Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating substance when stimulated with FSH

The effect of the different follicular cell types on resumption of meiosis was studied during stimulation with FSH. Cumulus enclosed oocytes (CEO), denuded oocytes (DO), and cumulus and mural granulosa cells were used. The resumption of meiosis and oocyte maturation were assessed by the determination of the germinal vesicle breakdown (GVBD) and polar body formation (PB) at the end of a 24 hr culture period in the presence of 4 mM hypoxanthine (HX). The effects of recombinant LH (r-LH) and hCG were also evaluated. Oocyte exposure to the gonadotrophins varied from 5 min to 24 hr (i.e., priming time). Oocytes were obtained from immature gonadotrophin-stimulated and -unstimulated mice. 1. FSH (1 IU/L-75 IU/L) provoked a dose-dependent increase in GVBD and PB in CEO, but not in DO, in stimulated and unstimulated mice. Eight IU/L was sufficient for inducing resumption of meiosis. In contrast, LH and hCG (both 1 IU/L-1500 IU/L) were without effect on GVBD and PB in CEO and DO of oocytes from stimulated and unstimulated mice. A combination of 8IU/L FSH and 4–8 IU/L hCG produced an additive effect, whereas combinations with LH and higher concentrations of hCG had no such effect. 2. A 2 hr priming with FSH (8 IU/L-75 IU/L) induced a dose-dependent oocyte maturation in CEO. Thirty minutes of priming with FSH (75 IU/L) was sufficient for induction of meiotic resumption in CEO. 3. Priming CEO with FSH for 2 hr followed by the separation and repooling of oocytes and cumulus cells induced oocyte maturation. GVBD of new, unprimed DO added to cumulus cells of primed CEO increased slightly but was significant, whereas GVBD in DO isolated from the primed CEO only increased marginally. DO cocultured with FSH-primed cumulus masses seem to be prevented from resuming meiosis. 4. Priming a coculture of granulosa cells and DO with FSH for 2 hr caused a significant increase in GVBD compared to the control, evaluated after 24 hr. In contrast, a 24 hr FSH-priming of a coculture of granulosa cells and DO was without effect on GVBD. 5. A spent medium in which unstimulated cumulus cells or mural granulosa cells had grown was without effect on GVBD in DO. However, a small fraction of the DO resumed meiosis after culture in a spent medium derived from a 2 hr priming of CEO and spent media from 24 hr priming of CEO induced a 2–3 times higher GVBD frequency in the DO compared to the controls. Heat treatment of spent media (70°C, 30 min) from a 24 hr FSH-priming of CEO still induced GVBD in naive DO. The results showed that FSH, in a concentration of as little as 8 IU/L, but not r-LH and hCG, induced within 30 minutes the cumulus cells to produce and after 2 hr to secrete a diffusible heat stable meiosis activating substance. This substance overcame, in a paracrine fashion, the inhibiting effect of HX and induced oocyte maturation directly in DO. The production of this substance, however, was dependent on the initial connection between the cumulus cells and the oocyte, indicating an important 2-way communication between these 2 cell types. The mural granulosa cells did not produce a meiosis inducing activity by stimulation with FSH, but significantly, more DO matured after coculture with the nonstimulated granulosa cells for 24 hr than for 2 hr. It is proposed that the heat stable meiosis activating component of the spent media from the FSH-stimulated CEO belongs to the meiosis activating sterols, MAS, previously isolated from human follicular fluid and from adult bull testes. Mol. Reprod. Dev. 46:296–305, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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。     

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     

Cumulus Cells Block Oocyte Meiotic Resumption via Gap Junctions in Cumulus Oocyte Complexes Subjected to DNA Double-Strand Breaks

During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.     

Stimulation and inhibition of rat oocyte meiosis by forskolin

The adenylate cyclase activator forskolin was used to study the role of cAMP for oocyte meiosis and follicular steroid secretion. Follicular and cumulus cAMP production was stimulated dose-dependently by forskolin, as was the follicular secretion of progesterone, testosterone and estradiol. Forskolin induced meiosis in follicle-enclosed oocytes with a maximal effect at 1 microM, with lower and higher concentrations being less effective. The spontaneous resumption of meiosis in isolated cumulus-enclosed oocytes was dose-dependently retarded by forskolin. Meiosis of cumulus-free oocytes was also retarded but only slightly. These data support the earlier hypothesis that a limited increase in follicular cAMP levels triggers meiosis, whereas sustained levels of cAMP in the oocyte itself prevent meiosis.     

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。     

Reducing CYP51 inhibits follicle-stimulating hormone induced resumption of mouse oocyte meiosis in vitro

Meiosis activating sterol, produced directly by lanosterol 14-α-demethylase (CYP51) during cholesterol biosynthesis, has been shown to promote the initiation of oocyte meiosis. However, the physiological significance of CYP51 action on oocyte meiosis in response to gonadotrophins’ induction remained to be further explored. Herein, we analyzed the role of CYP51 in gonadotrophin-induced in vitro oocyte maturation via RNA interference (RNAi). We showed that although both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) significantly induced meiotic resumption in follicle-enclosed oocytes (FEOs), the effect of LH on oocyte meiosis resumption in FEOs was weaker than FSH. Moreover, both FSH and LH were able to upregulate CYP51 expression in cultured follicular granulosa cells when examined at 8 h or 12 h posttreatments, respectively. Interestingly, whereas knockdown of CYP51 expression via small interference RNA (siRNA) moderately blocked (23% reduction at 24 h) FSH-induced oocyte maturation [43% germinal vesicle breakdown (GVBD) rate in RNAi vs. 66% in control, P < 0.05] in FEOs, similar treatments showed no apparent effects on LH-induced FEO meiotic maturation (58% GVBD rate in RNAi vs. 63% in control, P > 0.05). Moreover, the results in a cumulus-enclosed oocytes (CEOs) model showed that approximately 30% of FSH-induced CEOs’ meiotic resumption was blocked upon CYP51 knockdown by siRNAs. These findings suggest that FSH, partially at least, employs CYP51, and therefore the MAS pathway, to initiate oocyte meiosis.     

Meiosis activating sterols (MAS) and fertility in mammals and man.

In mammals two meiosis activating sterols (MAS) have been found to activate meiotic resumption in mouse oocytes, in vitro. FF-MAS (4, 4-dimethyl-5alpha-cholesta-8,14,24-triene-3beta-ol) was extracted from human preovulatory follicular fluid and T-MAS (4, 4-dimethyl-5alpha-cholest-8,24-diene-3beta-ol) from bull testicular tissue. Quite unexpected, these two sterols, which introduce the cholesterol biosynthetic pathway from lanosterol, may be locally acting substances with important physiological function for reproduction. FF-MAS and T-MAS are present in the preovulatory follicular fluid of different mammalian species and have the capacity to initiate resumption of meiosis in mouse oocyte cultured in the presence of hypoxanthine, a natural meiosis maturation inhibitor. FF-MAS is produced by the cumulus cells of intact oocyte-cumulus complexes upon FSH-stimulation and provides the oocyte with a go-signal for the resumption of meiosis. T-MAS constitutes the vast majority of MAS found in the mammalian testis and in the human ejaculate; in particular a high concentration is found in the spermatozoa. T-MAS may be produced by the spermatids and the presence of T-MAS in spermatozoa may suggest that T-MAS plays a role in fertilization by affecting the second meiotic division.J. Exp. Zool. (Mol. Dev. Evol.) 285:237-242, 1999.     

Fatty acid oxidation and meiotic resumption in mouse oocytes

We have examined the potential role of fatty acid oxidation (FAO) in AMP‐activated protein kinase (AMPK)‐induced meiotic maturation. Etomoxir and malonyl CoA, two inhibitors of carnitine palmitoyl transferase‐1 (CPT1), and thus FAO, blocked meiotic induction in dbcAMP‐arrested cumulus cell‐enclosed oocytes (CEO) and denuded oocytes (DO) by the AMPK activator, AICAR. C75, an activator of CPT1 and FAO, stimulated meiotic resumption in CEO and DO. This effect was insensitive to the AMPK inhibitor, compound C, indicating an action downstream of AMPK. Palmitic acid or carnitine also promoted meiotic resumption in DO in the presence of AICAR. Since C75 also suppresses the activity of fatty acid synthase (FAS), we tested another FAS inhibitor, cerulenin. Cerulenin stimulated maturation in arrested oocytes, but to a lesser extent, exhibited significantly slower kinetics and was effective in CEO but not DO. Moreover, etomoxir completely blocked C75‐induced maturation but was ineffective in cerulenin‐treated oocytes, suggesting that the meiosis‐inducing action of C75 is through activation of FAO within the oocyte, while that of cerulenin is independent of FAO and acts within the cumulus cells. Finally, we determined that long chain, but not short chain, fatty acyl carnitine derivatives were stimulatory to oocyte maturation. Palmitoyl carnitine stimulated maturation in both CEO and DO, with rapid kinetics in DO; this effect was blocked by mercaptoacetate, a downstream inhibitor of FAO. These results indicate that activation of AMPK stimulates meiotic resumption in mouse oocytes by eliminating a block to FAO. Mol. Reprod. Dev. 76: 844–853, 2009. © 2009 Wiley‐Liss, Inc.     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.     

Progesterone and 17 alpha-OH-progesterone in concentrations similar to that of preovulatory follicular fluid is without effect on resumption of meiosis in mouse cumulus enclosed oocytes cultured in the presence of hypoxanthine

Some intermediates in the cholesterol biosynthesis between lanosterol and cholesterol are capable of inducing resumption of meiosis in cultured mouse oocytes without the presence of gonadotropins. The mechanism by which these so-called Meiosis Activating Sterols (MAS) activate the meiotic process is unknown, and it is uncertain whether they participate in the physiological control of resumption of meiosis. Recently, it has been shown that accumulation of MAS occurs in a liver cell line and in rat testis tissue cultured in the presence of micromolar concentrations of progesterone and 17 alpha-OH-progesterone. Such high concentrations of progesterone and 17 alpha-OH-progesterone only occur in fluid of preovulatory follicles. In connection with the mid-cycle surge of gonadotropins, this may represent one mechanism whereby follicular accumulation of MAS takes place. In the present study, the effect of 10 micro M progesterone and 10 micro M 17 alpha-OH-progesterone on resumption of meiosis was evaluated using mouse cumulus enclosed oocytes (CEO) cultured in the presence of 4mM hypoxanthine. By the end of the 24-h culture period, the frequency by which oocytes had resumed meiosis was assessed by the determination of germinal vesicle breakdown (GVBD). Neither progesterone nor 17 alpha-OH-progesterone or a combination showed any effect on GVBD. In addition, progesterone and 17 alpha-OH-progesterone in combination with a sub-optimal dose of FSH (4 IU/l) did not affect GVBD. In conclusion, accumulation of MAS to an extent that allows resumption of meiosis to occur in CEO is unlikely to be induced by progesterone and 17 alpha-OH-progesterone or a combination.     

Towards a new understanding on the regulation of mammalian oocyte meiosis resumption

Mammalian oocytes reach prophase of first meiosis around the time of birth, and remain at this stage for months or years, depending on the species. Only after puberty will the fully-grown oocytes begin to resume meiosis which is stimulated by gonadotropin surge. It has long been known that a high level of intra-oocyte cyclic adenosine 3',5'-monophosphate (cAMP) prevents oocyte meiosis resumption as indicated by germinal vesicle breakdown (GVBD). Recently, guanosine triphosphate-binding (G) protein-coupled receptors/G proteins/adenyl cyclase pathway endogenous to the oocyte as well as cAMP diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that prevent oocytes from resuming meiosis. Another second messager molecule, guanosine 3',5'-cyclic monophosphate (cGMP), has also recently been found to play important roles in maintaining oocyte meiosis arrest. cGMP in the follicular somatic cells diffuses into the oocyte and causes an increase in oocyte cAMP, presumably by acting on phosphodiesterase 3 (PDE3). The cGMP level in the somatic compartment of the follicle decreases in response to luteinizing hormone (LH), and this change may be mediated through the epidermal growth factor (EGF)-like factors and specific cGMP-phosphodiesterase subtype activity. It is well known that gonadotropic stimulation of meiotic resumption depends on mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle; recent studies show that LH, through cAMP/protein kinase A (PKA) and protein kinase C (PKC) pathways, induces the synthesis of paracine factors such as EGF-like facors and meiosis activating sterol (MAS) to regulate oocyte GVBD via the MAPK pathway in follicle cells. A recent granulosa cell-specific knockout study has for the first time provided in vivo evidence for the important role of extracellular regulated kinase 1 and 2 (ERK1/2), two main forms of MAPK, and their downstream molecules in granulosa cells in oocyte meiosis resumption. Unresolved questions and future directions on research regarding signaling changes in follicle cells and oocytes as well their communication in response to the gonadotropin surge are addressed in this review.     

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.     

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.     

Influence of follicular components on oocyte meiosis in vitro

Oocytes and follicular components obtained from ovaries recovered from mature Hereford cows at slaughter were used to determine follicular influence on oocyte maturation. Some oocytes were fixed immediately to determine the stage of maturation. The remaining oocytes were cultured for 32 to 34 hr in various environments to determine the influences of the granulosum and follicular fluids on meiotic changes. All noncultured oocytes had dictyate nuclei except one in premetaphase. Oocytes cultured in 50 or 100% follicular fluid or in contact with stratum granulosum cells showed some meiotic inhibition both before and after germinal vesicle breakdown (GVB). The least resumption of meiosis occurred in oocytes cultured in their intact follicles.     

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。     

Fibronectin in reproduction

The fibronectin (FN) levels in human follicular fluids have been shown to correlate well with follicular size and oocyte maturity, suggesting a role of FN in oocyte maturation. When added to the culture medium, the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS), which specifically inhibits the cell-binding of FN, has been shown to inhibit both spontaneous resumption of meiosis and gonadotropin-releasing hormone-induced meiosis of the oocytes. In another set of experiments, GRGDS has been found to inhibit the in vitro cleavage of mouse embryos by a still unknown mechanism.