Cumulus Cell-enclosed Oocytes Research Articles

Effects of Paclitaxel Before Vitrification on the Nuclear Maturation and Development of Immature Porcine Oocytes

BACKGROUND: Cryopreservation of porcine oocytes is difficult compared with other species and immature oocytes particularly so compared to the meiotic stage. OBJECTIVE: To evaluate the efficacy of a pretreatment with 1μ M paclitaxel (PTX, 30 min exposure) before vitrification to promote the maturation of porcine immature oocytes. MATERIALS AND METHODS: Cumulus cell-enclosed oocytes (COs) aspirated from porcine ovaries were divided into three groups: i) nonpretreated with PTX and non-vitrified group (control group); ii) pretreated with PTX and vitrified group (PTX-V group); and iii) non-pretreated with PTX and vitrified group (nPTX-V group). RESULTS: The nuclear maturation rate up to the preovulatory stage was significantly lower (P<0.05) in the nPTX-V group than in the control group, but was similar in the PTX-V and control groups. No significant differences were observed in viability assessed by a normal CO morphology and the embryonic development of oocytes activated by the parthenogenetic stimulation between the PTX-V and control groups, but not the non-PTX-V group. CONCLUSION: PTX may promote the maturation of vitrified porcine immature oocytes.

L-Serine transport in growing and maturing mouse oocytes.

Serine has roles in cell metabolism besides protein synthesis including providing one-carbon units to the folate cycle. Since growing mouse oocytes undergo a burst of folate accumulation as they near full size, we have investigated whether oocytes transport serine. Substantial serine transport appeared in oocytes near the end of their growth. Serine transport continued when oocytes resumed meiosis but ceased partway through first meiotic metaphase, remaining quiescent in mature eggs in second meiotic metaphase. The serine transporter was sodium dependent and inhibited by alanine, cysteine, leucine, or histidine, and had a Michaelis-Menten constant (Km ) for serine of 200 µM. Unexpectedly, exposing cumulus cell-enclosed oocytes to the physiological mediator of meiotic arrest, natriuretic peptide precursor Type C, substantially stimulated serine transport by the enclosed oocyte. Finally, in addition to transport by the oocyte itself, cumulus cells also supply serine to the enclosed oocyte via gap junctions within intact cumulus-oocyte complexes.

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.

Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes.

In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO.

A Requirement for Fatty Acid Oxidation in the Hormone-Induced Meiotic Maturation of Mouse Oocytes1

We have previously shown that fatty acid oxidation (FAO) is required for AMP-activated protein kinase (PRKA)-induced maturation in vitro. In the present study, we have further investigated the role of this metabolic pathway in hormone-induced meiotic maturation. Incorporating an assay with (3)H-palmitic acid as the substrate, we first examined the effect of PRKA activators on FAO levels. There was a significant stimulation of FAO in cumulus cell-enclosed oocytes (CEO) treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and RSVA405. In denuded oocytes (DO), AICAR stimulated FAO only in the presence of carnitine, the molecule that facilitates fatty acyl CoA entry into the mitochondria. The carnitine palmitoyltransferase 1 activator C75 successfully stimulated FAO in CEO. All three of these activators trigger germinal vesicle breakdown. Meiotic resumption induced by follicle-stimulating hormone (FSH) or amphiregulin was completely inhibited by the FAO inhibitors etomoxir, mercaptoacetate, and malonyl CoA. Importantly, FAO was increased in CEO stimulated by FSH and epidermal growth factor, and this increase was blocked by FAO inhibitors. Moreover, compound C, a PRKA inhibitor, prevented the FSH-induced increase in FAO. Both carnitine and palmitic acid augmented hormonal induction of maturation. In a more physiological setting, etomoxir eliminated human chorionic gonadotropin (hCG)-induced maturation in follicle-enclosed oocytes. In addition, CEO and DO from hCG-treated mice displayed an etomoxir-sensitive increase in FAO, indicating that this pathway was stimulated during in vivo meiotic resumption. Taken together, our data indicate that hormone-induced maturation in mice requires a PRKA-dependent increase in FAO.

Perturbing microtubule integrity blocks AMP-activated protein kinase-induced meiotic resumption in cultured mouse oocytes

The oocyte meiotic spindle is comprised of microtubules (MT) that bind chromatin and regulate both metaphase plate formation and karyokinesis during meiotic maturation; however, little information is known about their role in meiosis reinitiation. This study was conducted to determine if microtubule integrity is required for meiotic induction and to ascertain how it affects activation of AMP-activated protein kinase (AMPK), an important participant in the meiotic induction process. Treatment with microtubule-disrupting agents nocodazole and vinblastine suppressed meiotic resumption in a dose-dependent manner in both arrested cumulus cell-enclosed oocytes (CEO) stimulated with follicle-stimulating hormone (FSH) and arrested denuded oocytes (DO) stimulated with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR). This effect coincided with suppression of AMPK activation as determined by western blotting and germinal vesicle immunostaining. Treatment with the MT stabilizer paclitaxel also suppressed meiotic induction. Targeting actin filament polymerization had only a marginal effect on meiotic induction. Immunolocalization experiments revealed that active AMPK colocalized with γ-tubulin during metaphase I and II stages, while it localized at the spindle midzone during anaphase. This discrete localization pattern was dependent on MT integrity. Treatment with nocodazole led to disruption of proper spindle pole localization of active AMPK, while paclitaxel induced excessive polymerization of spindle MT and formation of ectopic asters with accentuated AMPK colocalization. Although stimulation of AMPK increased the rate of germinal vesicle breakdown (GVB), spindle formation and polar body (PB) extrusion, the kinase had no effect on peripheral movement of the spindle. These data suggest that the meiosis-inducing action and localization of AMPK are regulated by MT spindle integrity during mouse oocyte maturation.

Fatty Acid Oxidation and Hormone-Induced Meiotic Resumption in Mouse Oocytes.

Previous results from our lab showed that fatty acid oxidation (FAO) is required for the pharmacological induction of meiotic maturation in mouse oocytes in vitro. The results indicated that treatment of meiotically arrested oocytes with AICAR, an activator of AMP-activated protein kinase (AMPK), led to germinal vesicle breakdown (GVB) by stimulating FAO. Since AMPK mediates the meiosis-inducing action of hormones, the present study was carried out to examine the role of FAO in hormonal induction of meiotic resumption. For all experiments, 3-week-old C57BL/6J X SJL/J F1 mice were primed with 5 IU PMSG and for most experiments cumulus-cell-enclosed oocytes (CEO) were harvested two days later for experimentation. The culture medium used for all experiments was MEM supplemented with BSA and pyruvate. CEO kept in meiotic arrest with dibutyryl cyclic AMP (dbcAMP) were stimulated to mature in vitro by FSH or Amphiregulin and exposed to increasing concentrations of Etomoxir, Mercaptoacetate or Malonyl CoA, all inhibitors of FAO. Whereas Etomoxir and Mercaptoacetate are pharmacological inhibitors, Malonyl CoA is a physiological inhibitor produced from acetyl CoA by the enzyme acetyl CoA carboxylase. In all cases, these inhibitors significantly blocked the stimulatory effect of FSH and Amphiregulin on meiotic resumption, in many cases, to control levels. FAO inhibitors also blocked GVB in cultured follicles stimulated with hCG. On the other hand, addition of the fatty acid palmitate increased the percentage of maturation in FSH-treated CEO. To further examine this relationship, we have developed a hanging drop assay for FAO that measures the production of 14CO2 from 14C-palmitate during 4.5 h culture. Initial results show activity in spontaneously maturing CEO that is significantly augmented by hormonal stimulation. Taken together, our data indicate that fatty acid oxidation is a vital component of meiotic maturation induced by physiological signals.

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.

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.

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

Meiosis activating sterol, produced directly by lanosterol 14-alpha-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.

Effect of Cysteamine Added to Maturation Medium on Nuclear Maturation, Glutathione and ATP Contents of Pig Oocytes

Abstract: The effect of cysteamine added to maturation medium on nuclear maturation, ATP content and glutathione (GSH) content of porcine oocytes was investigated. The nuclear maturation up to the metaphase stage of second meiotic division (the M-II stage) of cumulus cell-enclosed oocytes (COs) matured with various concentrations (0, 15, 45, 150, and 450 µM) of cysteamine was observed. The maturation rate of the 150 µM group was significantly higher (P < 0.05) than that of the control (0 µM) group. Although the ATP content of oocytes derived from COs matured with 150 µM cysteamine was the same as that of the immature group, the ATP content of COs matured without cysteamine was significantly higher (P < 0.05) than that of the immature group. Conversely, the GSH content of oocytes derived from COs matured with 150 µM cysteamine was significantly higher (P < 0.05) than that of the immature group. However, the content of the group matured without cysteamine was not significantly different from that of the imm...

Effect of β-hydroxybutyrate Added to Maturation Medium on Nuclear Maturation of Pig Oocytes

Abstract: The effects of β-hydroxybutyrate (BHB) added to a modified NCSU37 medium during in vitro maturation (IVM) on nuclear maturation, and ATP and glutathione (GSH) contents of porcine oocytes were investigated. Nuclear maturation up to the metaphase stage of the second meiotic division (the M-II stage) of cumulus cell-enclosed oocytes (COs) matured with various concentrations (0.0, 2.3, 3.1, 3.9 and 4.7 mM) of BHB was observed. The nuclear maturation rate of the oocytes of the 3.1 mM group was significantly higher (P < 0.05) than that of the control (0.0 mM) group. Both the ATP and GSH contents of the oocytes matured with or without 3.1 mM BHB and removed from cumulus cells after IVM were not significantly different. These results indicate that 3.1 mM BHB added to the maturation medium may stimulate the nuclear maturation of porcine oocytes without changing their ATP and GSH contents.

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.

The Effect of the Addition and Removal of Various Cryoprotectants on the Nuclear Maturation and ATP Content of Immature Porcine Oocytes

This study was undertaken to investigate the influence of exposure and removal of four different cryoprotectants (CPAs) on the ATP content of cumulus cell-enclosed (COs) and cumulus cell-denuded (DOs) immature porcine oocytes. The in vitro nuclear maturation of the COs, exposed to and removed from the CPAs was also assessed. Both COs and DOs were exposed to 1.5 M concentrations of each CPA (ethylene glycol (EG); propylene glycol (PG); dimethyl-sulfoxide (DMSO); and glycerol (G)) for durations of 5, 15, and 30 minutes at room temperature (23.5±1.5°C), and immersed in physiological saline supplemented with 20% (v/v) fetal bovine serum for 5 minutes (39°C) to remove each CPA. Before, during and after exposure to each CPA, the ATP content of both the COs and the DOs was measured. After removal from each CPA an aliquot of the COs was matured for 44±2 h, and their nuclear maturation rates were measured up to the metaphase stage of the second meiotic division (the M-II stage). The maturation rates up to the M-II stage were not significantly different between all the groups that were exposed to each CPA for 5 minutes. For 15 and 30 minute exposures, the maturation rates of the COs exposed to PG, DMSO and EG were almost the same as those of the control groups; however, the rates of G group exposed for 15 and 30 minutes were significantly lower (p<0.05) than the control group. These groups were also found to have a decrease in the ATP content of COs and DOs during and after exposure for the same periods (p<0.05). In addition, although the ATP contents of the COs after exposure to EG for any period were the same as the controls, the ATP content of the DOs exposed to EG for any period were significantly lower (p<0.05) than those of the controls. When the ATP content of the COs and DOs of each CPA were compared, the DOs exposed to PG were found to have a significantly greater level (p<0.05) than DOs exposed to G for any duration. In addition, the ATP content of DOs exposed to PG for 30 min and removal was also higher (p<0.05) than when exposed to DMSO for the same period. These findings indicate that PG may be a useful CPA for the cryopreservation of immature porcine oocytes.

Decreased Oocyte-Granulosa Cell Gap Junction Communication and Connexin Expression in a Type 1 Diabetic Mouse Model

In women, type 1 diabetes is associated with an increased risk of poor prenatal outcomes such as congenital anomalies and early miscarriage. In murine models of type 1 diabetes, impaired oocyte meiotic maturation, abnormal oocyte metabolism, and increased granulosa cell apoptosis have been noted. because gap junction communication is critical for the regulation of oocyte growth and meiotic maturation, we investigated the level of communication between the oocyte and surrounding cumulus cells in a streptozotocin-induced type 1 diabetic B6SJL/F1 mouse model and the expression of gap junction proteins known as connexins. Fluorescence recovery after photobleaching analyses of cumulus cell-enclosed oocytes (CEOs) from diabetic mice showed a 60% decrease in communication as compared with CEOs from nondiabetic mice. Real-time RT-PCR analyses confirmed the presence of Cx26, Cx37, and Cx57 mRNA and revealed a significant decrease in Cx37 mRNA expression in oocytes from diabetic mice compared with nondiabetic mice. Western analyses detected Cx26 expression in CEO but not denuded oocyte (DO) samples, and Cx37 in DO samples. Cx26 protein levels were decreased by 78% in CEOs from diabetic mice, and Cx37 protein levels were decreased 36% in DOs from diabetic mice. This decrease in connexin expression and gap junction communication in CEOs from diabetic mice may be responsible for the impaired oocyte meiotic maturation and poor pregnancy outcomes.

Effects of cumulus cells on rabbit oocyte in vitro maturation

Cumulus cells (CCs) are of great importance in oocyte development and maturation in many species, but detailed influence of CCs has not been extensively examined, especially on rabbit. The present study was designed to investigate the effects of CCs and the elongation of in vitro maturation (IVM) time on rabbit oocyte nuclear and ooplasmic maturation and survival. Cumulus oocyte complexes (COCs) and naked oocytes (NOs) were recovered directly from rabbits super-ovulated with eCG. Corona-enclosed oocytes (COs) and denuded oocytes (DOs) were obtained from COCs after removing a part or whole of CCs. The oocytes were cultured in the following seven groups. (i) Cumulus cell enclosed oocytes (CEOs) were cultured alone (CEOs); (ii) COs were cultured alone (COs); (iii) DOs were cultured alone (DOs); (iv) NOs were cultured alone; (v) DOs were co-cultured with COCs [DOs(COCs)]; (vi) DOs were co-cultured with CCs [DOs(CCs)]; (vii) NOs were co-cultured with CCs [NOs(CCs)]. After the oocytes were cultured for 24 and 30 h, the nuclear maturation was evaluated by first polar body (PB1) extrusion while the ooplasmic maturation was evaluated by the cleavage rate after parthenogenetic activation. The results showed that the nuclear maturation rate of CEOs, COs, DOs(COCs) and DOs(CCs) after 24 h incubation were significantly different from each other (p < or = 0.05), the rate of DOs(CCs) was similar to that of DOs (p > or = 0.05). The cleavage rates in the first two groups were significantly higher than those of the others (p < 0.05). For oocytes cultured for 30 h, the nuclear maturation rates were significantly different for each culture model (p < 0.05). The cleavage rates in first two groups were significantly higher than those of others (p < 0.05). Both the nuclear and cleavage rates significantly increased when the culture time of DOs(COCs) was prolonged from 24 to 30 h. DOs(CCs) nuclear maturation was significantly improved when the culture time was prolonged from 24 to 30 h, but the ooplasmic maturation was not. Few NOs incubated with or without CCs accomplished nuclear maturation (approximately 2% both), even when the culture time was prolonged from 24 to 30 h. The oocyte degeneration rates were significantly different for each culture model after both 24 and 30 h incubation (p < or = 0.05). There was no significant difference in oocyte degeneration in the same groups between 24 and 30 h incubation (p > 0.05). The results suggest that rabbit CCs affect oocyte nuclear and ooplasmic maturation, and their survival. The prolongation of the culture time of rabbit oocyte from 24 to 30 h improves the nuclear and ooplasmic maturation differently in the present system. Rabbit oocytes free of CCs, especially NOs, show weak meiotic resumption potential and compromised viability, which cannot be improved by co-culture with dispersed CCs. The degeneration mostly happens at early time of IVM.

P65. Dual effects of nitric oxide and different signaling pathways to maturation of cumulus cell-enclosed mouse oocytes

P65. Dual effects of nitric oxide and different signaling pathways to maturation of cumulus cell-enclosed mouse oocytes

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.

MOUSE OOCYTES PROMOTE STEROL BIOSYNTHESIS IN CUMULUS CELLS: MORE EVIDENCE FOR OOCYTEREGULATION OF METABOLIC COOPERATIVITY BETWEEN GRANULOSA CELLS AND OOCYTES

To evaluate the function(s) of oocyte-derived BMP15 and GDF9 on the development and function of cumulus cells before LH-stimulation of cumulus expansion, we previously compared the transcriptomes of cumulus cells from eCG-primed wildtype (WT), Bmp15−/−, and Bmp15−/−Gdf9+/− double mutant (DM) mice by micro-array analysis, and found that transcripts encoding enzymes of metabolic pathways in cumulus cells, particularly glycolysis, sterol biosynthesis, inositol metabolism, and pentose phosphate, were significantly affected. Here we have focused on the sterol biosynthesis pathway and studied its regulation in cumulus cells by oocytes. Oocytectomy (OOX) of WT cumulus cell-oocyte complexes reduced the expression in cumulus cells of 15 of 17 mRNAs encoding enzymes for sterol biosynthesis. This reduction was prevented when OOX cumulus cells were co-cultured with fully-grown oocytes (FGOs). Associated with the down-regulation of these transcripts' expression, the rate of de novo cholesterol biosynthesis was reduced by more than 90% in OOX cumulus cells. The reduction in cholesterol biosynthesis was recovered to only 50% of the control level by co-culture with FGOs. This suggests that contact between oocytes and cumulus cells is necessary for cumulus cell cholesterol biosynthesis. The expression of 6 mRNAs encoding enzymes in this pathway, Mvd, Fdps, Sqle, Lss, and Tm7sf2, was also significantly lower in cumulus cells of Bmp15−/− and DM, compared to that in WT mice, and these low mRNA levels were reflected in reduced levels of cholesterol synthesis (55% and 25% of WT level in Bmp15−/− and DM cumulus cells, respectively). These results suggest that the oocyte-derived paracrine factors, including GDF9 and BMP15, enhance sterol biosynthesis in cumulus cells by promoting the expression of mRNAs encoding the key enzymes in this pathway. Comparison of the expression of all the transcripts encoding enzymes in this pathway in cumulus cells and oocytes revealed that 7 mRNAs, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol, and Ebp, were robustly expressed in cumulus cells, but barely detectable in oocytes. Moreover, the level of de novo synthesized cholesterol in cumulus cell-enclosed oocytes was about 5-fold higher than that in denuded oocytes. We conclude that oocytes are deficient in synthesizing sterols and require cumulus cells to provide them with cholesterol and the other intermediates of the sterol biosynthetic pathway. Moreover, mouse oocyte-derived BMP15 and GDF9 profoundly affect the metabolic functions of cumulus cells before the LH surge. Supported by HD23839 (Y-QS, KS, JJE), HD21970 (KS, KW, JJE), HD33438 (SAP, MMM) and 5F32HD46335 (SAP). (platform)

AMP-ACTIVATED PROTEIN KINASE (AMPK) IS INVOLVED IN FOLLICLE-STIMULATING HORMONE (FSH)-INDUCED MOUSE OOCYTE MEIOTIC MATURATION

We have previously shown that AMPK activation can induce mouse oocyte meiotic resumption in vitro. The present study was carried out to determine whether AMPK activity is involved in follicle-stimulating hormone (FSH)-induced maturation. The upstream kinase of AMPK specifically phosphorylates AMPK on Thr172 of the catalytic subunit (PT172-AMPK) to activate the enzyme. Acetyl-CoA carboxylase (ACC) is a major substrate of AMPK, and its phosphorylation state is a common indicator of AMPK activity. By western analysis, a time course experiment was carried out to test phospho-ACC levels in FSH-treated oocytes. Cumulus cell-enclosed oocytes were denuded and extracted for western analysis after 3, 6, or 9 h culture with or without FSH in 300 μM dbcAMP. Within 3 h of FSH treatment, phospho-ACC levels were increased in germinal vesicle (GV)-stage oocytes when compared to nonstimulated controls and remained elevated in these oocytes throughout 9 h of culture. Interestingly, phospho-ACC returned to the basal level in oocytes undergoing GV breakdown by 9 h of FSH treatment. Using PT172 antibody (binds only to the activated kinase), western analysis showed that PT172-AMPK was elevated in FSH-treated GV-stage oocytes within 6 h. These results indicate that AMPK is activated in FSH-stimulated oocytes before meiotic resumption. Compound C, a potent small-molecule AMPK inhibitor, or adenine 9-beta-d-arabinofuranoside, a precursor of araATP (a competitive inhibitor of AMPK), dose-dependently prevented FSH-induced meiotic resumption and ACC phosphorylation in GV-stage oocytes, further supporting a role for AMPK in FSH-induced maturation. To directly observe active AMPK, oocytes were fixed, permeabilized and processed for immunofluorescent staining using anti-PT172 antiserum. Fluorescence was generally increased throughout the entire GV-stage oocyte after 6 h of FSH treatment compared to controls. A shift of active AMPK localization was observed after GVB, with accumulation around the condensed chromosomes. Compound C downregulated this FSHinduced increase in fluorescence. Taken together, these results support the idea that AMPK activation within the mouse oocyte plays a mediating role in FSH-stimulated meiotic resumption. Supported by funds from the NIH (HD040392). (platform)