Regulation of the G2/M Transition in Rodent Oocytes

Regulation of maturation in meiotically competent mammalian oocytes is a complex process involving the carefully coordinated exchange of signals between the somatic and germ cell compartments of the ovarian follicle via paracrine and cell–cell coupling pathways. This review highlights recent advances in our understanding of how such signaling controls both meiotic arrest and gonadotropin-triggered meiotic resumption in competent oocytes and relates them to the historical context. Emphasis will be on rodent systems, where many of these new findings have taken place. A regulatory scheme is then proposed that integrates this information into an overall framework for meiotic regulation that demonstrates the complex interplay between different follicular compartments

Porcine oocyte maturation in vitro : role of cAMP and oocyte-secreted factors: a practical approach

Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling

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

The effect of cadmium on the bovine in vitro oocyte maturation and early embryo development

Common pollutants such as heavy metals and cadmium is among those with high environmental concerns. In vivo studies had shown that cadmium (Cd) causes oocyte degeneration and embryo mortality, and lowers pregnancy rates in mammals. However, there is limited information available about direct effects of Cd on oocyte maturation and/or embryo development. This study was aimed to investigate if Cd has any effect on the oocyte maturation and/or embryo development in vitro. Bovine COCs were collected from the slaughter house and cultured for 24 h in serum-free media only (Controls) or supplemented with 0.2, 2.0 and 20.0 μM CdCl2. At 24 h cumulus cell expansion was assessed in all COCs. COCs were either denuded and stained for determination of nuclear maturation or fertilized for assessment of subsequent embryo development. Cd at the lowest concentration (0.2 μM) did not affect any of the parameters studied. However, at higher concentrations (2.0 and 20.0 μM) it significantly (P < 0.05) reduced the percentage of fully-expanded COCs and significantly (P < 0.05) increased the percentage of partially and/or non-expanded COCs compared to controls and 0.2 μM. Cadmium at higher concentrations (2.0 and 20.0 μM) also significantly (P < 0.01) reduced the percentage of oocytes reaching metaphase II stage compared to controls and 0.2 μM. Post-fertilization cleavage rate in presumptive zygotes and blastocyst development significantly (P < 0.05) reduced 0.2, 2.0 and 20.0 μM CdCl2 compared to the controls (0.0 μM). In conclusion, these results suggest that Cd had direct detrimental effects on the bovine oocyte maturation and its developmental competence

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

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 3H-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

AMPK Regulation of Mouse Oocyte Meiotic Resumption in Vitro

We have previously shown that the adenosine analog 5-aminoimidazole-4-carboxamide-1-β-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

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