MPF Activity Research Articles

MiR-155-5p improves oocyte maturation in porcine cumulus cells through connexin 43-mediated regulation of MPF activity

In this study, we aimed to investigate the effect of the expression of miR-155-5p and its target genes on oocyte maturation. We analyzed the expression of miR-155-5p and its target genes in cumulus cells and oocytes using quantitative real-time reverse-transcription polymerase chain reaction. Using carboxyfluorescein, porcine cumulus cells were transfected with mimics and inhibitors of ssc-miR-155-5p to induce in vitro maturation, and subsequently, cumulus expansion, oocyte maturation, and cleavage rate were measured. We found that miR-155-5p expression in cumulus cells at the metaphase II stage was significantly higher than that at the germinal vesicle (GV) stage, whereas Cx43 expression was significantly lower than that at the GV stage (P < 0.05). Compared with those in the negative control group, the cumulus diffusion area of cumulus oocyte complexes; oocyte maturation rate; cleavage rate; HAS2, PTGS2, CD44, PTX3, and TNFAIP6 expression in cumulus cells; and GDF9, BMP15, CyclinB1, and CDK1 expression in oocytes were significantly increased in the miR-155-5p mimics group (P < 0.05), whereas the mRNA and protein expression of CX43 were significantly decreased (P < 0.05). Compared with that in the negative control group, the protein expression of CyclinB1 and p-CDK1 (Thr14, Tyr15) in the miR-155-5p mimics group was significantly increased (P < 0.05). These results suggest that miR-155-5p regulates maturation promoting factor activity by targeting Cx43, which improves the in vitro maturation and cleavage rate of porcine oocytes.

High-dose zearalenone exposure disturbs G2/M transition during mouse oocyte maturation

Zearalenone is a mycotoxin produced by fungi of the genus Fusarium, which has severe toxicity on animal and human health including reproduction. Previous study showed that zearalenone exposure inhibited oocyte polar body extrusion, while in present study we found that high dose zearalenone disturbed oocyte meiosis resumption. Our results showed that a high concentration of 100 μM zearalenone reduced the rate of germinal vesicle (GV) breakdown in mouse oocytes. Further analysis indicated that zearalenone caused the decrease of Cyclin B1 and CDK1 expression, indicating MPF activity was affected, which further induced G2/M arrest, and this could be rescued by the inhibition of Wee1 activity. We found that the oocytes under high concentration of zearalenone showed lower γ-H2A.X expression, suggesting that DNA damage repair was disturbed, which further activated of DNA damage checkpoints. This could be confirmed by the altered expression of CHK1 and CHK2 after zearalenone treatment. Moreover, the organelles such as mitochondria, ribosome, endoplasmic reticulum and Golgi apparatus were diffused from germinal vesicle periphery after zearalenone exposure, indicating that zearalenone affected protein synthesis, modification and transport, which further induced the arrest of G2/M transition. Taken together, our results showed that high dose of zearalenone exposure induced G2/M transition defect by affecting organelle function-related CHK1/2-Wee1-MPF pathway.

Electrofusion Stimulation Is an Independent Factor of Chromosome Abnormality in Mice Oocytes Reconstructed via Spindle Transfer.

Oocytes reconstructed by spindle transfer (ST) are prone to chromosome abnormality, which is speculated to be caused by mechanical interference or premature activation, the mechanism is controversial. In this study, C57BL/6N oocytes were used as the model, and electrofusion ST was performed under normal conditions, Ca2+ free, and at room temperature, respectively. The effect of enucleation and electrofusion stimulation on MPF activity, spindle morphology, γ-tubulin localization and chromosome arrangement was compared. We found that electrofusion stimulation could induce premature chromosome separation and abnormal spindle morphology and assembly by decreasing the MPF activity, leading to premature activation, and thus resulting in chromosome abnormality in oocytes reconstructed via ST. Electrofusion stimulation was an independent factor of chromosome abnormality in oocytes reconstructed via ST, and was not related to enucleation, fusion status, temperature, or Ca2+. The electrofusion stimulation number should be minimized, with no more than 2 times being appropriate. As the electrofusion stimulation number increased, several typical abnormalities in chromosome arrangement and spindle assembly occurred. Although blastocyst culture could eliminate embryos with chromosomal abnormalities, it would significantly decrease the number of normal embryos and reduce the availability of embryos. The optimum operating condition for electrofusion ST was the 37°C group without Ca2+.

Altered redox homeostasis in steroid-depleted follicles attenuates hCG regulation of follicular events: Cross-talk between endocrine and IGF axis in maturing oocytes

Steroids and insulin-like growth factors (Igfs) are indispensable for folliculogenesis and reproductive fitness in the vertebrate ovary. The intrafollicular redox balance is also of immense importance for ovarian follicles wherein low levels of ROS are being utilized for cell signalling and regulation of gene expression; its excess may interfere with normal physiological processes leading to ovotoxicity. However, the functional relevance of ovarian steroidogenesis in maintaining the follicular microenvironment with coordinated redox homeostasis and intra-ovarian growth factors axis is relatively less understood. Using zebrafish full-grown (FG) ovarian follicles in vitro, our study shows that blocking steroid biosynthesis with anti-steroidal drugs, DL-aminoglutethimide (AG) or Trilostane (Trilo), prevents hCG (LH analogue)-induced StAR expression concomitant with a robust increase in intrafollicular ROS levels. Congruent with heightened intracellular levels of superoxide anions (O2•–) and hydrogen peroxide (H2O2), priming with AG or Trilo abrogates the transcript abundance of major antioxidant enzyme genes (SOD1, SOD2, and CAT) in hCG-stimulated follicles. Significantly, blocking steroidogenesis attenuates transcript abundance of HSP70 but elevates NOX4 expression potentially through ERα-mediated pathway. Importantly, disrupted redox balance in AG/Trilo pre-incubated FG follicles negatively impacts hCG-mediated activation of PKA/CREB signaling and transcriptional activation of igf ligands. Elevated ROS attenuation of antioxidant defense parameters and impaired endocrine and autocrine/paracrine homeostasis converge upon reduced p34cdc2 (Thr-161) phosphorylation, a reliable marker for MPF activation, and resumption of meiotic G2-M1 transition in hCG-treated follicles. Collectively, altered redox homeostasis in steroid-depleted follicles has a significant negative influence on GTH (LH) regulation of follicular events, specifically Igf synthesis, meiotic maturational competence and ovarian fitness.

Inhibition of CDK4/6 kinases causes production of aneuploid oocytes by inactivating the spindle assembly checkpoint and accelerating first meiotic progression

Cyclin D-CDK4/6 complex mediates the transition from the G1 to S phase in mammalian somatic cells. Meiotic oocytes pass through the G2/M transition and complete the first meiosis to reach maturation at the metaphase of meiosis II without intervening S phase, while Cyclin D-CDK4/6 complex is found to express during meiotic progression. Whether Cyclin D-CDK4/6 complex regulates meiotic cell cycle progression is not known. Here, we found its different role in oocyte meiosis: Cyclin D-CDK4/6 complex served as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Inhibition of CDK4/6 kinases disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, but unexpectedly accelerated meiotic progression by inactivating SAC, consequently resulting in production of aneuploid oocytes. Further studies showed that the MPF activity decrease before first polar body extrusion was accelerated probably by inactivation of the SAC to promote ubiquitin-mediated cyclin B1 degradation. Taken together, these data reveal a novel role of Cyclin D-CDK4/6 complex in mediating control of the SAC in female meiosis I.

FBXO34 Regulates the G2/M Transition and Anaphase Entry in Meiotic Oocytes.

There are two important events in oocyte meiotic maturation, the G2/M transition and metaphase I progression. Thousands of proteins participate in regulating oocyte maturation, which highlights the importance of the ubiquitin proteasome system (UPS) in regulating protein synthesis and degradation. Skp1–Cullin–F-box (SCF) complexes, as the best characterized ubiquitin E3 ligases in the UPS, specifically recognize their substrates. F-box proteins, as the variable adaptors of SCF, can bind substrates specifically. Little is known about the functions of the F-box proteins in oocyte maturation. In this study, we found that depletion of FBXO34, an F-box protein, led to failure of oocyte meiotic resumption due to a low activity of MPF, and this phenotype could be rescued by exogenous overexpression of CCNB1. Strikingly, overexpression of FBXO34 promoted germinal vesicle breakdown (GVBD), but caused continuous activation of spindle assembly checkpoint (SAC) and MI arrest of oocytes. Here, we demonstrated that FBXO34 regulated both the G2/M transition and anaphase entry in meiotic oocytes.

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†.

The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4hpa (P<0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1hpa and IVF increased p-CDK1Thr14-Tyr15 at 17hours postfertilization (hpf) (P<0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4hpa (P<0.05) and its activity at 4 and 1hpa, respectively (P<0.05). Meanwhile, IVF increased p-ERK1/2 at 6hpf (P<0.05); however, its kinase activity decreased at 6hpf (P<0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4hpa (P<0.05). p-P38 was only observed at 1hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

Chronic cadmium exposure causes oocyte meiotic arrest by disrupting spindle assembly checkpoint and maturation promoting factor

Cadmium (Cd) is a bioaccumulative heavy metal element with potential toxicity on the female reproductive system, but the exact molecular mechanisms have not yet been clearly defined. In this study, female mice were exposed to 0.5 mg/kg/day of CdCl2 for 60 consecutive days. We found that chronic Cd exposure significantly decreased the fecundity of female mice by affecting oocyte meiotic progression as indicated by disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, consequently resulting in aneuploid oocytes. Further studies showed that the periodic fluctuations of MPF activity and cyclin B1 expression were disturbed in Cd-exposed oocytes probably by affecting the spindle assembly checkpoint protein Bub3. In addition, Cd exposure induced oxidative stress as indicated by an increased level of reactive oxygen species and apoptosis in oocytes, leading to oocyte quality deterioration. Taken together, these data suggest that Cd exposure causes disrupted molecular events of meiotic progression and deterioration of oocyte quality via oxidative stress, leading to decrease of female fertility.

Managing the Oocyte Meiotic Arrest-Lessons from Frogs and Jellyfish.

During oocyte development, meiosis arrests in prophase of the first division for a remarkably prolonged period firstly during oocyte growth, and then when awaiting the appropriate hormonal signals for egg release. This prophase arrest is finally unlocked when locally produced maturation initiation hormones (MIHs) trigger entry into M-phase. Here, we assess the current knowledge of the successive cellular and molecular mechanisms responsible for keeping meiotic progression on hold. We focus on two model organisms, the amphibian Xenopus laevis, and the hydrozoan jellyfish Clytia hemisphaerica. Conserved mechanisms govern the initial meiotic programme of the oocyte prior to oocyte growth and also, much later, the onset of mitotic divisions, via activation of two key kinase systems: Cdk1-Cyclin B/Gwl (MPF) for M-phase activation and Mos-MAPkinase to orchestrate polar body formation and cytostatic (CSF) arrest. In contrast, maintenance of the prophase state of the fully-grown oocyte is assured by highly specific mechanisms, reflecting enormous variation between species in MIHs, MIH receptors and their immediate downstream signalling response. Convergence of multiple signalling pathway components to promote MPF activation in some oocytes, including Xenopus, is likely a heritage of the complex evolutionary history of spawning regulation, but also helps ensure a robust and reliable mechanism for gamete production.

Effects of Ferrocenyl 4-(Imino)-1,4-Dihydro-quinolines on Xenopus laevis Prophase I - Arrested Oocytes: Survival and Hormonal-Induced M-Phase Entry.

Xenopus oocytes were used as cellular and molecular sentinels to assess the effects of a new class of organometallic compounds called ferrocenyl dihydroquinolines that have been developed as potential anti-cancer agents. One ferrocenyl dihydroquinoline compound exerted deleterious effects on oocyte survival after 48 h of incubation at 100 μM. Two ferrocenyl dihydroquinoline compounds had an inhibitory effect on the resumption of progesterone induced oocyte meiosis, compared to controls without ferrocenyl groups. In these inhibited oocytes, no MPF (Cdk1/cyclin B) activity was detected by western blot analysis as shown by the lack of phosphorylation of histone H3. The dephosphorylation of the inhibitory Y15 residue of Cdk1 occurred but cyclin B was degraded. Moreover, two apoptotic death markers, the active caspase 3 and the phosphorylated histone H2, were detected. Only 7-chloro-1-ferrocenylmethyl-4-(phenylylimino)-1,4-dihydroquinoline (8) did not show any toxicity and allowed the assembly of a histologically normal metaphase II meiotic spindle while inhibiting the proliferation of cancer cell lines with a low IC50, suggesting that this compound appears suitable as an antimitotic agent.

SKP1 drives the prophase I to metaphase I transition during male meiosis.

The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.

Survivin regulates chromosome segregation by modulating the phosphorylation of Aurora B during porcine oocyte meiosis

ABSTRACTSURVIVIN is an essential chromosomal passenger complex (CPC) subunit and participates in cell division. In this study, we used porcine oocyte as a model to investigate the roles of Survivin during porcine oocyte maturation. Survivin was highly expressed in germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages oocytes, mainly localized in the GV at GV stage and on the chromosomes after GVBD. We have used RNA interference to specifically deplete Survivin in oocytes during in vitro maturation (IVM). Immunofluorescence assay showed that Survivin-depleted oocytes failed to produce polar body in meiosisⅠ (failed to complete cytokinesis), and they were arrested in metaphaseⅠwith misaligned chromosomes. The homologous chromosomes in Survivin-depleted oocytes could not be separated normally. Moreover, both the phosphorylation levels of Aurora B and the mRNA level of Mad2L1 related to spindle assembly checkpoint (SAC) was decreased in Survivin-depleted oocytes, which thus inhibited the degradation of Cyclin B1 (CCNB1) to complete meiosis. Taken together, we conclude that Survivin is an important mediator of centromere and midbody docking of Aurora-B as well as its activity and regulates SAC and MPF activity during meiosis in porcine oocytes.

Increased Expression of Maturation Promoting Factor Components Speeds Up Meiosis in Oocytes from Aged Females

The rate of chromosome segregation errors that emerge during meiosis I in the mammalian female germ line are known to increase with maternal age; however, little is known about the underlying molecular mechanism. The objective of this study was to analyze meiotic progression of mouse oocytes in relation to maternal age. Using the mouse as a model system, we analyzed the timing of nuclear envelope breakdown and the morphology of the nuclear lamina of oocytes obtained from young (2 months old) and aged females (12 months old). Oocytes obtained from older females display a significantly faster progression through meiosis I compared to the ones obtained from younger females. Furthermore, in oocytes from aged females, lamin A/C structures exhibit rapid phosphorylation and dissociation. Additionally, we also found an increased abundance of MPF components and increased translation of factors controlling translational activity in the oocytes of aged females. In conclusion, the elevated MPF activity observed in aged female oocytes affects precocious meiotic processes that can multifactorially contribute to chromosomal errors in meiosis I.

Developmental competence of buffalo (Bubalus bubalis) denuded oocytes cocultured with cumulus cells: Protective role of cumulus cells

The objectives of the present study were to evaluate the developmental competence of buffalo denuded oocytes (DOs) cocultured with cumulus cells (CCs) during in vitro maturation, and to investigate the mechanisms by which CCs promote oocyte maturation and development. Buffalo oocytes were matured in vitro for 24 h in three groups: (1) intact cumulus–oocyte complexes (COCs) (2) DOs cocultured with CCs (DOsCC), and (3) DOs cultured alone (DOs). Matured oocytes were used to determine the relative mRNA abundance of Gdf-9, Bmp15, Zar1, Caspase-3, Bcl-2, Zp2, Zp3, Cd9 and Pde3a by Rt-qPCR and CASPASE-3 protein expression by immunofluorescence. The intracellular content of cGMP, cAMP and MPF activity and the rate of embryonic development were also assessed. Results of the present study showed that in DOs, the relative mRNA abundance of Gdf-9, Bmp15, and Cd9 significantly (P < 0.05) decreased, whereas Caspase-3 (mRNA and protein levels), Bcl-2, and Pde3a exhibited higher expression than DOsCC and COCs. However, there was no significant difference among the groups in the expression level of Zar-1, Zp2, and Zp3. The intracellular content of cAMP and MPF activity was notably higher (P < 0.05) in DOs compared to COCs and DOsCC. There was no significant difference between COCs and DOsCC in cGMP content, which was significantly lower (P < 0.05) in DOs. Moreover, the cleavage and blastocyst rates were 58.4 ± 1.8%, 43.7 ± 1.1%, 18.4 ± 0.9% and 18.0 ± 1.3%, 11.0 ± 0.9% and 4.5 ± 0.6% in COCs, DOsCC and DOs groups, respectively. In conclusion, the presence of CCs protects buffalo DOs from apoptosis and promotes maturation through regulation of the intracellular content of cAMP and MPF activity and improves the fertilizing capacity of oocytes through modulation of the gamete fusion gene, Cd9.

Bistability analysis in mathematical model of M-phase control in Xenopus oocyte extracts

To gain more insight into the molecular mechanism that regulates eukaryotic cell cycle, we propose a modification to an existing Mathematical model of mitosis-promoting factor control in Xenopus oocyte extract. To achieve this, we redirect how cell exits from mitosis using ubiquitin ligase enzyme APCP:Cdc20 which is acting as a modifier in cyclin degradation. We present a modified wiring map using combination of mass action kinetics and Michaelis–Menten kinetics to represent the biochemical processes. This results in a system of non-linear ordinary differential equations. The equilibria of the system were analyzed analytically. Using MPF as a bifurcation parameter, we analytically derived conditions under which bistability can occur in the MPF activation module. We find that for bistability to occur, it is necessary that the rate of phosphorylation of Wee1 must be greater than the rate of its dephosphorylation and that the concentration of MPF must lie in certain range of parameter values. Numerical simulations conducted show that the time taken to exit the cell cycle is shorter than those published in some reports and the sensitivity analysis results suggest that the rate of synthesis of cyclin is the most sensitive parameter in the dynamics of cell cycle. Through numerical experiments, we found new parameter sets that will give rise to bistability in the MPF activation module.

Toxic effects of atrazine on porcine oocytes and possible mechanisms of action.

Because atrazine is a widely used herbicide, its adverse effects on the reproductive system have been extensively researched. In this study, we investigated the effects of atrazine exposure on porcine oocyte maturation and the possible mechanisms. Our results showed that the rates of oocyte maturation significantly decreased after treatment with 200 μM atrazine in vitro. Atrazine treatment resulted in abnormal spindle morphology but did not affect actin distribution. Atrazine exposure not only triggered a DNA damage response but also decreased MPF levels in porcine oocytes. Our results also revealed that atrazine worsened porcine oocyte quality by causing excessive accumulation of superoxide radicals, increasing cathepsin B activity, and decreasing the GSH level and mitochondrial membrane potential. Furthermore, atrazine decreased developmental competence of porcine oocytes up to the blastocyst stage and changed some properties: cell numbers, apoptosis, and related gene expression levels. Collectively, our results indicate that porcine oocyte maturation is defective after atrazine treatment at least through disruption of spindle morphology, MPF activity, and mitochondrial function and via induction of DNA damage, which probably reduces developmental competence.

Combined Treatment with Demecolcine and 6-Dimethylaminopurine during Postactivation Improves Developmental Competence of Somatic Cell Nuclear Transfer Embryos in Pigs

ABSTRACTThis study determined the effects of postactivation treatment with demecolcine and/or 6-dimethylaminopurine (6-DMAP) on in vivo and in vitro developmental competence of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were treated for 4 hours with 0.4 µg/mL demecolcine, 2 mM 6-DMAP, or both after electric activation, then transferred to surrogate pigs or cultured for 7 days. The formation rate of SCNT embryos with a single pronucleus was higher in combined treatment with demecolcine and 6-DMAP (95.2%) than treatment with demecolcine alone (87.1%). Blastocyst formation of SCNT embryos was significantly increased in combined treatment with demecolcine and 6-DMAP (48.7%) compared with demecolcine (22.2%) or 6-DMAP alone (37.3%). Fluctuation of maturation promoting factor activity showed different patterns among various postactivation treatments. Pregnancy was established in 1 of 5 surrogates after transfer of SCNT embryos that were treated with demecolcine and 6-DMAP. The pregnant surrogate delivered one healthy live piglet. The results of our study demonstrated that postactivation treatment with demecolcine and 6-DMAP together improved preimplantation development and supported normal in vivo development of SCNT pig embryos, probably influencing MPF activity and nuclear remodeling, including induction of single pronucleus formation after electric activation.

Quercetin delays postovulatory aging of mouse oocytes by regulating SIRT expression and MPF activity.

If no fertilization occurs at an appropriate time after ovulation, oocyte quality deteriorates rapidly as a process called postovulatory aging. Because the postovulatory aging of oocytes has detrimental effects on embryo development and offspring, many efforts have been made to prevent oocyte aging. Here we showed that quercetin prevented the decline in oocyte quality during postovulatory aging of oocytes. Quercetin treatment reduced aging-induced morphological changes and reactive oxygen species accumulation. Moreover, quercetin attenuated the aging-associated abnormalities in spindle organization and mitochondrial distribution, preventing decrease of SIRT expression and histone methylation. Quercetin also ameliorated the decrease in maturation-promoting factor activity and the onset of apoptosis during postovulatory aging. Furthermore, quercetin treatment during postovulatory aging improves early embryo development. Our results demonstrate that quercetin relieves deterioration in oocyte quality and improves subsequent embryo development.

The Role of Glucose Metabolism on Porcine Oocyte Cytoplasmic Maturation and Its Possible Mechanisms.

In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 μM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 μM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity.

CenpH regulates meiotic G2/M transition by modulating the APC/CCdh1-cyclin B1 pathway in oocytes

ABSTRACTMeiotic resumption (G2/M transition) and progression through meiosis I (MI) are two key stages for producing fertilization-competent eggs. Here, we report that CenpH, a component of the kinetochore inner plate, is responsible for G2/M transition in meiotic mouse oocytes. Depletion of CenpH by morpholino injection decreased cyclin B1 levels, resulting in attenuation of maturation-promoting factor (MPF) activation, and severely compromised meiotic resumption. CenpH protects cyclin B1 from destruction by competing with the action of APC/CCdh1. Impaired G2/M transition after CenpH depletion could be rescued by expression of exogenous cyclin B1. Unexpectedly, blocking CenpH did not affect spindle organization and meiotic cell cycle progression after germinal vesicle breakdown. Our findings reveal a novel role of CenpH in regulating meiotic G2/M transition by acting via the APC/CCdh1-cyclin B1 pathway.