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.     

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.     

AMPK regulation of mouse oocyte meiotic resumption in vitro

We have previously shown that the adenosine analog 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulates an increase in AMPK activity and induces meiotic resumption in mouse oocytes [Downs, S.M., Hudson, E.R., Hardie, D.G., 2002. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev. Biol, 245, 200-212]. The present study was carried out to better define a causative role for AMPK in oocyte meiotic maturation. When microinjected with a constitutively active AMPK, about 20% of mouse oocytes maintained in meiotic arrest with dibutyryl cAMP (dbcAMP) were stimulated to undergo germinal vesicle breakdown (GVB), while there was no effect of catalytically dead kinase. Western blot analysis revealed that germinal vesicle (GV)-stage oocytes cultured in dbcAMP-containing medium plus AICAR possessed elevated levels of active AMPK, and this was confirmed by AMPK assays using a peptide substrate of AMPK to directly measure AMPK activity. AICAR-induced meiotic resumption and AMPK activation were blocked by compound C or adenine 9-beta-d-arabinofuranoside (araA, a precursor of araATP), both inhibitors of AMPK. Compound C failed to suppress adenosine uptake and phosphorylation, indicating that it did not block AICAR action by preventing its metabolism to the AMP analog, ZMP. 2'-deoxycoformycin (DCF), a potent adenosine deaminase inhibitor, reversed the inhibitory effect of adenosine on oocyte maturation by modulating intracellular AMP levels and activating AMPK. Rosiglitazone, an anti-diabetic agent, stimulated AMPK activation in oocytes and triggered meiotic resumption. In spontaneously maturing oocytes, GVB was preceded by AMPK activation and blocked by compound C. Collectively, these results support the proposition that active AMPK within mouse oocytes provides a potent meiosis-inducing signal in vitro.     

MEK inhibitors block AICAR-induced maturation in mouse oocytes by a MAPK-independent mechanism

The present study was carried out to assess the possible role of mitogen-activated protein kinase (MAPK) in the meiosis-inducing action of the AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured 4 hr in Eagle's minimum essential medium containing dbcAMP plus increasing concentrations of AICAR or okadaic acid (OA). OA is a phosphatase inhibitor known to stimulate both meiotic maturation and MAPK activation and served as a positive control. Both OA and AICAR were potent inducers of meiotic resumption in mouse oocytes and brought about the phosphorylation (and thus, activation) of MAPK, but by different kinetics: MAPK phosphorylation preceded GVB in OA-treated oocytes, while that resulting from AICAR treatment appeared only after GVB. The MEK inhibitors, PD98059 and U0126, blocked the meiotic resumption induced by AICAR but not that induced by OA. Although the MEK inhibitors suppressed MAPK phosphorylation in both OA- and AICAR-treated oocytes, meiotic resumption was not causally linked to MAPK phosphorylation in either group. Furthermore, AICAR-induced meiotic resumption in Mos-null oocytes (which are unable to stimulate MAPK) was also abrogated by PD98059 treatment. A non-specific effect of the MEK inhibitors on AICAR accessibility to the oocyte was discounted by showing that they failed to suppress either nucleoside uptake or AICAR-stimulated phosphorylation of acetyl CoA carboxylase (ACC), a substrate of AMPK. The suppression of AICAR-induced maturation by MEK inhibitors must, therefore, be occurring by actions unrelated to MEK stimulation of MAPK; consequently, it would be prudent to consider this possible non-specific action of the inhibitors when they are used to block MAPK activation in mouse oocytes.     

Role of AMPK throughout meiotic maturation in the mouse oocyte: Evidence for promotion of polar body formation and suppression of premature activation

This study was conducted to assess the role of AMPK in regulating meiosis in mouse oocytes from the germinal vesicle stage to metaphase II. Exposure of mouse cumulus cell‐enclosed oocytes (CEO) and denuded oocytes (DO) during spontaneous maturation in vitro to AMPK‐activating agents resulted in augmentation of the rate and frequency of polar body formation. Inhibitors of AMPK had an opposite, inhibitory effect. In addition, the AMPK inhibitor, compound C (Cmpd C) increased the frequency of oocyte activation. The stimulatory action of the AMPK‐activating agent, AICAR, and the inhibitory action of Cmpd C were diminished if exposure was delayed, indicating an early action of AMPK on polar body formation. The frequency of spontaneous and Cmpd C‐induced activation in CEO was reduced as the period of hormonal priming was increased, and AMPK stimulation eliminated the activation response. Immunostaining of oocytes with antibody to active AMPK revealed an association of active kinase with chromatin, spindle poles, and midbody during maturation. Immunolocalization of the α1 catalytic subunit of AMPK showed an association with condensed chromatin and the meiotic spindle but not in the spindle poles or midbody; α2 stained only diffusely throughout the oocyte. These data suggest that AMPK is involved in a regulatory capacity throughout maturation and helps promote the completion of meiosis while suppressing premature activation. Mol. Reprod. Dev. 77:888–899, 2010. © 2010 Wiley‐Liss, Inc.     

AMP-activated protein kinase is involved in hormone-induced mouse oocyte meiotic maturation in vitro

We have previously shown that AMP-activated protein kinase (AMPK) can induce the resumption of meiosis in mouse oocytes maintained in meiotic arrest in vitro. The present study was carried out to determine whether AMPK activation is involved in hormone-induced maturation. Follicle-stimulating hormone (FSH) and the EGF-like peptide, amphiregulin (AR), are potent inducers of maturation in cumulus cell-enclosed oocytes (CEO). Within 3 h of FSH treatment, phospho-acetyl CoA carboxylase (ACC) levels were increased in germinal vesicle (GV)-stage oocytes when compared to non-stimulated controls and remained elevated throughout 9 h of culture, indicating AMPK activation. A similar response to AR was observed after 6 h of culture. Using anti-PT172 antibody (binds only to activated AMPK), Western analysis demonstrated active AMPK in both FSH- or AR-treated GV-stage oocytes within 6 h. The AMPK inhibitors, compound C and adenine 9-beta-d-arabinofuranoside (araA), blocked FSH- or AR-induced meiotic resumption and ACC phosphorylation, further supporting a causal role for AMPK in hormone-induced meiotic resumption. Immunocytochemistry using anti-PT172-AMPK antibody showed an increased diffuse cytoplasmic staining and more intense punctate staining in the germinal vesicles of oocytes following treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or with FSH or AR, and this staining was eliminated by compound C or a blocking peptide for the anti-PT172 antibody. Staining of oocytes from hCG-stimulated mice with the anti-PT172 antibody also showed pronounced label in the germinal vesicles within 1-2 h. Furthermore, in oocytes from all groups, active AMPK was always observed in association with the condensed chromosomes of maturing oocytes. Taken together, these results support a role for AMPK in FSH and AR-induced maturation in vitro and hCG-induced maturation in vivo.     

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.     

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.     

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 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.     

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.     

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。     

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.     

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)     

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.     

Maturation of the mouse oocyte-cumulus cell complex: stimulation by lectins.

We have used carbohydrate-binding proteins, or lectins, as tools to investigate the physiological phenomena associated with the preovulatory maturation of the oocyte-cumulus cell complex. Certain lectins are mitogens, and since other mitogenic agents such as growth factors are known to stimulate meiotic maturation and cumulus expansion, we tested the ability of lectins to provoke these physiological responses. Cumulus cell-enclosed oocytes (CEO) from primed mice were maintained in meiotic arrest in vitro with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) and treated with one of eleven different lectins. With the exception of pokeweed mitogen (PWM), all of the mitogenic lectins tested were able to induce germinal vesicle breakdown (GVB) in meiotically arrested oocytes, and this action required the presence of the somatic cumulus cells; in fact, either there was no effect or maturation was suppressed when cumulus cell-free oocytes (denuded oocytes; DO) were treated with lectins. None of the nonmitogenic lectins stimulated meiotic maturation in either CEO or DO. The mitogenic lectin concanavalin A (Con A) also induced maturation in CEO when meiotic arrest was maintained with hypoxanthine, guanosine, or 3-isobutyl-1-methylxanthine. The kinetics of spontaneous oocyte maturation in inhibitor-free medium were not altered by Con A. Only the mitogenic lectins that induced meiotic maturation stimulated cumulus expansion, with Con A the most active lectin. The actions of Con A on the maturation of the oocyte-cumulus cell complex were inhibited by methyl-alpha-D-mannopyranoside as predicted by its sugar-binding specificity. These results demonstrate that (1) lectins can stimulate maturation of the mouse oocyte-cumulus cell complex; (2) mitogenicity is associated with the positive activity of the lectins; and (3) cumulus cells mediate the stimulatory action of lectins on oocyte maturation, while inhibition of GVB occurs at the oocyte level. These data support the idea that common signals mediate the mitogenic and maturation-inducing actions of lectins.     

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。