhibitor present in granulosa/cumulus cells.     

Possible role for phosphatidylinositol 3-kinase in regulating meiotic maturation of bovine oocytes in vitro

In this study 2 phosphatidylinositol 3-kinase (PI 3-kinase)-specific inhibitors, wortmannin and 2-[4-Morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002), were used to investigate whether PI 3-kinase is involved in the signal transduction that leads to bovine oocyte maturation. Bovine follicular oocytes were cultured in vitro for 24 h in a basic medium consisting of tissue culture medium-199 supplemented with LH, FSH, fetal cow serum, Na-pyruvate and gentamicin. The oocytes were then examined for the stage of meiotic progression and degree of cumulus expansion. In Experiment 1, in cumulus-oocyte complexes (COCs), wortmannin, at any level tested (10(-8) M, 10(-7) M or 10(-6) M), had no effect on resumption of meiosis as judged by germinal vesicle breakdown and progression to prometaphase I or metaphase I. However, wortmannin significantly (P < 0.01) decreased the proportion of oocytes developing to metaphase II in a dose-dependent manner. In Experiment 2, when denuded oocytes were cultured with wortmannin at 0, 10(-7) M and 10(-6) M concentrations, the same pattern of response for COCs was observed, with no effect on meiotic resumption and a significant (P < 0.01) decrease in the proportion of oocytes reaching metaphase II. In Experiment 3, half of the recovered COCs were denuded and both denuded and intact COCs were cultured in the presence of 0, 2.5 x 10(-5) M, 5.0 x 10(-5) M and 7.5 x 10(-5) M LY 294002 before being examined for meiotic progression. Whereas LY294002, at any examined level, had no effect on the percentage of oocytes developing to metaphase I, it significantly (P < 0.01) decreased the proportion of metaphase II oocytes when used at 5.0 x 10(-5) or 7.5 x 10(-5) M for both intact COCs and denuded oocytes. In Experiment 4, no significant difference in the degree of cumulus expansion was scored after the COCs were cultured in the presence of wortmannin or LY294002 or in the absence of either treatment. These results provide indirect evidence for a role of PI 3-kinase in the bovine oocyte itself in regulating meiotic progression beyond metaphase I.     

Effect of dibutyryl cyclic adenosine monophosphate on granulosa cell proliferation, oocyte growth and meiotic maturation in isolated mouse primary ovarian follicles cultured in collagen gels.

Isolated primary follicles from 10-day-old mice were cultured in a collagen gel matrix for 6 days in Minimum Essential Medium + foetal calf serum, followed by culture in unsupplemented medium (control) or in medium containing hypoxanthine (2 mM) or dibutyryl cyclic adenosine monophosphate (dbcAMP, 0.25 mM) for a further 3 or 6 days. Less than 10% of oocytes resumed meiosis during the culture period in all groups. At recovery, the diameter of oocytes at the germinal vesicle stage was recorded and their ability to resume meiosis was determined. Hypoxanthine had little effect on oocyte growth and meiotic competence, but culture in dbcAMP resulted in oocytes that were larger (60.2 +/- 0.6 microns) than those of controls (55.8 +/- 0.5 microns) and more competent to resume meiosis than were controls (42.9% and 10.8%, respectively). The addition of dbcAMP to the culture medium induced a 4-5-fold increase in the number of granulosa cells oocyte compared with controls (3757 +/- 423 and 838 +/- 93, respectively). These results indicate that increased oocyte growth and meiotic competence is primarily mediated via dbcAMP effects on the granulosa cells.     

Effects of dibutyryl cyclic AMP on oocyte maturation and ovulation in the perfused rabbit ovary

The involvement of cyclic AMP (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and in-vitro perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without dibutyryl cyclic AMP [Bu)2cAMP) at 10(-3), 10(-4) or 10(-5) M for 4-12 h. At 4 h spontaneous meiotic maturation was significantly inhibited by (Bu)2cAMP (P less than 0.025). With prolonged incubation, spontaneous maturation progressed despite exposure to (Bu)2cAMP. When ovaries were continuously perfused in vitro for 12 h with (Bu)2cAMP (10(-3) M) or medium alone, (Bu)2cAMP stimulated ovarian progesterone production, but did not affect ovulation or maturation of follicular oocytes. When ovaries were perfused in vitro with or without (Bu)2cAMP (10(-3), 10(-4) or 10(-5) M) for the first 2 h and then transferred to medium without (Bu)2cAMP for an additional 10 h, ovulation did not occur, but transient exposure to (Bu)2cAMP stimulated a dose-related increase in maturation of follicular oocytes. Degeneration of follicle-enclosed oocytes and cumulus-oocyte complexes was not affected by exposure to (Bu)2cAMP. These results suggest that transient, but not continuous, elevation of cAMP after the gonadotrophin surge may be required for the initiation of oocyte maturation.     

Effects of gonadotropin-releasing hormone agonists on meiotic maturation of follicle-enclosed oocytes in rabbits

The effects of GnRH agonists on in vitro maturation of rabbit follicle-enclosed oocytes were studied. Rabbit preovulatory follicles were cultured with or without hCG (10(2) ng/ml), buserelin (10(2)-10(5) ng/ml), or leuprolide (10(2)-10(5) ng/ml) for 14 hours in vitro. GnRH agonists induced the resumption of meiosis in the follicle-enclosed oocytes in a dose-dependent manner. The percentage of oocytes achieving GVBD following treatment with 10(5) ng/ml buserelin (87.9 +/- 6.3%) or 10(5) ng/ml leuprolide (86.0 +/- 4.1%) did not differ significantly from hCG-treated control (87.3 +/- 3.8%). Mature oocytes initially were detected within 2 hours of GnRH agonist exposure. Concomitant addition of a GnRH antagonist at 10(4) ng/ml significantly blocked the stimulatory effect of GnRH agonist on oocyte maturation. GnRH agonists significantly stimulated both prostaglandin (PG) E2 (PGE2) and PGF2 alpha production by preovulatory follicles (p less than 0.01), but secreted prostanoid levels did not differ significantly among different concentrations of GnRH agonists. Meiotic maturation of follicle-enclosed oocytes following GnRH agonist exposure began 2 hours earlier than production of PGs. PG production stimulated by GnRH agonists was reduced significantly by indomethacin. However, oocyte maturity in the presence of GnRH agonist plus indomethacin did not differ significantly from that of GnRH agonist alone. GnRH agonistic analogues induce the resumption of meiosis in follicle-enclosed oocytes in rabbits by a mechanism other than PG stimulation.     

Realization pathways of prolactin modulating effect on the cAMP-dependent mechanism of meiosis regulation in bovine oocytes

Cyclic AMP is one of the key regulators of mammalian meiosis. In the present work, realization pathways of the previously revealed modulating effect of prolactin (PRL) on the cAMP-dependent mechanism of meiosis regulation in bovine oocytes were studied. A comparative investigation of individual and combined effects of PRL (50 ng/ml) and an activator of adenylate cyclase forskolin (FK, 20 μM) on the meiotic reinitiation and completion of nuclear maturation in cumulus-surrounded and cumulus-free oocytes was performed. It has been shown that the pattern of the effects of PRL on the meiotic resumption in oocytes devoid of cumulus cells depends on the presence of FK in the culture medium. Furthermore, the realization of this effect is not associated with the activation of cytoplasmic isoforms of protein kinase C. It has also been found that PRL inhibits the retarding action of FK on the completion of oocyte nuclear maturation both in the presence and absence of cumulus cells. These findings suggest that PRL may modulate the functioning of the cAMP-dependent mechanism of meiosis regulation by the direct action on bovine oocytes, with realization of this action being independent of the metabolic coupling of oocytes with cumulus cells.