Resumption Of Meiosis In Oocytes Research Articles

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals.

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.

276 PHOSPHOINOSITIDE 3-KINASE REGULATES EXPRESSION OF KEY ENZYMES AND CELLULAR TRANSPORTERS OF GLUCOSE METABOLISM IN CUMULUS CELLS OF BOVINE COCs CULTURED IN VITRO

The aim was to analyse the function of the PI3K pathway during oocyte maturation in bovine by use of the specific inhibitor, LY294002. Genes studied in cumulus cells (CC) were PDH, G6PDH, GLUT1, and GLUT4. PDH is an important enzyme for oxidative metabolism, G6PDH is related to resumption and progression of oocyte meiosis, and GLUT1 and GLUT4 are glucose transporters. This study was performed in defined medium (MIV B) in absence or presence of 10 ng mL–1 of FSH. Polar body extrusion was analysed after culture and correlated with gene expression. Experimental methods were bovine COC (n = 35–40/well, n = 3 replicates) collected from ovaries obtained from abattoirs (DF, Brazil) and cultivated in either 400 µL of medium MIV B, or MIV B + 100 µM of LY294002, or MIV B + 10 ng mL–1 of FSH; or MIV B + 10 ng mL–1 of FSH + 100 µM of LY294002 during 22 to 24 h. After culture, COC were mechanically denuded and CC from 20 COC/group were isolated. Gene expression of GLUT1, GLUT4, G6PDH, and PDH were measured by real time PCR. The CC of immature COC were also collected and analysed as the calibrator group. Student-Newman-Keuls was performed as a statistical test. The percentage of oocytes that extruded the polar body was determined. Two-way ANOVA, followed by Bonferroni test, and t-test were performed to determine statistical significance. In MIV B, the polar body extrusion rate was 25.48 ± 7.64%, while FSH increased it up to 74.52 ± 10.58% (P < 0.05). The extrusion of polar body was inhibited by LY294002 in the absence or presence of FSH (67.51 ± 6.13 and 31.02 ± 16.97%, respectively, P < 0.05). Gene expression of PDH was not altered by any culture medium in contrast to GLUT1, GLUT4, and G6PDH expression (Table 1). Only G6PDH expression showed the same pattern as the polar body extrusion, in absence or presence of FSH. In conclusion, PI3-K inhibition affects polar body extrusion and expression of genes related to glucose metabolism in CC. Lower G6PDH expression in CC may be related to low rates of polar body extrusion in treated oocytes. Table 1.Gene expression of GLUT1, GLUT4, and G6PDH in CC of COC after culture The authors thank FAP-DF (193.000.577/2009), CNPq, CAPES and Ponte Alta abattoir, Brasilia.

278 INFLUENCE OF NITRIC OXIDE AND PHOSPHODIESTERASE INHIBITORS ON CYCLIC NUCLEOTIDES AND MEIOSIS RESUMPTION IN BOVINE OOCYTES IN VITRO MATURED

Nitric oxide (NO) is a local action mediator of essential hormones and neurotransmitters that regulate several physiological processes in reproduction. Nitric oxide activates soluble guanylate cyclase (sGC), and results in the production of cyclic guanosine monophosphate (cGMP). This nucleotide is involved in oocyte maturation and, consequently, in the success of fertilization. In addition to cGMP, another cyclic nucleotide, cAMP, is also related to maturation. The levels of these nucleotides are balanced by synthesis and degradation is made by phosphodiesterases (PDE). The aim of this study was to analyse the effect of NO and PDE inhibitors on cyclic nucleotides concentrations and meiosis resumption in bovine oocytes matured in vitro. In experiment 1, cumulus-oocyte complexes (COC) were cultured in maturation medium with a NO donor (0.1 µM S-nitroso-N-acetylpenicillamine; SNAP) associated or not with a PDE5 inhibitor (10 µM sildefanil; SILD) for 1 h; after this period, cGMP levels (40 COC/group) were measured in COC using commercial enzyme immunoassay kits (EIA). Immature (0 h) and control COC (untreated) were measured as well. In experiment 2, COC were cultured for 9 h in maturation medium with an NO donor (0.1 µM SNAP) associated or not with inhibitors the PDE5 (10 µM SILD), PDE3 (20 µM cilostamide; CIL), or PDE8 (50 µM dipyridamole; DIP) and assessed for meiosis resumption (%GVBD; ± 100 COC/treatment). In experiment 3, COC were cultured as in experiment II and cAMP levels (10 COC/group) were measured after 1 h in culture using commercial EIA kits. Immature COC were also measured. Statistical analyses were performed using the SAS system. Data were tested for normal distribution and were transformed (arcsine; GVBD). The percentages of GVBD were analysed by one-way ANOVA followed by Bonferroni post-hoc test (4 replicates). The cGMP and cAMP levels were analysed by two-way ANOVA followed by Bonferroni post-hoc testing (5 replicates). Differences with probabilities of P < 0.05 were considered significant. In experiment 1, cGMP levels decreased from 0 to 1 h in control and SNAP groups (P < 0.05). When SNAP was associated with SILD, cGMP levels were similar to immature oocytes (P > 0.05%). In experiment 2, all treatments delayed meiosis by decreasing GVBD rates (24.7 ± 2.8 to 56.9 ± 8.6%) when compared to controls (77.1 ± 1.8%, P < 0.05). However, SNAP + CIL had the lowest GVBD rates (24.7 ± 2.8%, P < 0.05). In experiment 3, cAMP levels declined from imature COC (93 ± 7 fmol/COC) to 1 h in all treatments (21 ± 6 to 10 ± 5 fmol/COC; P < 0.05). The results indicate that NO and PDE inhibitors during maturation were ineffective in preventing the decrease in cyclic nucleotides levels during the first hour of maturation, but were still effective in delaying meiosis resumption, and the effectiveness depends on the PDE isoform which is inhibited (SNAP + CIL was most effective).

Effects of AY9944 A-7 on gonadotropin-induced meiotic resumption of oocytes and development of parthenogenetic embryos in sheep

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. Follicular fluid meiosis-activating sterol is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this research was to investigate the specific contribution of AY9944 A-7 on gonadotropin-induced meiotic resumption and its interactive effects with FSH or LH on meiotic maturation of oocytes and preimplantation development of parthenogenetic embryo in sheep by addition of AY9944 A-7 during IVM to cause accumulation of FF-MAS. First, ovine cumulus–oocyte complexes (COCs) were cultured in the presence of FSH (10 μg/mL), LH (10 μg/mL), AY9944 A-7 (20 μmol/L), FSH (10 μg/mL)+AY9944 A-7 (20 μmol/L), or LH (10 μg/ml) + AY9944 A-7 (20 μmol/L) with an inhibitor hypoxanthine (Hx) to prevent spontaneous meiosis of oocytes. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. The kinetics of gonadotropin and AY9944 A-7–induced meiotic resumption in vitro was also evaluated in the study. The numbers of oocytes resuming meiosis and undergoing germinal vesicle breakdown were counted after the COCs were cultured for 2, 4, 8, 12, 16, 20, and 24 hours. Matured oocytes extruding PBI were selected for parthenogenetic activation, and the percentages developing to the two-cell stage and blastocyst stage were recorded as indicators of parthenogenetic embryo developmental competence. It was observed that FSH could induce the resumption of meiosis of ovine COCs cultured in the presence of Hx, but LH could not. AY9944 A-7 had a synergistic effect with FSH on nuclear maturation and developmental competence of embryos produced by parthenogenetic activation, whereas it had no added advantage on LH action. However, the kinetics of meiotic resumption after AY9944 A-7 stimulation was remarkably delayed when compared with FSH-induced maturation. In conclusion, the current study suggested that AY9944 A-7 supplementation in IVM medium optimized the beneficial effects of FSH on meiotic maturation of ovine oocytes and subsequent developmental competence of embryos produced by parthenogenetic activation. This work had important potential for developing a novel technique in IVM of ovine oocytes.

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes.

In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.

Reduction of nitric oxide level leads to spontaneous resumption of meiosis in diplotene-arrested rat oocytes cultured in vitro.

The present study was aimed to investigate whether a decrease of nitric oxide (NO) level is beneficial for sponateous resumptiom of meiosis in diplotene-arrested oocytes cultured in vitro. For this purpose, diplotene-arrested oocytes were collected from ovary of immature female rats after a single subcutaneous injection of 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h. In vitro effects of S-nitroso-l-acetyl penicillamine (SNAP; an NO donor) and aminoguanidine (AG; an inducible NOS [iNOS] inhibitor), intracellular NO, cyclic guanosine monophosphate (cGMP), Cdc25B, Thr-14/Tyr-15 and Thr-161 phosphorylated cyclin-dependent kinase-1 (CDK1), and cyclin B1 levels were analyzed. The SNAP inhibited spontaneous meiotic resumption form diplotene arrest in a concentration-dependent manner, while AG-induced meiotic resumption form diplotene in 0.1 mmol/L 3-isobutyl-1-methylxanthine (IBMX)-treated oocytes in a concentration-dependent manner. The intracellular NO as well as cGMP levels were decreased significantly during spontaneous meiotic resumption from diplotene arrest. The reduction of Cdc25B expression level was associated with the accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. However, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels were reduced significantly during meiotic resumption from diplotene arrest. Taken together, these data suggest that the inhibition of iNOS expression leads to a decrease of NO and cGMP levels thereby decreasing Cdc25B level. The reduced CDC25 B level leads to accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. As a result, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels are decreased leading to maturation-promoting factor (MPF) inactivation. The inactive MPF finally induced meiotic resumption from diplotene stage in rat oocytes cultured in vitro.

A moderate increase of hydrogen peroxide level is beneficial for spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

Hydrogen peroxide (H2O2) acts as a signaling molecule and modulates various aspects of cell functions in a wide variety of cells including mammalian germ cells. We examined whether a decreased level of intra-oocyte cyclic 3′,5′-adenosine monophosphate (cAMP) leads to accumulation of H2O2, and if so, whether a moderate increase of H2O2 inactivates maturation promoting factor (MPF) during spontaneous resumption of meiosis in rat oocytes cultured in vitro. Removal of cumulus cells and culture of denuded oocytes in vitro significantly decreased oocyte cAMP level and led to spontaneous meiotic resumption from diplotene arrest. The reduced oocyte cAMP level was associated with an increased oocyte H2O2 level and reduced catalase activity. Exogenous supplementation of H2O2 induced meiotic resumption from diplotene arrest in a concentration- and time-dependent manner in oocytes treated with 0.1 mM of 3-isobutyl-1-methylxanthine, while dibutyryl-cAMP and 3-t-butyl-4-hydroxyanisole inhibited the stimulatory effect of exogenous H2O2. The increased intra-oocyte H2O2 level induced Thr-14/Tyr-15 phosphorylation of CDK1, while Thr-161 phosphorylated CDK1 and cyclin B1 levels were reduced significantly. These results suggest that a decreased level of intra-oocyte cAMP is associated with an increased level of H2O2. The increased level of H2O2 was associated with high phosphorylation of Thr-14/Tyr-15 and dephosphorylation of the Thr-161 residue of CDK1 and reduced the cyclin B1 level, which eventually inactivated MPF. The MPF inactivation triggered spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

Tribbles expression in cumulus cells is related to oocyte maturation and fatty acid metabolism.

BackgroundIn mammals, the Tribbles family includes widely expressed serine-threonine kinase-like proteins (TRIB1, TRIB2 and TRIB3) that are involved in multiple biological processes including cell proliferation and fatty acid (FA) metabolism. Our recent studies highlighted the importance of FA metabolism in cumulus cells (CC) during oocyte maturation in vertebrates and reported a higher TRIB1 expression in CC surrounding in vivo mature oocytes as compared to immature ooocytes in mice and cows. The objective was to investigate Tribbles expression patterns in bovine CC during in vitro maturation (IVM) and to examine their roles in the cumulus-oocyte complex.MethodsTribbles gene expression was analyzed in bovine and murine CC using quantitative RT-PCR. Proteins were detected using Western blot and intracellular localization was assessed by immunofluorescence. Bovine COCs were treated with etomoxir, an inhibitor of FA oxidation (FAO) which blocks CPT1 activity, during 6 h and 18 h IVM. Oocyte meiotic stage was assessed and expression of Tribbles and lipid metabolism genes was quantified in CC.Results and discussionTRIB1 and TRIB3 were more strongly expressed whereas TRIB2 was less expressed in CC surrounding the oocytes from preovulatory follicles than in CC of immature ones. In CC, Tribbles were located in the cytoplasm and nucleus; in mitotic cells TRIB2 and TRIB3 were detected in the spindle. In the oocyte, Tribbles proteins were detected in the ooplasm; also TRIB2 and TRIB3 were more accumulated in the germinal vesicle. In bovine CC, expression of TRIB1 and TRIB3 was transiently increased at a time preceding oocyte meiosis resumption in vitro. Treatment with etomoxir (150 μM) during IVM resulted in a significant reduction of oocyte maturation rate and decreased MAPK3/1 phosphorylation in the oocytes. In CC, 18 h IVM of etomoxir treatment significantly increased expression of TRIB1, TRIB3, CPTA1 (enzyme regulating FA entry in mitochondria for FAO) and CD36 (thrombospondin receptor involved in FA transport). Under the same conditions, expression of TRIB2 and ACACA (Acetyl coenzyme A carboxylase involved in FA synthesis) decreased in CC.All considered, Tribbles family members may be involved in cell proliferation and in FAO signaling in CC and participate in oocyte meiotic resumption regulation.

Phosphorylation of ARPP19 by protein kinase A prevents meiosis resumption in Xenopus oocytes

During oogenesis, oocytes are arrested in prophase and resume meiosis by activating the kinase Cdk1 upon hormonal stimulation. In all vertebrates, release from prophase arrest relies on protein kinase A (PKA) downregulation and on the dephosphorylation of a long-sought but still unidentified substrate. Here we show that ARPP19 is the PKA substrate whose phosphorylation at serine 109 is necessary and sufficient for maintaining Xenopus oocytes arrested in prophase. By downregulating PKA, progesterone, the meiotic inducer in Xenopus, promotes partial dephosphorylation of ARPP19 that is required for the formation of a threshold level of active Cdk1. Active Cdk1 then initiates MPF autoamplification loop that occurs independently of both PKA and ARPP19 phosphorylation at serine 109 but requires the Greatwall-dependent phosphorylation of ARPP19 at serine 67. Therefore, ARPP19 stands at a crossroads in the meiotic M-phase control network by integrating differential effects of PKA and Greatwall, two essential kinases for meiosis resumption.

Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential.

BackgroundEvaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle’s/Hank’s’ M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic suppression.MethodsExperiment 1 - Equine oocytes processed immediately or held in EH at uncontrolled temperature (22 to 27°C) were evaluated for initial chromatin configuration, in vitro maturation (IVM) rates and mitochondrial energy/redox potential. Experiment 2 - We then investigated the effect of holding temperature (25°C, 30°C, 38°C) on initial chromatin status of held oocytes, and subsequently repeated mitochondrial energy/redox assessment of oocytes held at 25°C vs. immediately-evaluated controls.ResultsEH holding at uncontrolled temperature was associated with advancement of germinal vesicle (GV) chromatin condensation and with meiotic resumption, as well as a lower maturation rate after IVM. Holding did not have a significant effect on mitochondrial distribution within chromatin configurations. Independent of treatment, oocytes having condensed chromatin had a significantly higher proportion of perinuclear/pericortical mitochondrial distribution than did other GV configurations. Holding did not detrimentally affect oocyte energy/redox parameters in viable GV-stage oocytes. There were no significant differences in chromatin configuration between oocytes held at 25°C and controls, whereas holding at higher temperature was associated with meiosis resumption and loss of oocytes having the condensed chromatin GV configuration. Holding at 25°C was not associated with progression of mitochondrial distribution pattern and there were no significant differences in oocyte energy/redox parameters between these oocytes and controls.ConclusionsMitochondrial distribution in equine GV-stage oocytes is correlated with chromatin configuration within the GV. Progression of chromatin configuration and mitochondrial status during holding are dependent on temperature. EH holding at 25°C maintains meiotic arrest, viability and mitochondrial potential of equine oocytes. This is the first report on the effects of EH treatment on oocyte mitochondrial energy/redox potential.

90 CHARACTERIZATION OF A PRIMARY CULTURE OF OVIDUCTAL CELLS IN THE BITCH

The oviduct is of particular importance in canine reproduction as it supports oocyte meiosis resumption, sperm capacitation and storage, fertilization and embryo development to the morula/blastocyst stage for 8 to 10 days post-ovulation. A long-time co-culture with oviducal cells could be employed to improve the yield of reproductive biotechnologies in this species, but no characterisation of canine oviduct cells in vitro has been reported to date. The objectives of this study were to (1) evaluate the viability and proportion of epithelial/fibroblast cells in a primary culture of canine oviducal cells collected around ovulation; (2) study the responsiveness of the cultured cells to steroids. Beagle bitches (n = 9) were ovariectomized between Day –1 and Day +1 around ovulation, and their oviducts were sectioned at the ampulla-isthmus junction. Mucosal cells (including stromal and epithelial cells) were collected by squeezing from the ampulla and isthmus sections and cultured separately at a concentration of 5 × 105 cells/well in 500 μL of M199 + 10% FCS at 39°C for 9 days. At Days 3 and 6, 1 × 106 cells were stimulated with 17β-oestradiol (E2, 20 pg mL–1) or progesterone (P4, 20 ng mL–1) for 6 h. At Days 3, 6, and 9 of culture, the viability of the cells was evaluated using the Live/Dead kit (Invitrogen, Carlsbad, CA, USA), and proportions of fibroblast and epithelial ciliated cells were evaluated by immuno-cytochemistry using anti-vimentin and anti-tubulin antibodies, respectively. At Days 0, 3 and 6, the total RNA was extracted from cells and mRNA levels of the oviduct-specific glycoprotein (OVGP, synthesised by nonciliated epithelial cells), E2 (ERα, ERβ) and P4 (PR) receptors were evaluated by RT-qPCR. The effects of the day of culture and of steroid exposure on mRNA levels were analysed by ANOVA followed by a Tukey test. Cell confluence was observed around Day 6 of culture and more than 90% of cells survived during the 9-day culture period. From Day 3 to Day 9, the proportion of vimentin-positive (fibroblast) cells was greater than 68% in both ampulla and isthmus cells. In contrast, the proportion of epithelial ciliated cells was low at Day 3 (9% in ampulla, 12% in isthmus) and null at Days 6 and 9 in both regions. The mRNA levels of OVGP, ER, and PR decreased significantly after 3 days of culture, and then remained stable in both ampulla and isthmus cells (P < 0.001). The steroid exposure had no effect on gene expression, except for ERα mRNA levels at Day 3, which was increased by E2 and reduced by P4 (P < 0.05). In conclusion, the method of collection did not allow us to collect a high proportion of epithelial oviducal cells. However, the relatively stable gene expression of PR and ER during the culture period provides us with a useful tool to study the steroid regulation of canine oviduct mucosal cell functions.

Effect of nitric oxide on the cyclic guanosine monophosphate (cGMP) pathway during meiosis resumption in bovine oocytes

Nitric oxide (NO) is a chemical messenger involved in the control of oocyte maturation. It stimulates guanylate cyclase to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases that may interfere with cAMP levels, a nucleotide also involved in meiosis resumption. The aim of this study was to determine the role played by NO on the cGMP/cAMP pathway during meiosis resumption in bovine oocytes. The effects of increasing NO generated by S-nitroso-N-acetylpenicillamine (SNAP; 10−7–10−3 mol/L) and of other drugs that may affect the NO/cGMP pathway (proptoporfirin IX and 8-Br-cGMP) on meiosis resumption were investigated in bovine cumulus-oocyte complexes (COCs) matured for 9 hours in a semidefined medium (TCM199 + 3 mg/mL BSA). The COCs matured with 10−7 mol/L SNAP associated or not with 100 μmol/L oxadiazole-one quinoxaline, a guanylate cyclase inhibitor, also had their cGMP and cAMP levels measured during the first hours of maturation (1, 3, and 6 hours). Quantitative polymerase chain reaction was performed by real-time polymerase chain reaction to determine the effects of NO on expression of genes encoding for enzymes of the NO/guanylate cyclase/cGMP and cAMP pathways during the first 9 hours of oocyte maturation. Increasing NO levels using 10−7 mol/L SNAP resulted in lower rate of germinal vesicle breakdown (36% germinal vesicle breakdown; P < 0.05) at 9 hours IVM, whereas control group and the treatments with 10−9 and 10−8 mol/L SNAP showed about 70% germinal vesicle breakdown (P > 0.05). A temporary increase in cGMP levels was also observed with the same treatment (4.51 pmol/COC) at 1 hour IVM, which was superior to the control group (2.97 pmol/COC; P < 0.05) and was reversed by inhibiting guanylate cyclase activity with 100 μmol/L oxadiazole-one quinoxaline. Neither cAMP levels nor gene expression were affected by NO. These results suggest that NO acts via guanylate cyclase/cGMP and that even a temporary increase in cGMP levels leads to a delay in meiosis resumption, even when cAMP levels have declined. Nitric oxide does not act on oocyte maturation by affecting cAMP levels or the expression of genes related to the NO/guanylate cyclase/cGMP and cAMP pathways. Also, to our knowledge this is the first report to detect PKG1, PKG2, phosphodiesterase-5A, ADCY3, ADCY6, and ADCY9 transcripts in bovine oocytes.

AY9944 A-7 promotes meiotic resumption and preimplantation development of prepubertal sheep oocytes maturing in vitro

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. FF-MAS is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this study was to determine the effects of AY9944 A-7 supplementation to oocyte maturation media on prepubertal sheep oocyte meiotic resumption and subsequent preimplantation development of embryos. Prepubertal sheep oocytes isolated at the germinal vesicle stage from their follicles were cultured with 0, 10, 20, 30, and 40 μM AY9944 A-7 for 24 hours in media with or without a meiotic inhibitor hypoxanthine (Hx, 4 mM). The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. After maturation for 24 hours, oocytes with PBI were inseminated in vitro, and the percentages developing to the two-cell stage and blastocyst stage were measured as indicators of early embryonic developmental competence. AY9944 A-7 induced maturation of sheep cumulus-oocyte complexes with optimal concentrations of 10 and 20 μM both in Hx-inhibited meiotic maturation and spontaneous maturation, whereas AY9944 A-7 with any concentrations had no significant effect on that of denuded oocytes and split cumulus-oocyte complexes. Furthermore, maturing oocytes treated with either 10 or 20 μM AY9944 A-7 dramatically increased the percentages of ovine embryos developing to the two-cell stage and blastocyst stage. Higher concentrations of AY9944 A-7, 30 and 40 μM, were detrimental to oocytes and led to their degeneration. The present findings indicated for the first time that AY9944 A-7 was not only able to promote meiotic maturation, both Hx-inhibited and spontaneous, but also enhanced preimplantation developmental competence of prepubertal sheep oocytes maturing in vitro.

Cyclin O Regulates Germinal Vesicle Breakdown in Mouse Oocytes1

It is well accepted that oocyte meiotic resumption is mainly regulated by the maturation-promoting factor (MPF), which is composed of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2). Maturation-promoting factor activity is regulated by the expression level of CCNB1, phosphorylation of CDC2, and their germinal vesicle (GV) localization. In addition to CCNB1, cyclin O (CCNO) is highly expressed in oocytes, but its biological functions are still not clear. By employing short interfering RNA microinjection of GV-stage oocytes, we found that Ccno knockdown inhibited CDC2 (Tyr15) dephosphorylation and arrested oocytes at the GV stage. To rescue meiotic resumption, cell division cycle 25 B kinase (Cdc25b) and Ccnb1 were overexpressed in the Ccno knockdown oocytes. Unexpectedly, we found that Ccno knockdown did not affect CDC25B entry into the GV, and overexpression of CDC25B was not able to rescue resumption of oocyte meiosis. However, GV breakdown (GVBD) was significantly increased after overexpression of Ccnb1 in Ccno knockdown oocytes, indicating that GVBD block caused by cyclin O knockdown can be rescued by cyclin B1 overexpression. We thus conclude that cyclin O, as an upstream regulator of MPF, plays an important role in oocyte meiotic resumption in mouse oocytes.

Characterization of the effects of metformin on porcine oocyte meiosis and on AMP-activated protein kinase activation in oocytes and cumulus cells

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 activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro

Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37°C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 ± 205, n = 8) to mature MII oocytes (744 633 ± 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge.

Insulin-like growth factor II (IGF-II) and follicle stimulating hormone (FSH) combinations can improve the in vitro development of grown oocytes enclosed in caprine preantral follicles

ObjectiveEvaluate the possible role of IGF-II alone or in association with FSH on in vitro development of isolated caprine preantral follicles. MethodsPreantral follicles (≥150μm) were isolated from goat ovaries and cultured for 18days in basic αMEM medium (control) or supplemented with IGF-II alone at 20 or 50ng/ml, named IGF20 and IGF50, respectively, or in combination with recombinant FSH (FSH, IGF20F or IGF50F). During in vitro culture, the follicles were analyzed by using morphology criteria, antrum formation and growth rate as parameters. After 18days of follicular culture, oocytes equal to or larger than 110μm were used for in vitro maturation (IVM). Oocyte viability and meiosis resumption were assessed by fluorescence microscopy after labeling with calcein-AM, ethidium homodimer and Hoechst 33342. ResultsThe IGF20 treatment was the only treatment capable of maintaining the percentage of morphologically normal follicles from D0 until D6 and from D12 to D18 (p>0.05), while in all other treatments the percentage of morphologically normal follicles decreased progressively during 18days of in vitro culture (p<0.05). At D18, all treatments with IGF-II or FSH resulted in a significantly higher percentage of normal follicles when compared to αMEM alone. The IGF50F treatment provided a significantly higher early antrum formation rate when compared to αMEM and FSH alone. The addition of IGF-II alone (20 or 50ng/ml) or in combination with FSH prevented oocyte degeneration after IVM. Moreover, the FSH treatment demonstrated a lower percentage of oocyte degeneration when compared to control (4.35% vs. 26.3%, respectively; p<0.05). Regarding meiosis resumption, the IGF20F treatment was the only treatment that significantly differed from αMEM alone. All treatments except the control (αMEM alone) presented oocytes at metaphase II. ConclusionIGF-II associated with FSH stimulated in vitro follicular development, oocyte viability and meiotic resumption of caprine oocytes after IVM.

Immunolocalization of Progesterone Receptors in the Canine Oviduct around Ovulation

In the bitch, oocyte maturation, sperm storage, fertilization and early embryo development take place within the oviducts under high and increasing circulating progesterone concentrations. To investigate the potential effects of progesterone on the canine oviduct, nuclear progesterone receptors (PR) were localized. Oviducts were collected by ovariectomy from adult Beagle bitches during anestrus, after the onset of proestrus but prior to the Luteinizing Hormone (LH) peak (Pre-LH), after the LH peak but before ovulation (Pre-ov) and on Days 1, 4 and 7 after ovulation (n = 3 bitches per stage). The cellular distribution of PR was studied by immunohistochemistry (IHC) in the ampulla, isthmus and tubal part of the utero-tubal junction (UTJ). Plasma progesterone and 17β-oestradiol were assayed on the day of surgery. PR were specifically expressed in the nuclei of epithelial, stromal and muscular cells in the ampulla, isthmus and UTJ. The IHC scores did not vary from one oviductal region to another. However, the epithelium displayed higher scores than the stroma at anestrus, Pre-ov, Days 4 and 7, and also higher scores than muscle at Days 4 and 7 (p < 0.05). Immunohistochemistry scores in the stroma and muscle decreased at Days 4 and 7 compared with previous stages (p < 0.05). Furthermore, muscular IHC scores were positively correlated with circulating 17β-oestradiol concentrations and negatively correlated with circulating progesterone concentrations (p < 0.05). In conclusion, PR were identified in the canine oviduct, with differences in expression between tissues and times around ovulation, suggesting that progesterone may regulate tubal functions and reproductive events in this species.

Are Oocytes from the Anestrous Bitch Competent for Meiosis?

In the bitch, oocyte meiosis resumption takes place in the oviduct. Using oocytes from anestrous bitches, in vitro maturation (IVM) generally gives very poor results. To investigate the contribution of oocyte competence to the low IVM yield, we compared in vivo maturation in an optimal environment with conventional IVM. A total of 418 grade 1 cumulus-oocyte complexes (COCs) from 10 anestrous bitches were transferred into the oviducts of recipient bitches either on Day -1 (n = 3 recipients), Day 0 (n = 2) or on Day +1 (n = 2) relative to ovulation. For each donor bitch, 20 grade 1 COCs were also cultured in vitro. After 72 h of in vivo or IVM, the nuclear stage of oocytes was determined after DNA and tubulin staining. Of the 154 oocytes recovered and examined after intratubal transfer, only 2% reached the metaphase I or II stage and 38.3% were degenerated. Oocytes cultured in vitro displayed a higher metaphase rate (7.6%, n = 170) and lower degeneration rate (12.9%) compared with transferred oocytes (p < 0.001). These results clearly demonstrate that the oocyte competence is the major limiting factor of IVM efficiency in the dog. Mimicking the tubal environment may thus not be sufficient to increase IVM yield in this species.

Aurora Kinase A Drives MTOC Biogenesis but Does Not Trigger Resumption of Meiosis in Mouse Oocytes Matured In Vivo1

Aurora kinase A (AURKA) is an important mitotic kinase involved in the G2/M transition, centrosome maturation and separation, and spindle formation in somatic cells. We used transgenic models that specifically overexpress in mouse oocytes either wild-type (WT-AURKA) or a catalytically inactive (kinase-dead) (KD-AURKA) AURKA to gain new insights regarding the role of AURKA during oocyte maturation. AURKA activation occurs shortly after hCG administration that initiates maturation in vivo. Although AURKA activity is increased in WT-AURKA oocytes, resumption of meiosis is not observed in the absence of hCG administration. Control oocytes contain one to three microtubule organizing centers (MTOCs; centrosome equivalent) at prophase I. At the time of germinal vesicle breakdown (GVBD), the first visible marker of resumption of meiosis, the MTOC number increases. In WT-AURKA oocytes, the increase in MTOC number occurs prematurely but transiently without GVBD, whereas the increase in MTOC number does not occur in control and KD-AURKA oocytes. AURKA activation is biphasic with the initial activation not requiring CDC25B-CDK1 activity, whereas full activation, which is essential for the increase in MTOCs number, depends on CDK1 activity. AURKA activity also influences spindle length and regulates, independent of its protein kinase activity, the amount of MTOC associated with gamma-tubulin. Both WT-AURKA and KD-AURKA transgenic mice have normal fertility during first 6 mo of life. These results suggest that although AURKA is not a trigger kinase for G2/M transition in mouse oocytes, it regulates MTOC number and spindle length, and, independent of its protein kinase activity, gamma-tubulin recruitment to MTOCs.