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.     

小鼠年龄及卵丘—卵母细胞间联接的解离对卵母细胞体外成熟的影响

实验研究了小鼠卵母细胞体外过程中卵丘－卵母细胞间的相互作用。实验小鼠为雌性Ｂ６Ｄ２杂交一代。激素处理４８小时后分离出卵后天和卵母细胞复合体,并培养在含有次黄嘌呤的培养液中。２４小时后检查卵母细胞核成熟情况。     

Effect of glucocorticoids on spontaneous and follicle-stimulating hormone induced oocyte maturation in mouse oocytes during culture

Several studies have indicated that glucocorticoids are involved in maturation of mammalian oocytes. Recently, maturation of porcine oocytes in culture was shown to be inhibited by glucocorticoids in a time- and dose-dependent manner. In addition, levels of cortisol available for biological action in fluid of preovulatory follicles are higher than that present in circulation. The present study evaluates the effect of cortisol and dexamethasone on mouse cumulus enclosed oocytes (CEO) undergoing spontaneous- and FSH-induced maturation during a 24 h culture period using breakdown of the germinal vesicle (GVBD) as end-point. FSH-induced oocyte maturation was studied using media containing 4.5 mM hypoxanthine to maintain levels of cAMP elevated, whereas spontaneous oocyte maturation was studied in a medium without hypoxanthine. In the presence of FSH (25 IU/l) the rate of GVBD was significantly elevated compared to the control. Dexamethasone (1–20 μg/ml) in combination with FSH resulted in a rate of GVBD similar to FSH alone. Cortisol (0.1–10 μg/ml) resulted in a significant higher rate of GVBD in combination with a physiological concentration of FSH (10 IU/l) as compared to the control but similar to that caused by FSH alone. Nearly all CEO that matured spontaneously resumed meiosis irrespective of whether or not cortisol was present. In conclusion, these results indicate that glucocorticoids have little or no influence on the regulation of oocyte maturation in the mouse. Species differences between mouse and pig oocytes may exist.     

体外培养条件下促性腺激素对昆明白小鼠卵母细胞成熟及卵丘扩展的影响

研究促卵泡激素（FSH）,人绒毛膜促性腺激素（hCG)对昆明白小鼠卵母细胞成熟和卵丘扩展的影响,以及体外培养时卵丘扩展与卵母细胞成熟之间的关系,FSH可以明显促进次黄嘌吟（HX）抑制条件下的卵丘－卵母细胞复合体CEO卵母细胞成熟及卵丘扩展,其最佳作用剂量为100IU／L,且FSH作用30分钟即可以使CEO获得恢复减数分裂的信息,在HX存在的条件下,FSH处理后10hr,CEO卵丘明显扩展,而生发泡破裂（GVBD）则在16－20hr明显增加,所有卵丘未扩展的CEO中卵母细胞均未发生GVBD,低剂量hCG单独或与FSH共同存在,对CEO卵母细胞成熟及卵丘扩展均无明显影响;高剂量hCG可以部分抑制FSH对卵母细胞成熟的促进作用,结果表明：FSH明显促进CEO卵母细胞成熟及卵丘扩展,而hCG却不具有此作用,体外培养条件下（含次黄嘌呤）,卵丘扩展是卵母细胞成熟的前提条件,但卵母细胞成熟并不需要卵丘完全扩展。     

FSH诱导卵丘细胞分泌一种促减数分裂物质(英文)

本实验利用卵母细胞的体外培养模型,将小鼠卵丘－卵母细胞复合体（ＣＥＯ）和去卵丘卵母细胞（ＤＯ）在体外培养,系统研究了促性腺激素（ＦＳＨ、ｈＣＧ）诱导小鼠卵母细胞减数分裂的机制。结果显示,ＦＳＨ能剂量依赖性地诱导ＣＥＯ恢复减数分裂（Ｆｉｇ．１）,但对ＤＯ无影响;ｈＣＧ对 ＣＥＯ、 ＤＯ皆无效果（Ｆｉｇ．２）;用 ＦＳＨ预处理ＣＥＯ时间达到１小时后,就能显著诱导卵母细胞成熟,２小时后作用达到最大;不再增强（Ｆｉｇ．３）;用 ＦＳＨ处理ＣＥＯ ２小时及２４小时的培养液,能诱导ＤＯ恢复减数分裂,但预处理卵丘细胞２４小时的培养液,并不能诱导ＤＯ恢复减数分裂（Ｆｉｇ．４Ａ）;这种培养液在７０℃下３０分钟后,仍能刺激ＤＯ成熟（Ｆｉｇ．４Ｂ）;甾醇类物质合成抑制剂酮康唑,可剂量依赖性地抑制ＦＳＨ的促减数分裂恢复作用（Ｆｉｇ．５）。这些结果说明, ＦＳＨ可能诱导卵丘－卵母细胞复合体中的卵丘细胞分泌一种促减数分裂恢复物质;该物质作用于卵母细胞,诱导其恢复减数分裂而成熟;这种物质可能是一种甾醇类物质。     

The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation

The concentration of hypoxanthine in mouse follicular fluid has been estimated to be 2-4 mM, and although this concentration maintains meiotic arrest in fully grown mouse oocytes in vitro, oocyte maturation in vivo is not induced by a decrease in the concentration of this purine in follicular fluid (J. J. Eppig, P. F. Ward-Bailey, and D. L. Coleman, Biol. Reprod. 33, 1041-1049, 1985). In the present study, the effect of 2 mM hypoxanthine on oocyte growth and development in vitro was assessed and the ability of gonadotropins to stimulate oocyte maturation in the continued presence of hypoxanthine was determined. Oocyte-granulosa cell complexes were isolated from 10- to 11-day-old mice and cultured in the presence or absence of 2 mM hypoxanthine. Oocytes from 10- to 11-day-old mice are in mid-growth phase and, without further development, are incompetent of undergoing meiotic maturation. During a 12-day culture period the granulosa cell-enclosed oocytes approximately doubled in size and, regardless of the presence or absence of hypoxanthine, 50-70% developed competence to undergo germinal vesicle breakdown (GVBD). Hypoxanthine promoted the continued association of oocytes with their companion granulosa cells during the 12-day culture period, and therefore had a beneficial effect on oocyte development. Most of the oocytes that acquired GVBD competence in the absence of hypoxanthine underwent spontaneous GVBD. In contrast, 95% of the GVBD-competent oocytes were maintained in meiotic arrest by hypoxanthine. Following withdrawal of the hypoxanthine after the 12-day culture, 75% of the GVBD-competent oocytes underwent GVBD. These results show that hypoxanthine, and/or its metabolites, maintains meiotic arrest in oocytes that grow and acquire GVBD competence in vitro. Follicle-stimulating hormone (FSH), but not luteinizing hormone or human chorionic gonadotropin, induced oocyte GVBD in the continued presence of hypoxanthine. FSH stimulated oocyte maturation at a significantly (P less than 0.01) higher frequency than coculture of the granulosa cell-denuded oocytes with granulosa cells in the continued presence of hypoxanthine. FSH did not induce the maturation of denuded oocytes cocultured with granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Transforming growth factor-alpha augments meiotic maturation of cumulus cell-enclosed mouse oocytes

Growth factors have been shown to play an important role in the regulation of ovarian function. In this study, we examined the effects of transforming growth factor-alpha (TGF-alpha) on the meiotic maturation of immature mouse oocytes in vitro. Cumulus cell-enclosed oocytes were exposed to TGF-alpha with or without the meiotic inhibitor hypoxanthine (HX), and oocyte maturation was assessed by germinal vesicle breakdown (GVBD). Likewise, mechanically denuded oocytes were examined for GVBD following exposure to HX and TGF-alpha. When cumulus cell-enclosed oocytes were exposed to TGF-alpha (1 microgram/ml) in the presence of HX (4 mM), an increase in GVBD was observed first after 5 hours of culture. Maximal stimulation was reached at 24 hours when 70% of the oocytes underwent maturation in the presence of TGF-alpha and HX as compared to 33% with HX only. Concentrations of TGF-alpha as low as 0.1 ng/ml produced a similar stimulatory response after 24 hours of culture. Spontaneous maturation in the presence of TGF-alpha, but without HX, was also enhanced. The stimulation of GVBD by TGF-alpha showed an increase over time both with and without HX. When denuded oocytes were exposed to TGF-alpha in the presence of HX, no effect was observed. Our results suggest that TGF-alpha is a potent stimulator of mouse oocyte maturation in vitro and that its effect is mediated by the surrounding cumulus cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic peptide negatively regulates follicle-stimulating hormone-induced porcine oocyte maturation and cumulus expansion via cGMP-dependent protein kinase pathway

This study examined the effect of atrial natriuretic peptide (ANP) on porcine cumulus-enclosed oocyte (CEO) maturation and cumulus expansion. ANP negatively regulated follicle-stimulating hormone (FSH)-stimulated germinal vesicle breakdown (GVBD; 90.1, 81.2 and 68.2% for FSH, FSH+10nM ANP and FSH+1 microM ANP, respectively), first polar body emission (PB1; 86.1, 75.3 and 53.3% for FSH, FSH+1 nM ANP and FSH+1 microM ANP, respectively) and cumulus expansion (CEI; 3.47, 3.16 and 2.43 for FSH, FSH+1 nM ANP and FSH+1 microM ANP, respectively) in a dose-dependent manner when CEOs were cultured in the maturation medium containing porcine follicular fluid (pFF). This negative effect showed a time-dependent manner after preincubation with 100 nM ANP for 5h (78.4% PB1), 10h (81.7% GVBD and 74.1% PB1), 20 h (78.5% GVBD and 68.9% PB1), and 44 h (75.3% GVBD and 60.5% PB1), respectively. ANP also significantly inhibited FSH-induced porcine oocyte GVBD (47.6% versus 83.8%) and PB1 emission (22.4% versus 45.2%) when CEOs were cultured in pFF-free maturation medium. cGMP analog 8-Br-cGMP (10 microM to 1mM) mimicked the effects of ANP on GVBD, PB1, and CEI. The negative effect of ANP was completely reversed by KT5823 (a specific inhibitor of cGMP-dependent protein kinase), while C-ANP-(4-23) (an analogue of ANP and specific binder for natriuretic peptide receptors-C) was ineffective in oocyte maturation. Neither ANP nor C-ANP-(4-23) had an effect on spontaneous porcine oocyte maturation and cumulus expansion. These results suggested that ANP negatively regulates FSH-activated porcine oocyte meiotic resumption, meiotic maturation and cumulus expansion. The function of ANP on porcine oocyte maturation is via the cGMP dependent protein kinase (PKG) pathway.     

绵羊卵泡液和次黄嘌呤对卵母细胞体外减数分裂的影响

目的　利用在培养液中添加绵羊卵泡液和次黄嘌呤 ,抑制卵母细胞GVBD发生 ,延长转录活性 ,从而使卵母细胞真正成熟 ,提高胚胎质量及生产效率。方法　利用体外成熟技术对有屠宰采集的绵羊卵母细胞进行培养 ,培养液中添加卵泡液及次黄嘌呤 ,检查成熟效果。结果　将卵母细胞培养在 5 0 %和 10 0 %的卵泡液中 ,2 4h后处于GV期的卵母细胞分别为 19% (8 4 2 )和 33 3% (13 39)。在含有 4mmol L次黄嘌呤的培养液中 ,2 4h后有2 1 6 % (16 74 )的卵母细胞处GV期 ,而对照组中只有 6 % (3 5 0 ) ,经过次黄嘌呤处理的卵母细胞多数都停滞于PⅠ期(44 6 % ,33 74 )。在 4mmol L次黄嘌呤培养液中添加FSH并未使受到抑制的卵母细胞诱导成熟。结论　卵泡液和次黄嘌呤只能在有限的程度上抑制减数分裂的重新启动 ,并对减数分裂的全过程都有影响 ,这种影响程度与抑制因子的浓度相关 ,存在明显的剂量效应。     

Glutamine and the maintenance of meiotic arrest in mouse oocytes: influence of culture medium,glucose, and cumulus cells

The selection of culture media and supplements therein has a tremendous impact on the regulation of oocyte maturation in vitro. In the present study, we have evaluated how altering the levels of glutamine in the presence or absence of glucose affects meiotic arrest in cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) when cultured in either the simple medium M16 or the more complex Eagle's minimum essential medium (MEM). We have also tested the effectiveness of follicle-stimulating hormone (FSH) in triggering germinal vesicle breakdown (GVB) and purine de novo synthesis in differing MEM culture conditions. When DO were cultured 17-18 hr in hypoxanthine (HX)- or dbcAMP-supplemented M16 medium, neither glucose nor glutamine had any effect on oocyte maturation, with dbcAMP the more effective inhibitor. In the absence of glutamine, cumulus cells promoted meiotic resumption, since significantly lower levels of meiotic arrest were maintained in CEO than in DO by either HX or dbcAMP, but addition of the amino acid dose-dependently decreased the maturation percentage in CEO below that observed in DO. In MEM, glutamine and glucose again had little effect on the maturation of DO, although the percentage of maturing DO in HX-supplemented medium was about 20% lower than that in M16 medium. In the absence of glucose, high levels of maturation were observed in CEO in glutamine-free medium that were dose-dependently lowered by the amino acid. However, when glucose was present, CEO were as effectively arrested as DO when glutamine was absent, with no further effect of the amino acid. This inhibitory action of glucose was dependent on the essential amino acids present in MEM. The effects of glutamine were not due to changes in metabolic coupling between the oocyte and cumulus cells. Measurement of purine de novo synthesis indicated that the maintenance of meiotic arrest as well as FSH induction of meiotic resumption were associated with increases in purine synthesis. We conclude that glucose and glutamine act cooperatively to promote the synthesis of new purine compounds within the somatic compartment and that the timing and duration of such synthesis determines whether meiotic resumption will be suppressed or promoted.     

Protein kinase C and intracellular calcium are involved in follicle-stimulating hormone-mediated meiotic resumption of cumulus cell-enclosed porcine oocytes in hypoxanthine-supplemented medium

The present experiments were conducted to examine the hypothesis that follicle-stimulating hormone (FSH) can stimulate the hydrolysis of phosphoinositide, generating the intracellular second messengers to activate protein kinase C and mobilizing intracellular calcium, thus inducing oocyte meiotic resumption. Pig cumulus cell-enclosed oocytes (CEO) were cultured for 24 hr in 4 mM hypoxanthine (HX)-supplemented medium and treated with different agents in the following designs: (1) CEO were treated with neomycin (an inhibitor of phosphoinositide hydrolysis) in the presence of FSH or only treated with 7,12-dimethylbenzin(a) anthracene (DMBA, a tumor promoter which can cause phosphorylation of phospholipase C (PLC), formation of inositol triphophate, and mobilization of intracellular calcium) to mimic the direct activation of PLC; (2) CEO were challenged by FSH, together with sphingosine or staurosporine (two kinds of PKC inhibitors); or treated with phorbol myristate acetate (PMA, an activator of PKC) separately; (3) CEO were primed with BAPTA/AM (an intracellular calcium chelator) or BAPTA/AM +FSH for 60 min, and then transferred into a new culture medium supplemented with FSH but without BAPTA/AM; total culture time was 24 hr. At the end of the culture, the incidence of germinal vesicle breakdown (GVBD) was calculated. The results showed that: (1) FSH (100 U/liter) could stimulate pig CEO to override the arrest of HX and resume meiosis; DMBA (10(-8)-10(-5) M) itself also had such a kind of effect; whereas neomycin, at the level of 10-20 mM, could dramatically inhibit the stimulatory effect of FSH. (2) Staurosporine (10(-9)-10(-6) M) or sphingosine (10(-8)-10(-5) M) could also inhibit the effect of FSH in a dose-dependent manner on stimulating CEO to resume meiosis. However, PMA (10(-8)-10(-5) M) alone had a dual effect on the meiotic resumption of pig CEO. PMA, at the level of 10(-8)-10(-6) M, could stimulate CEO to resume meiosis, and at high concentration of 10(-5) M , it could even enhance the inhibitory effect of HX. (3) Priming CEO with BAPTA/AM only or BAPTA/AM +FSH for 60 min could significantly inhibit the effect of FSH in a dose-dependent manner. These results indicate that in the process of ligand-mediated meiotic resumption of pig CEO, FSH can stimulate the hydrolysis of phosphoinositide leading to the activation of PKC and mobilization of intracellular calcium; and suggest that multiple signaling pathways and signal interaction are involved in this process.     

抗真菌药物影响生殖细胞发育机理的研究

目的：探讨抗真菌药物对促性腺激素诱导的卵母细胞成熟的机理。方法：用小鼠卵母细胞体外培养模型,将小鼠卵母细胞培养在含有次黄嘌呤（ＨＸ,卵母细胞成熟抑制物）的培养注中,观察促卵泡生成素（ＦＳＨ）、两性毒素Ｂ、酮康唑对卵母细胞减数分裂恢复的影响。结果：①ＦＳＨ（１０￣２００ＩＵ／Ｌ）显著刺激卵丘－卵母细胞复合体（ＣＥＯ）克服ＨＸ的抑制而恢复减数分裂,该作用具有剂量依赖性（Ｐ〈０．０５）;②两性霉素Ｂ（０     

FSH-stimulated follicular secretions enhance oocyte maturation in pigs

Follicular secretions can support cytoplasmic maturation in vitro in the pig. The effects of follicular secretions stimulated in vitro by different combinations of gonadotropins and over different culture periods on cytoplasmic maturation of the pig oocyte were studied. In Experiment 1, follicular shells (including theca and mural granulosa cells) from 5 to 7-mm follicles were cultured in vitro under the stimulation of different combinations of gonadotropins for 48 h, and then the obtained conditioned media were used for oocyte maturation. Oocytes cultured in conditioned medium harvested after treatment of follicular shells with 2.5 mug/ml FSH (FSH-stimulated conditioned medium) yielded a higher percentage of male pronuclear formation than those matured in conditioned medium harvested after culture of follicular shells with a combination of hormones (2.5mug/ml FSH, 2.5 mug/ml LH and 20 ng/ml PRL, FSH-LH-PRL-stimulated conditioned medium; 54.1 vs 28.5%; P=0.001). Addition of the combination of FSH, LH and PRL during the period of oocyte maturation marginally improved male pronuclear formation rates (41.3 vs 55.6%; P=0.06). In Experment 2, follicular shells were cultured under the stimulation of FSH only. Conditioned media were harvested after the first 24 h and the second 24 h of culture. The rates of male pronuclear formation in oocytes matured in these 2 conditioned media did not differ (P=0.65), but were higher than those of oocytes matured in fresh control medium (P<0.03). It is concluded that factors secreted by follicular cells stimulated by FSH alone provide better support for full oocyte maturation in the pig than by combined FSH, LH and PRL treatment.     

Resumption of meiosis in pig oocytes cultured with cumulus and parietal granulosa cells: the effect of protein synthesis inhibition

In denuded and cumulus-enclosed pig oocytes, puromycin at concentrations 5, 10, and 25 micrograms/ml did not lower the rate of germinal vesicle breakdown (GVBD) after 24 h of culture. GVBD was prevented in 50, 75, and 100 micrograms/ml of puromycin. After 40 h of culture, 5 and 10 micrograms puromycin/ml impaired significantly incidence of metaphase II (42 and 30%), respectively. Concentrations of 25 and 50 micrograms puromycin/ml absolutely prevented the first polar body (I PB) expulsion. The results indicated that GVBD in pig oocytes is far less sensitive to puromycin than I PB expulsion. Culture of cumulus-enclosed pig oocytes isolated with a piece of membrana granulosa (C + P oocytes) did not allow GVBD after 24 and 32 h in control medium. After 24 h of culture, GVBD occurred in 43 and 56% of C + P oocytes in the medium supplemented with 17 and 25 micrograms puromycin/ml. GV was broken down in 80 and 68% of C + P oocytes cultured in 17 and 25 micrograms puromycin/ml for 32 h. It is concluded that inhibition of protein synthesis by puromycin released pig oocytes from the block exerted by granulosa cells.     

褪黑素对FSH诱导的小鼠卵母细胞体外成熟的影响

通过次黄嘌呤(HX)阻滞、FSH诱导体外培养模型研究了褪黑素(MT)对小鼠卵母细胞成熟的影响,探讨褪黑素(MT)是否影响小鼠卵母细胞的体外成熟。0.1mg/mL和0.02mg/mL两有效浓度的MT能显著抑制促性腺激素(FSH)诱导的HX阻滞的CEOsGVBD的发生(P<0.05),对PBl的排出虽有一定的抑制作用,但没有统计学意义;MT和次黄嘌呤(HX)对CEOs的自发成熟有协同抑制作用(P<0.01),但在裸卵(DO)自发成熟的阻滞中没有协同效应。MT是调节哺乳动物卵母细胞成熟的重要激素之一,其作用机制可能是通过卵丘细胞实现的。     

Protein kinase C modulators influence meiosis kinetics but not fertilizability of mouse oocytes.

The role of protein kinase C (PKC) in the successive steps of mouse oocyte meiotic process was investigated. We have used either OAG, an analog of diacylglycerol, or mezerein, a nonphorbol ester diterpene, less tumor promoting than phorbol esters, as PKC activators, and staurosporine as PKC inhibitor. Cumulus-free oocytes were cultured in minimum essential medium with each of these PKC modulators and maturation stages were screened every two hours until the end of the process. Both PKC activators prevented GVBD at each tested dose for 4 hr (OAG) and 8 hr (mezerein), and decreased the frequencies of PB oocytes. The inhibitory effects of both activators were dose dependent and reversible. The addition of OAG to the culture medium after GVBD occurrence (i.e., after 4 hrs) did not affect PB extrusion whereas similar addition of mezerein significantly decreased the frequency of PB oocytes. Inhibition of PKC by staurosporine accelerated GVBD and increased the frequency of PB extrusion. When staurosporine was added after GVBD, PB extrusion occurred earlier but PB oocyte frequency was not increased. Fertilizability was not affected when oocyte maturation occurred in the presence of any of these substances despite the delay in maturation process. These results clearly indicate that the PKC pathway is involved in mouse oocyte meiotic process: activation of the enzyme would arrest meiotic process whereas its inhibition would participate in meiosis induction.     

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.     

Differential response of cumulus cell-enclosed and denuded mouse oocytes in a meiotic induction model system

In this study we have examined the effects of denuded oocyte coculture with dissociated cumulus cells (CC) or intact oocyte-CC complexes on meiotic resumption. When denuded oocytes (DO) or cumulus cell-enclosed oocytes (CEO) were cultured in 40-microl drops of medium under oil, and held in meiotic arrest with 4 mM hypoxanthine plus 25 microM dbcAMP, they underwent germinal vesicle breakdown (GVB) at similar frequencies (34%-35%). Coculture of DO with complexes or dissociated CCs stimulated maturation (50% and 61% GVB, respectively), with no effect of DO on maturation of cocultured CEO (32% GVB). This coculture effect was increased with the number of CCs added to the culture drop. When either glucose or glutamine was eliminated from the medium, no meiotic induction resulted from cocultured CCs. When CEO were cultured alone in microdrops, increasing their number from 10 to 50 significantly lowered the percentage resuming maturation, an effect also reduced by removing glucose and/or glutamine from the medium. This effect was not observed with DO. When inhibitory medium was conditioned overnight with complexes, subsequent culture with DO led to higher maturation percentages than culture in unconditioned medium; however, when CEO were cultured in conditioned medium, there was either no effect or increased inhibition of maturation. Assay of glucose and pyruvate in spent medium showed that DO cultured alone consumed glucose and pyruvate, but under CC coculture conditions more glucose was consumed and significant amounts of pyruvate accumulated in the medium, changes that led to an increase in the maturation of DO. Further experiments showed that DO were more sensitive than CEO to the meiosis-inducing effect of pyruvate. These results demonstrate different responsiveness of DO and CEO to coculture conditions and question the physiological relevance of denuded oocyte/CC coculture to study meiotic induction.