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The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.

The efficiency of bovine in vitro embryo production has remained low despite extensive effort to understand the effects of culture conditions, media composition and supplementation. As bovine oocytes resume meiosis spontaneously when cultured, it was hypothesized that preventing meiosis in vitro before in vitro maturation (IVM) and in vitro fertilization (IVF) would allow more oocytes to acquire developmental competence. This article reviews some of the factors involved in meiotic arrest as well as the effects of meiotic inhibition before IVM on bovine oocytes developmental competence following IVF. Follicular components and cAMP-elevating agents can delay or inhibit meiosis in various proportions of oocytes; however, few studies have examined their effects on development following IVM and IVF because they are not practical (follicular components) or have a transient effect on meiosis (cAMP-elevating agents). Protein synthesis or phosphorylation inhibition prevented meiosis in high percentages of oocytes; however, these non-specific inhibitions led to lower developmental competence compared with non-arrested oocytes. Maturation promoting factor (MPF) inhibition with specific inhibitors has been examined in several studies. Despite faster maturation following removal from inhibition and some structural damage to the oocytes, MPF inhibition generally led to blastocyst rates similar to control, non-arrested oocytes. Future work will involve evaluating the effects on arrested oocytes of molecules that can improve developmental competence in non-arrested oocytes. It is also anticipated that new IVM systems that take into consideration new knowledge of the mechanisms involved in the control of meiosis will be developed. Moreover, global gene expression analysis studies will also provide clues to the culture conditions required for optimal expression of developmental competence., (© Her Majesty the Queen in Right of Canada 2011. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.)

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures., (Copyright © 2011 Wiley Periodicals, Inc.)

The adenosine monophosphate-activated protein kinase (AMPK) activators, 5'-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) and metformin (MET), inhibit resumption of meiosis in bovine cumulus-enclosed oocytes (CEO) and denuded oocytes (DO). The objectives of this study were to: (1) examine the effects of AMPK inhibitors on bovine oocyte meiosis in vitro; and (2) determine if AICAR or MET activates oocyte and/or cumulus cell AMPK. The AMPK inhibitor compound C (CC; 0.5, 1, 5, and 10 μM) did not reverse the inhibitory effects of AICAR (1 mM) and MET (2 mM) on bovine oocyte meiosis. Additionally, CC (5 and 10 μM) inhibited meiosis (p < 0.05) in CEO and DO cultured for 7 h. Okadaic acid (1 μM) reversed the inhibitory effect of MET (2 mM) and CC (5 μM; p < 0.05) but not of AICAR (1 mM). Phosphorylation of the alpha subunit of AMPK on Thr172 is required for activation. Based on western blot analysis, AICAR, MET and CC did not affect Thr172 phosphorylation levels in DO and oocytes from complexes (p > 0.05). In cumulus cells, Thr172 phosphorylation decreased after 3 h of culture (p < 0.05), regardless of the presence of AMPK modulators in the culture medium. Higher concentrations of AICAR (2 mM) and MET (10 mM) did not affect Thr172 phosphorylation, but phosphorylation on Ser79 of ACC, a substrate of AMPK, was increased in response to MET (p < 0.05). In conclusion, we inferred that the inhibitory effect of AICAR and MET on bovine oocyte meiosis was probably not mediated through activation of AMPK. Moreover, these compounds probably inhibited meiosis through different pathways.

The objective of this study was to examine the effects of manipulating the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway on bovine oocyte nuclear maturation in vitro. Cumulus-enclosed oocytes (CEO) were recovered from abattoir-derived ovaries and cultured in M199+FCS for 7 or 21h in the presence of various molecules affecting the NO/cGMP pathway, and then fixed and stained for evaluation of the stage of nuclear maturation. Cyclic GMP levels were also measured in cumulus-oocyte complexes after 3 and 6 h of culture. The iNOS inhibitor, aminoguanidine (AG, 10 and 50 mM) and the NO donor sodium nitroprusside (SNP, 100 and 500 microM) significantly inhibited GVBD after 7h of culture. However, a lower concentration of SNP (0.01 microM) stimulated GVBD. The inhibitory effects of AG and SNP were reversible, indicating that they were not toxic effects. Although SNP (500 microM) increased cGMP levels in cumulus-oocyte complexes after 3 h of culture, the inhibitor of soluble guanylate cyclase ODQ and the protein kinase G (PKG) inhibitor KT5823 did not reverse the inhibitory effect of SNP on meiosis, suggesting that SNP does not inhibit meiosis through the cGMP/PKG pathway. Similarly, an analogue of cGMP (8-Bromo-cGMP 0.5, 1, 3, and 6 mM), as well as activation of guanylate cyclase with Protoporphyrin IX or atrial natriuretic peptide, or inhibition of the enzyme with ODQ, did not have any significant effect on GVBD after 7 h of culture, supporting the idea that the effects of AG and SNP were not due to altered cGMP levels. Atrial natriuretic peptide, Protoporphyrin IX and SNP 500 microM increased cGMP levels after 3 h but not 6 h of culture. In conclusion, soluble and particulate guanylate cyclases could be activated in bovine cumulus-oocyte complexes, but accumulation of cGMP was probably not responsible for the effects of NO on meiosis.

The influence of the culture medium and energy sources on spontaneous nuclear maturation and inhibition of maturation in bovine cumulus-enclosed oocytes (CEO) was examined. CEO were cultured in Medium 199, minimum essential medium, M16, or synthetic oviduct fluid (SOF), all containing 3 mg/mL bovine serum albumin (BSA), and SOF without BSA, alone or supplemented with hypoxanthine (HYPO, 4 mM) or forskolin (FSK, 100 microM) for 21 h. More CEO remained at the GV stage in M16 compared to other media (P < 0.05). Supplementation with HYPO increased and FSK reduced the percentage of CEO remaining at the GV stage (P < 0.05) only in M16. The effects of energy sources, in the absence or presence of HYPO or FSK, were examined in CEO cultured in M16 salts+PVA. Glucose (0.5 and 5.5 mM), pyruvate (0.32 and 3.2 mM), lactate (3.3 mM) and glutamine (1.3 mM) significantly reduced the percentage of CEO remaining at the GV stage compared to M16 salts alone; only glutamine significantly increased the percentage of CEO at the MII stage compared to M16 salts. In M16 salts+HYPO, glucose (0.5 mM), pyruvate (0.32 mM), lactate (3.3 mM) and glutamine (1.3 mM) significantly reduced the percentage of GV and degenerate oocytes and increased the percentage of CEO at the MI stage. In M16 salts+FSK, the energy sources significantly decreased the percentage of oocytes with condensed chromosomes and increased the percentage of CEO reaching metaphase I. In conclusion, meiotic inhibitors had different effects in different culture media and glucose, pyruvate, lactate and glutamine were stimulatory to nuclear maturation. It was noteworthy that some of the results obtained were contrary to previous findings in mouse oocytes.

It was previously demonstrated that inhibition of cAMP degradation with phosphodiesterase type 3 (PDE3) inhibitors resulted in the maintenance of bovine cumulus-oocyte complexes (COC) and denuded oocytes (DO) in meiotic arrest, while a PDE4 inhibitor was without effect. In this study, different inhibitors of PDE3 and PDE4 were tested for their effects on bovine oocyte nuclear maturation. Bovine COC and DO were cultured in TCM-199+10% fetal bovine serum (FBS) with or without different concentrations of the PDE inhibitors. The PDE3 inhibitor trequinsin significantly increased the percentage of COC remaining at the germinal vesicle (GV) stage after 7h of culture (19.3, 60.3, and 67.8% GV for control and trequinsin 10 and 50 nM, respectively) while Ro 20-1724 (a PDE4 inhibitor) was without effect. In DO, only trequinsin at 10 nM had a significant effect after 7h of culture (51.3 and 86.1% GV for control and trequinsin 10 nM, respectively). Trequinsin reduced the percentage of COC reaching the mature phase after 22h, but was without effect on DO. The protein kinase A (PKA) inhibitor H-89 reversed the inhibitory effect of trequinsin in COC and DO, indicating that inhibition of nuclear maturation by trequinsin involves activation of PKA. Trequinsin increased cAMP concentrations in COC but not in DO, suggesting that cumulus cells may also contain a PDE3 isoenzyme.

The effect of adenylate cyclase stimulation via the components of the enzyme on nuclear maturation in bovine cumulus-enclosed and zona-free oocytes was examined. The stimulating agents were cholera toxin, pertussis toxin, forskolin, sodium fluoride and prostaglandin E2. Cyclic AMP contents were measured in cumulus-oocyte complexes, cumulus-enclosed oocytes and in zona-free oocytes after stimulation, to establish the relationship between cumulus cell and oocyte cAMP concentrations and the meiotic status of the oocyte. In cumulus-enclosed oocytes, forskolin alone and 3-isobutyl-1-methylxanthine (IBMX), at 0.5 mmol l-1, inhibited the resumption of meiosis after 8 h of culture; the other agents were without effect. After 24 h of culture, IBMX at 0.5 mmol l-1 was without effect, but at 2 mmol l-1 reduced the percentage of oocytes at the mature stage (51 versus 82% in control medium). Forskolin alone reduced the proportion of oocytes at the mature stage from 82 to 58%. Forskolin plus IBMX at 2 mmol l-1 and sodium fluoride plus IBMX at 2 mmol l-1 significantly diminished the maturation rate (6 and 17% mature oocytes, respectively). Cholera toxin (with IBMX) and forskolin (alone or with IBMX) stimulated the synthesis of high amounts of cAMP in complexes, but only forskolin had a significant effect on the cAMP contents of oocytes derived from complexes. Forskolin was more effective in zona-free oocytes than in cumulus-enclosed oocytes in inhibiting nuclear maturation (24% mature oocytes versus 73% in control medium) even after 24 h of culture; its effect was potentiated by IBMX; forskolin also stimulated cAMP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

This study was undertaken to create an in-vitro model using granulosa cell monolayers to replace the role of the follicle in the maturation of bovine oocytes. Cumulus-oocyte complexes were co-incubated with fresh or 7-day granulosa cell cultures (with new or conditioned medium) or with conditioned medium alone, in the presence or absence of IBMX (isobutylmethylxanthine), adenosine or heparin. Progression to the metaphase-II stage was significantly affected by the co-culture of oocytes with bovine granulosa cell monolayers and to a lesser degree when cultured with supernatant alone (conditioned medium). The oocytes attached rapidly to the monolayer, suggesting that the intimate contact between the granulosa cells and the cumulus-oocyte complexes is an important signal for the maintenance of meiotic arrest. Heparin did not prevent maturation itself, but prevented attachment of cumulus-oocyte complexes to monolayers, thereby reducing their inhibitory effect. Adenosine prevented cumulus expansion and reduced maturation and IBMX was an effective inhibitor only in the presence of additional granulosa cells.