Metabolic coupling and ligand-stimulated meiotic maturation in the mouse oocyte-cumulus cell complex.

Cumulus cells are metabolically coupled to the mammalian oocyte via heterologous gap junctions. One function attributed to the gap junctional communications is the transfer of regulatory signals that direct the meiotic state of the oocyte. However, the precise role of these junctions in meiotic maturation is still unclear. The aim of this study was to test the hypothesis that meiotic resumption is induced by the transfer of a stimulatory signal(s) from the cumulus cells to the oocyte through the gap junctional coupling pathway. We have previously shown that the mitogenic lectin concanavalin A (Con A) induces oocyte maturation in isolated cumulus cell-enclosed oocytes (CEO) when meiotic arrest is maintained with a number of different inhibitory agents [Biol Reprod 1990; 42:413-423]. In the present study, Con A stimulated maturation in dibutyryl cAMP (dbcAMP)-arrested CEO but not in denuded oocytes cocultured with cumulus cells. Heptanol, a known gap junction uncoupler, effectively prevented Con A- and FSH-induced maturation of intact CEO and dramatically reduced metabolic coupling between cumulus cells and the oocyte. However, this alcohol had no effect on denuded oocytes (DO) or on dbcAMP-arrested CEO in the absence of stimulating ligand. Con A and FSH produced only a minimal loss of coupling. When the effects of heptanol were compared with those of the n-alkanols hexanol and decanol, the efficacies of these agents as suppressors of Con A-stimulated oocyte maturation was directly related to their relative abilities to suppress metabolic coupling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C and meiotic regulation in isolated mouse oocytes

In this study, the possible role of protein kinase C (PKC) in mediating both positive and negative actions on meiotic maturation in isolated mouse oocytes has been examined. When cumulus cell-enclosed oocytes (CEO) were cultured for 17-18 hr in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, each of the five different activators and five different antagonists of PKC stimulated germinal vesicle breakdown (GVB) in a dose-dependent fashion. One of the activators, phorbol-12-myristate 13-acetate (PMA), also triggered GVB in CEO arrested with isobutylmethylxanthine or guanosine, but not in those arrested with dibutyryl cyclic AMP. When denuded oocytes (DO) were cultured for 3hr in inhibitor-free medium, all PKC activators suppressed maturation (<10% GVB compared to 94% in controls), while the effect of PKC antagonists was negligible. Four of the five antagonists reversed the meiosis-arresting action of HX in DO. PMA transiently arrested the spontaneous maturation of both CEO and DO, with greater potency in DO. The stimulatory action of PMA in HX-arrested oocytes was dependent on cumulus cells, because meiotic induction occurred in CEO but not DO. PKC activators also preferentially stimulated cumulus expansion when compared to antagonists. A cell-cell coupling assay determined that the action of PMA on oocyte maturation was not due to a loss of metabolic coupling between the oocyte and cumulus oophorus. Finally, Western analysis demonstrated the presence of PKCs alpha, beta1, delta, and eta in both cumulus cells and oocytes, but only PKC epsilon was detected in the cumulus cells. It is concluded that direct activation of PKC in the oocyte suppresses maturation, while stimulation within cumulus cells generates a positive trigger that leads to meiotic resumption.     

Gonadotropin-induced murine oocyte maturation in vivo is not associated with decreased cyclic adenosine monophosphate in the oocyte-cumulus cell complex

The cyclic adenosine monophosphate (cAMP) content of intact oocyte-cumulus cell complexes at various times after the induction of oocyte maturation in mice in vivo was correlated with the time of commitment by the oocytes to undergo germinal vesicle breakdown (GVB) and metabolic coupling between the oocyte and cumulus cells. Seventy-nine percent of the oocytes either underwent GVB or were committed to do so by 2 h after injection of human chorionic gonadotropin (hCG). This occurred without a decrease in the coupling between cumulus cells and the oocyte and with increasing cAMP levels in the oocyte-cumulus cell complex. Maintenance of threshold levels of cAMP within mammalian oocytes appears essential for the maintenance of meiotic arrest, but data presented here suggest that oocyte maturation in mice is induced by gonadotropins in nonatretic follicles in vivo by some mechanism other than one which decreases the cAMP content of the intact oocyte-cumulus cell complex.     

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.     

A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes

Cyclic adenosine monophosphate (cAMP) has been implicated as an important regulator of meiotic maturation in mammalian oocytes. A decrease in cAMP, brought about by the action of cAMP phosphodiesterase (PDE), is thought to initiate germinal vesicle breakdown (GVB) by the inactivation of cAMP-dependent protein kinase. However, the product of PDE activity, 5'-AMP, is a potent activator of an important regulatory enzyme, AMP-activated protein kinase (AMPK). The aim of this study was to evaluate a possible role for AMPK in meiotic induction, using oocytes obtained from eCG-primed, immature mice. Alpha-1 and -2 isoforms of the catalytic subunit of AMPK were detected in both oocytes and cumulus cells. When 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICA riboside), an activator of AMPK, was tested on denuded oocytes (DO) and cumulus cell-enclosed oocytes (CEO) maintained in meiotic arrest by dbcAMP or hypoxanthine, GVB was dose-dependently induced. Meiotic induction by AICA riboside in dbcAMP-supplemented medium was initiated within 3 h in DO and 4 h in CEO and was accompanied by increased AMPK activity in the oocyte. AICA riboside also triggered GVB when meiotic arrest was maintained with hypoxanthine, 8-AHA-cAMP, guanosine, or milrinone, but was ineffective in olomoucine- or roscovitine-arrested oocytes, indicating that it acts upstream of maturation-promoting factor. Adenosine monophosphate dose-dependently stimulated GVB in DO when meiotic arrest was maintained with dbcAMP or hypoxanthine. This effect was not mimicked by other monophosphate or adenosine nucleotides and was not affected by inhibitors of ectophosphatases. Combined treatment with adenosine and deoxycoformycin, an adenosine deaminase inhibitor, stimulated GVB in dbcAMP-arrested CEO, suggesting AMPK activation due to AMP accumulation. It is concluded that phosphodiesterase-generated AMP may serve as a transducer of the meiotic induction process through activation of AMPK.     

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.     

Mouse versus rat: Profound differences in meiotic regulation at the level of the isolated oocyte

Cumulus cell-enclosed oocytes (CEO), denuded oocytes (DO), or dissected follicles were obtained 44-48?hr after priming immature mice (20-23 days old) with 5?IU or immature rats (25-27 days old) with 12.5?IU of equine chorionic gonadotropin, and exposed to a variety of culture conditions. Mouse oocytes were more effectively maintained in meiotic arrest by hypoxanthine, dbcAMP, IBMX, milrinone, and 8-Br-cGMP. Atrial natriuretic peptide, a guanylate cyclase activator, suppressed maturation in CEO from both species, but mycophenolic acid reversed IBMX-maintained meiotic arrest in mouse CEO with little activity in rat CEO. IBMX-arrested mouse, but not rat, CEO were induced to undergo germinal vesicle breakdown (GVB) by follicle-stimulating hormone (FSH) and amphiregulin, while human chorionic gonadotropin (hCG) was ineffective in both species. Nevertheless, FSH and amphiregulin stimulated cumulus expansion in both species. FSH and hCG were both effective inducers of GVB in cultured mouse and rat follicles while amphiregulin was stimulatory only in mouse follicles. Changing the culture medium or altering macromolecular supplementation had no effect on FSH-induced maturation in rat CEO. The AMP-activated protein kinase (AMPK) activator, AICAR, was a potent stimulator of maturation in mouse CEO and DO, but only marginally stimulatory in rat CEO and ineffective in rat DO. The AMPK inhibitor, compound C, blocked meiotic induction more effectively in hCG-treated mouse follicles and heat-treated mouse CEO. Both agents produced contrasting results on polar body formation in cultured CEO in the two species. Active AMPK was detected in germinal vesicles of immature mouse, but not rat, oocytes prior to hCG-induced maturation in vivo; it colocalized with chromatin after GVB in rat and mouse oocytes, but did not appear at the spindle poles in rat oocytes as it did in mouse oocytes. Finally, cultured mouse and rat CEO displayed disparate maturation responses to energy substrate manipulation. These data highlight significant differences in meiotic regulation between the two species, and demonstrate a greater potential in mice for control at the level of the cumulus CEO.     

EGF-like peptides mediate FSH-induced maturation of cumulus cell-enclosed mouse oocytes

This study was carried out to examine the participation of epidermal growth factor (EGF)-like peptides in the induction of germinal vesicle breakdown (GVB) in mouse cumulus cell-enclosed oocytes (CEO). The EGF-like peptide, amphiregulin (AR), dose-dependently stimulated meiotic resumption in CEO, but not denuded oocytes (DO) maintained in meiotic arrest with 300 microM dbcAMP. The EGF receptor (EGFR) kinase inhibitor, AG1478, blocked meiotic resumption induced by FSH and AR in CEO, but had no effect in DO. FSH-induced maturation was also suppressed by antisera to both EGFR and EGF. Maturation occurred with slightly faster kinetics in AR-stimulated CEO when compared to FSH-stimulated CEO. When CEO were maintained in meiotic arrest with a low level of dbcAMP, FSH was initially inhibitory to maturation and later stimulatory; the stimulatory phase was prevented by AG1478, indicating mediation by EGF-like peptides. Pulsing CEO with high levels of dbcAMP also stimulated GVB and could be blocked by AG1478. Treatment of arrested CEO with PKC agonists stimulated maturation and this was prevented with AG1478 as well as antibodies to EGFR. FSH-induced maturation of dbcAMP-arrested CEO was blocked by bisindolylmaleimide I (BIM-I), an inhibitor of PKC, implicating PKC in FSH action. EGF-stimulated CEO failed to resume maturation in the presence of glycerrhetinic acid, a gap junction inhibitor, suggesting transfer of positive signal through the cell-cell coupling pathway. These data support the idea that EGF-like peptides provide a common pathway mediating the meiosis-inducing influence of FSH, cAMP pulsing, and PKC activation in mouse CEO by a gap junction-dependent process.     

Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes

This study examined the effects of three different cellular stresses on oocyte maturation in meiotically arrested mouse oocytes. Cumulus-cell enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured for 17-18 h in dbcAMP-containing medium plus increasing concentrations of the metabolic poison, sodium arsenite, or the free radical-generating agent, menadione. Alternatively, oocytes were exposed to osmotic stress by pulsing with sorbitol and returned to control inhibitory conditions for the duration of culture. Arsenite and menadione each dose-dependently induced germinal vesicle breakdown (GVB) in both DO and CEO. DO, but not CEO, pulsed for 60 min with 500 mM sorbitol were stimulated to resume maturation. The lack of effect in CEO suggests that the cumulus cells may be playing a protective role in osmotic stress-induced GVB. The AMP-activated protein kinase (PRKA; formerly known as AMPK) inhibitors, compound C and araA, completely blocked the meiosis-stimulating effects of all the tested stresses. Western blots showed that acetyl-CoA carboxylase, an important substrate of PRKA, was phosphorylated before GVB, supporting a role for PRKA in stress-induced maturation. Together, these data show that a variety of stresses stimulate GVB in meiotically arrested mouse oocytes in vitro and suggest that this effect is mediated through activation of PRKA.     

The meiotic response of cumulus cell-enclosed mouse oocytes to follicle-stimulating hormone in the presence of different macromolecules.

Experiments were carried out to determine the effect of different macromolecules on the follicle-stimulating hormone (FSH)-induced maturation of mouse oocytes in culture. Cumulus cell-enclosed oocytes (CEO) were isolated from gonadotropin-primed mice and maintained in meiotic arrest for 17-18 h with the cAMP analogue, dibutyryl cAMP (dbcAMP). Germinal vesicle breakdown (GVB) was stimulated by the addition of FSH. Medium was supplemented with either no macromolecule or with varying concentrations of polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), crystallized bovine serum albumin (BSA), or fetal bovine serum (FBS). Oocyte maturation in all FSH-free cultures occurred at a frequency of about 30% or below. High frequencies of maturation were achieved when FSH was added to macromolecule-free medium or to cultures containing PVP, PVA, or BSA. Crystallized BSA was the most effective of these in supporting stimulation of maturation (94% GVB at 3 mg/ml, compared with 72-74% with synthetic polymer-supplemented or macromolecule-free media). The BSA effect was not due to contaminating fatty acids, and a less pure fraction V BSA was not as effective in supporting FSH-induced maturation. FBS suppressed FSH stimulation of maturation in a dose-dependent fashion. Sera from pigs, goats, horses, and rats were also inhibitory, but bovine calf serum (BCS) permitted a high maturation frequency (80% GVB). When added to medium containing either FBS or BCS, crystallized BSA had no effect on FSH-stimulated maturation, but fraction V BSA suppressed maturation in both serum-supplemented media. Under no conditions did FSH stimulate maturation in cumulus cell-free oocytes. These results demonstrate that hormone-induced oocyte maturation is supported in vitro by nonprotein polymers as well as BSA and that the behavior of the oocyte-cumulus cell complex depends on the purity of the BSA sample. In addition, serum contains inhibitory factors that suppress the positive response to FSH. Thus, the choice of macromolecular supplement is of critical importance when testing the hormone responsiveness of isolated cumulus cell-enclosed oocytes in culture.     

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G-proteins.

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.     

Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes

The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors [CX, emetine (EM), and puromycin (PUR)] each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of epidermal growth factor action on maturation of the murine oocyte and cumulus oophorus in vitro

Experiments were carried out to determine the specificity of growth factor action on maturation of the oocyte-cumulus cell complex in vitro. Cumulus cell-enclosed oocytes (CEO) from primed mice were maintained in meiotic arrest in vitro with hypoxanthine (HX) and treated with one of ten different growth-promoting factors. The percentage of germinal vesicle breakdown (GVB) in the HX controls ranged from 44 to 64.7% after 21-22 h. Oocytes responded to treatment with growth-promoting factors in one of three ways: (1) no response; (2) low response; or (3) high response. The nonresponding groups included transforming growth factor-beta, platelet-derived growth factor, bombesin, sodium orthovanadate, nerve growth factor, and insulin-like growth factors I and II, each of which had no statistically significant effect on GVB. Insulin and fibroblast growth factor were members of the low response group and stimulated increases in GVB of 21.2 24.9%. Epidermal growth factor (EGF) was the only factor that produced a high frequency of maturation in the CEO; 100% of the arrested CEO were stimulated to undergo GVB in response to EGF treatment (a 51% increase over controls). No interaction was observed when EGF was tested with follicle-stimulating hormone (FSH) on hormone-induced GVB. When tested for an action on cumulus cell expansion, EGF was the only growth-promoting factor that triggered this response and did so more effectively than FSH. Heparin suppressed cumulus expansion in both EGF- and FSH-treated CEO, but did not prevent GVB stimulated by either hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

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。     

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。     

腺嘌呤对体外小鼠卵母细胞减数分裂的抑制

本文研究了嘌呤类物质对小鼠卵母细胞减数分裂的影响。于卵母细胞的生发泡内显微注射腺嘌呤和腺嘌呤的类似物苄基腺嘌呤可显著抑制卵母细胞的分裂的重新启动。同时发现在腺嘌呤的作用过程中,腺苷酸环化酶的激活剂氟化钠可增强其对卵母细胞的抑制作用,表明cAMP途径在小鼠卵母细胞减数分裂成熟过程中起重要作用。腺嘌呤在不同培养液中的抑制效果不一,次黄嘌呤在DMEM和EMEM中对小鼠的卵丘细胞-卵母细胞复合体(COC)和无卵丘细胞的裸卵(DO)均具有明显的抑制效应。但腺嘌呤在DMEM比在EMEM中对COC的抑制效果更强,而且腺嘌呤在DMEM中与次黄嘌呤具有协同效应,这些差别可能是由于两种培养液中不同成分如谷氨酰胺造成卵母细胞对腺嘌呤吸收差异而引起的。     

Maintenance of murine oocyte meiotic arrest: uptake and metabolism of hypoxanthine and adenosine by cumulus cell-enclosed and denuded oocytes

To analyze the potential mechanisms by which hypoxanthine and adenosine maintain meiotic arrest in mouse oocytes this study focused on: the uptake and metabolism of hypoxanthine and adenosine; the effect of inhibitors of inosine monophosphate (IMP) dehydrogenase on purine-mediated meiotic arrest; and the role of adenosine metabolism on the maintenance of meiotic arrest. Although the denuded oocyte can take up radiolabeled hypoxanthine and adenosine, an intact cumulus oophorus greatly augments uptake of these molecules (and/or metabolites). Both of these compounds were completely metabolized during incubation in vitro: hypoxanthine was apparently metabolized to uric acid and adenosine was metabolized to ADP; a small amount of each compound was also converted to inosine by cumulus cells and transferred to the oocyte. The IMP dehydrogenase inhibitors, bredinin and mycophenolic acid (MA), induced, in a dose-dependent manner, the resumption of maturation in cumulus cell-enclosed oocytes maintained in meiotic arrest by hypoxanthine but had no effect on denuded oocytes. MA did not induce maturation when meiotic arrest was maintained by guanosine. Nor did MA alter the uptake of hypoxanthine by cumulus cell-enclosed oocytes. The poorly metabolized analog of adenosine, 2-chloroadenosine, was as effective as adenosine in its synergistic action with hypoxanthine in maintaining meiotic arrest. It is concluded that hypoxanthine and adenosine are metabolized within the oocyte-cumulus cell complex; xanthyl and/or guanyl compounds are produced by oocyte-cumulus cell complexes in the presence of hypoxanthine and play an important role in the maintenance of meiotic arrest; and adenosine need not be metabolized to act synergistically with hypoxanthine in maintaining meiotic arrest.     

A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes

This study was carried out to compare the possible role of a secreted paracrine factor versus that of a gap-junction-transmitted signal in mediating meiotic induction in isolated mouse oocytes from PMSG-primed, immature mice. In the first set of experiments, oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 2 mM dbcAMP or FSH, washed, and the oocytes then cultured for 17-18 h in 40 microl drops containing either 300 microM dbcAMP or 4 mM hypoxanthine (HX). Each set of pretreated oocytes was cultured under three different conditions: (1) intact cumulus-cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured alone after removal of cumulus cells; and (3) co-cultured cumulus cells and oocytes (CC/DO), where the cumulus cells were removed in the same drop with a mouth-operated pipette and cultured alongside the oocytes. When pretreated with high dbcAMP or FSH, maturation was stimulated in CEO when cultured in either inhibitor (by 41.4-53.7%). Pretreatment failed to affect the maturation rate in DO. DO maturation was not altered appreciably by co-cultured cumulus cells when arrest was maintained with dbcAMP. However, an increase in maturation of 21-23% was observed in CC/DO in the HX-containing cultures that was not dependent on prior treatment with a meiosis-inducing stimulus. When DO were co-cultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the stimulated complexes, despite the fact that oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18alpha-glycyrrhetinic acid (GA), was utilised. An initial experiment determined that GA dose-dependently blocked OCC metabolic coupling (0.2% coupling at 10 microM compared with 13.6% in controls). When HX-arrested CEO and DO were cultured for 17-18 h in medium containing increasing concentrations of GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a positive stimulus in the absence of functional gap junctional communication. No effect of GA was seen in dbcAMP-arrested oocytes. A kinetics experiment showed that when CEO were cultured in dbcAMP +/- FSH, meiotic induction was initiated after 3 h and germinal vesicle breakdown reached 60% by 6 h. When GA was added to the cultures at different times after the initiation of culture (0, 2, 3, 4 and 5 h), meiotic induction was immediately blocked. In addition, measurement of OCC coupling revealed that no reduction in coupling occurred during this induction period in the absence of GA. It is concluded that cumulus cells can secrete a positive factor, but that this is normally overridden by inhibitory influences transmitted through the gap junction pathway in intact complexes. Furthermore, upon exposure of complexes to a meiosis-inducing stimulus, a positive gap-junction-mediated signal now predominates to trigger germinal vesicle breakdown, and this signal is utilised throughout the induction period.     

Induction of meiotic maturation in mouse oocytes by adenosine analogs

In this study we have examined the meiosis-inducing influence of adenosine analogs in mouse oocytes. When a varied group of nucleosides and nucleotides were tested on overnight cultures of hypoxanthine-arrested, cumulus cell-enclosed oocytes (CEO), halogenated adenosine nucleosides, but not native adenosine, exhibited a significant meiosis-inducing capability. When tested under a variety of conditions, meiotic induction by 8-bromo-adenosine (8-Br-Ado) and a second adenosine analog, methylmercaptopurine riboside (MMPR), was especially potent in denuded oocytes (DO) compared to CEO and was not dependent on the type of inhibitor chosen to maintain meiotic arrest. Germinal vesicle breakdown (GVB) was stimulated with rapid kinetics and was preceded by an increase in AMP-activated protein kinase (AMPK) activity. Moreover, compound C, an inhibitor of AMPK, blocked the meiosis-inducing activities of both adenosine analogs. When tested for an effect on meiotic progression to metaphase II (MII) in spontaneously maturing CEO, 8-Br-Ado and the AMPK activator, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), increased the percentage of MII-stage oocytes, but MMPR decreased this number. Adenosine and inhibitors of de novo purine synthesis had no effect on the completion of maturation, while compound C suppressed this process. These results support the proposition that oocyte AMPK mediates the positive influence of AICAR and 8-Br-Ado on both the initiation and completion of meiotic maturation. The role of AMPK in MMPR action is less clear.     

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.