Maturation-promoting Factor Activity Research Articles

G-Protein-Coupled Receptor Kinase 2 Inhibition Induces Meiotic Arrest by Disturbing Ca2+ Release in Porcine Oocytes.

G-protein-coupled receptor kinase 2 (GRK2) interacts with Gβγ and Gαq, subunits of G-protein alpha, to regulate cell signalling. The second messenger inositol trisphosphate, produced by activated Gαq, promotes calcium release from the endoplasmic reticulum (ER) and regulates maturation-promoting factor (MPF) activity. This study aimed to investigate the role of GRK2 in MPF activity during the meiotic maturation of porcine oocytes. A specific inhibitor of GRK2 (βi) was used in this study. The present study showed that GRK2 inhibition increased the percentage of oocyte arrest at the metaphase I (MI) stage (control: 13.84 ± 0.95%; βi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; βi: 65.40 ± 1.14%). The level of phospho-GRK2 decreased in the treated group, suggesting that GRK2 activity was reduced upon GRK2 inhibition. Furthermore, the addition of βi decreased Ca2+ release from the ER. The protein levels of cyclin B and cyclin-dependent kinase 1 were higher in the treatment group than those in the control group, indicating that GRK2 inhibition prevented a decrease in MPF activity. Collectively, GRK2 inhibition induced meiotic arrest at the MI stage in porcine oocytes by preventing a decrease in MPF activity, suggesting that GRK2 is essential for oocyte meiotic maturation in pigs.

Fangchinoline inhibits mouse oocyte meiosis by disturbing MPF activity

Fangchinoline (FA) is an alkaloid derived from the traditional Chinese medicine Fangji. Numerous studies have shown that FA has a toxic effect on various cancer cells, but little is known about its toxic effects on germ cells, especially oocytes. In this study, we investigated the effects of FA on mouse oocyte maturation and its potential mechanisms. Our results showed that FA did not affect meiosis resumption but inhibited the first polar body extrusion. This inhibition is not due to abnormalities at the organelle level, such as chromosomes and mitochondrial, which was proved by detection of DNA damage and reactive oxygen species. Further studies revealed that FA arrested the oocyte at the metaphase I stage, and this arrest was not caused by abnormal kinetochore-microtubule attachment or spindle assembly checkpoint activation. Instead, FA inhibits the activity of anaphase-promoting complexes (APC/C), as evidenced by the inhibition of CCNB1 degeneration. The decreased activity of APC/C may be due to a reduction in CDC25B activity as indicated by the high phosphorylation level of CDC25B (Ser323). This may further enhance Maturation-Promoting Factor (MPF) activity, which plays a critical role in meiosis. In conclusion, our study suggests that the metaphase I arrest caused by FA may be due to abnormalities in MPF and APC/C activity.

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.

Effect of Growth Factors and Hormones during In Vitro Growth Culture of Cumulus-Oocyte-Complexes Derived from Small Antral Follicles in Pigs

This study evaluated the effect of various growth factors and hormones in an in vitro growth (IVG) medium on the in vitro maturation (IVM) and developmental competence of oocytes derived from small antral follicles (SAFs) in pigs. Cumulus-oocyte complexes (COCs) derived from SAFs were either untreated or treated with epidermal growth factor (EGF), insulin-like factor-1 (IGF-1), insulin, or growth hormone (GH) for 2 days of IVG. Following IVG, COCs were cultured for maturation, and IVM oocytes were induced for parthenogenesis (PA). During IVG, the nuclear maturation of oocytes was significantly increased by the insulin treatment compared to other treatments. Moreover, the insulin treatment significantly increased blastocyst formation after PA relative to the No-IVG, control, EGF, and GH treatments. The cumulus expansion score after IVG-IVM was significantly higher in the insulin group than in the other groups. The glutathione (GSH) contents in IVM oocytes were increased through treatment with IGF, insulin, and GH compared to those of No-IVG oocytes. The level of reactive oxygen species (ROS) in IVM oocytes in all treatment groups was significantly lower after IVG culture than in the No-IVG group. The maturation-promoting factor (MPF) activity after IVM in the insulin-treated oocytes was significantly higher than that of the oocytes treated with EGF, IGF-1, and GH. In conclusion, this study demonstrates that insulin treatment during IVG culture improves the maturational and developmental competence of oocytes derived from SAFs in pigs through its effect on cumulus cell expansion and cytoplasmic microenvironments, such as GSH, ROS, and MPF activity.

Beneficial effects of fibroblast growth factor 10 supplementation during in vitro maturation of buffalo cumulus-oocyte complexes

Fibroblast growth factor 10 (FGF10) is an important regulator of the mammalian cumulus-oocyte complex that plays a crucial role in oocyte maturation. In this study, we investigated the effects of FGF10 supplementation on the in vitro maturation (IVM) of buffalo oocytes and its related mechanisms. During IVM, the maturation medium was supplemented with a range of concentrations of FGF10 (0, 0.5, 5, and 50 ng/mL) and the resulting effects were corroborated using aceto-orcein staining, TUNEL apoptosis assay, detection of Cdc2/Cdk1 kinase in oocytes, and real-time quantitative PCR. In matured oocytes, the 5 ng/mL-FGF10 treatment resulted in a significantly increased nuclear maturation rate, which increased the activity of maturation-promoting factor (MPF) and enhanced buffalo oocyte maturation. Furthermore, it treatment significantly inhibited the apoptosis of cumulus cells, while simultaneously promoting its proliferation and expansion. This treatment also increased the absorption of glucose in cumulus cells. Thus, our results indicate that adding an appropriate concentration of FGF10 to a maturation medium during IVM can be beneficial to the maturation of buffalo oocytes and improve the potential of embryo development.

Effects of postovulatory oviduct changes and female restraint stress on aging of mouse oocytes.

Postovulatory oocyte aging is one of the major causes for human early pregnancy loss and for a decline in the population of some mammalian species. Thus, the mechanisms for oocyte aging are worth exploring. While it is known that ovulated oocytes age within the oviduct and that female stresses impair embryo development by inducing apoptosis of oviductal cells, it is unknown whether the oviduct and/or female stress would affect postovulatory oocyte aging. By comparing aging characteristics, including activation susceptibility, maturation-promoting factor activity, developmental potential, cytoplasmic fragmentation, spindle/chromosome morphology, gene expression, and cumulus cell apoptosis, this study showed that oocytes aged faster in vivo in restraint-stressed mice than in unstressed mice than in vitro. Our further analysis demonstrated that oviductal cells underwent apoptosis with decreased production of growth factors with increasing time after ovulation, and female restraint facilitated apoptosis of oviductal cells. Furthermore, mating prevented apoptosis of oviductal cells and alleviated oocyte aging after ovulation. In conclusion, the results demonstrated that mouse oviducts underwent apoptosis and facilitated oocyte aging after ovulation; female restraint facilitated oocyte aging while enhancing apoptosis of oviductal cells; and copulation ameliorated oviductal apoptosis and oocyte aging.

Positive Feedback Stimulation of Ccnb1 and Mos mRNA Translation by MAPK Cascade During Mouse Oocyte Maturation.

In mammalian species, both the maturation promoting factor (MPF) and the mitogen-activated protein kinase (MAPK) cascade play critical roles in modulating oocyte meiotic cell-cycle progression. MPF is a critical heterodimer composed of CDK1 and cyclin B1. Activation of MPF and ERK1/2 requires the activation of maternal Ccnb1 and Mos mRNAs translation, respectively. The phosphorylation and degradation of CPEB1 that triggered by ERK1/2 is a principal mechanism of activating maternal mRNA translation. However, the interplay of these two key kinases in mediating mammalian translational activation of cytoplasmic mRNAs during oocyte maturation is unclear. We prove evidence that the translational activation of Ccnb1 transcripts containing a long 3′-UTR during meiotic resumption works in an ERK1/2-dependent way. A low level of ERK1/2 activation was detected prior to meiotic resumption. Precocious activation of MAPK signaling in germinal vesicle stage oocytes promotes the translation of Ccnb1 mRNA and meiotic maturation. Inhibition or precocious activation of CDK1 activity has an appreciable effect on the translation of Ccnb1 mRNA, suggesting that both kinases are required for Ccnb1 mRNA translational activation. CDK1 triggers phosphorylation, but not degradation, of CPEB1 in oocytes; the degradation of CPEB1 was only triggered by ERK1/2. Moreover, the translational activation of Mos mRNA is regulated by ERK1/2 and cytoplasmic polyadenylation elements too. Taken together, the cooperation and positive feedback activation of ERK1/2 and CDK1 lead to the fine-tuning of mRNA translation and cell-cycle progression during mouse oocyte maturation.

Modulation of phosphatidylinositol 3-kinase activity during in vitro oocyte maturation increases the production of bovine blastocysts.

This study aimed to evaluate the effect of regulating phosphatidylinositol 3-kinase (PI3K) activity on the kinetics of oocyte nuclear maturation and the blastocyst rate. To evaluate oocyte viability, nuclear maturation rate and in vitro embryo production, cumulus-oocyte complexes (COCs) were maintained for 0, 10 min, 6 h or 22 h in TCM 199 medium supplemented with 20 nM wortmannin, an inhibitor of PI3K. After each period, COCs were transferred to the same medium without wortmannin and kept under the same conditions until completion of 22 h of in vitro maturation (IVM). To evaluate the effect of time on progression of nuclear maturation, COCs cultivated with 20 nM wortmannin was maintained for 22, 28 or 34 h of IVM. To determine the effect of wortmannin on the activity of maturation-promoting factor (MPF), COCs were kept under IVM conditions in the presence of the inhibitor for 0, 1, 3, 6, or 8 h. Exposure of COCs to wortmannin decreased (P < 0.05) the percentage of oocytes that reached metaphase II (MII) up to 22 h, MPF activity and reduced PI3K activity by 30%. However, after 28 and 34 h, 70% of oocytes reached the MII stage in the presence of inhibitor Moreover, COCs matured in the presence of wortmannin showed an increase (P < 0.05) in the blastocyst rate. These findings suggested that the regulation of the PI3K activity during IVM of bovine COCs interfered with the meiotic progression due to control of MPF activity, positively affecting the blastocyst rate.

Role of AMP-activated protein kinase during postovulatory aging of mouse oocytes†

Studies suggested that postovulatory oocyte aging might be prevented by maintaining a high maturation-promoting factor (MPF) activity. Whether AMP-activated protein kinase (AMPK) plays any role in postovulatory oocyte aging is unknown. Furthermore, while activation of AMPK stimulates meiotic resumption in mouse oocytes, it inhibits meiotic resumption in pig and bovine oocytes. Thus, the species difference in AMPK regulation of oocyte MPF activities is worth in-depth studies. This study showed that AMPK activation with metformin or 5-aminoimidazole- 4-carboxamide- 1-beta-d- ribofuranoside and inactivation with compound C significantly increased and decreased, respectively, the activation susceptibility (AS) and other aging parameters in aging mouse oocytes. While AMPK activity increased, MPF activity and cyclic adenosine monophosphate (cAMP) decreased significantly with time post ovulation. In vitro activation and inactivation of AMPK significantly decreased and increased the MPF activity, respectively. MPF upregulation with MG132 or downregulation with roscovitine completely abolished the effects of AMPK activation or inactivation on AS of aging oocytes, respectively. AMPK facilitated oocyte aging with increased reactive oxygen species (ROS) and cytoplasmic calcium. Furthermore, treatment with Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitors significantly decreased AS and AMPK activation. Taken together, the results suggested that AMPK facilitated oocyte aging through inhibiting MPF activities, and postovulatory oocyte aging activated AMPK with decreased cAMP by activating CaMKs via increasing ROS and cytoplasmic calcium.

CENP-T regulates both the G2/M transition and anaphase entry by acting through CDH1 in meiotic oocytes.

Oocyte meiotic maturation failure is one of the major causes for female infertility. Meiotic resumption (the G2/M transition) and progression through metaphase I (MI) are two critical stages of oocyte meiotic maturation. Here, we report that centromere protein T (CENP-T), an internal kinetochore protein, plays a critical role in meiotic resumption of mouse oocytes. Depletion of CENP-T by siRNA injection increased the CDH1 (also known as FZR1) level, resulting in increased activity of the anaphase-promoting complex (APC)-CDH1 complex, and further leading to decreased levels of the cyclin protein CCNB1, attenuated maturation-promoting factor (MPF) activity, and finally severely compromised meiotic resumption. The impaired meiotic resumption caused by CENP-T depletion could be rescued by overexpression of exogenous CCNB1 or knockdown of endogenous CDH1. Overexpression of exogenous CENP-T resulted in decreased CDH1 levels, which accelerated the progression of G2/M transition, and accelerated meiotic cell cycle progression after germinal vesicle breakdown (GVBD). Unexpectedly, spindle organization after GVBD was not affected by the overexpression, but the distribution of chromosomes was affected. Our findings reveal a novel role for CENP-T in regulating meiotic progression by acting through CDH1.

The chromatin remodeler Snf2h is essential for oocyte meiotic cell cycle progression.

Oocytes are indispensable for mammalian life. Thus, it is important to understand how mature oocytes are generated. As a critical stage of oocytes development, meiosis has been extensively studied, yet how chromatin remodeling contributes to this process is largely unknown. Here, we demonstrate that the ATP-dependent chromatin remodeling factor Snf2h (also known as Smarca5) plays a critical role in regulating meiotic cell cycle progression. Females with oocyte-specific depletion of Snf2h are infertile and oocytes lacking Snf2h fail to undergo meiotic resumption. Mechanistically, depletion of Snf2h results in dysregulation of meiosis-related genes, which causes failure of maturation-promoting factor (MPF) activation. ATAC-seq analysis in oocytes revealed that Snf2h regulates transcription of key meiotic genes, such as Prkar2b, by increasing its promoter chromatin accessibility. Thus, our studies not only demonstrate the importance of Snf2h in oocyte meiotic resumption, but also reveal the mechanism underlying how a chromatin remodeling factor can regulate oocyte meiosis.

Microcystin-LR influences the in vitro oocyte maturation of zebrafish by activating the MAPK pathway

Harmful cyanobacteria and their production of microcystins (MCs) exert significant toxicity on reproduction of fish, especially the process of oogenesis. Our previous studies demonstrated that MCs have negative impacts on the quantity and quality of mature oocytes in female zebrafish. However, the underlying mechanisms of MCs disrupting oocyte maturation (OM) have been rarely reported. In the present study, in vitro oocytes (immature) were separated from zebrafish and treated with 1, 10, 100 μg/L MC-LR. The serine/threonine protein phosphatase 2A (PP2A) activity was downregulated significantly in oocytes exposed to 10 and 100 μg/L MC-LR for both 2 and 4 h. The phosphorylation levels of mitogen-activated protein kinase (MAPK) were detected without noticeable change in all oocytes treated with MC-LR for 2 h, whereas the activated levels of MAPK subtypes (ERK, p38 and JNK) increased remarkably in the 100 μg/L MC-LR treatment of 4 h. In the oocytes exposed to 100 μg/L MC-LR for 4 h, germinal vesicle breakdown (GVBD) rates changed abnormally and maturation-promoting factor (MPF) activity increased significantly, in accordance with the upregulation of Cyclin B protein levels. Moreover, the MAPK inhibitors (10 μM) were applied to explore the role of MAPK subtypes during MC-LR influencing OM and results showed that ERK inhibitor U0126 and p38 inhibitor SB203580 mitigated the effects of 100 μg/L MC-LR-induced MAPK hyper-phosphorylation and elevated GVBD in the oocytes. In conclusion, the present study indicates that microcystins disrupt the meiotic maturation by the pathway of MC-PP2A-MAPK-OM due to the phosphorylation disorder in oocytes.

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development.

NEK5, a member of never in mitosis-gene A-related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA-mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin-dependent kinase 1 in oocytes, resulting in a decrease of maturation-promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1-cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.

Thiamethoxam inhibits blastocyst expansion and hatching via reactive-oxygen species–induced G2 checkpoint activation in pigs

Thiamethoxam (TMX) is a neonicotinoid insecticide. It has specific high toxicity to insects. Residues of TMX have been detected in various crops. Early embryo quality is vital for fertility. Excessive production of reactive oxygen species (ROS) can override embryonic antioxidant defenses, producing oxidative stress that triggers apoptosis, necrosis, and/or permanent DNA damage responses in the early embryo. Comparative studies have indicated that TMX hepatotoxicity is significant in mammals in acute tests, but little is known about accumulated chronic toxicity in early embryonic development. Porcine embryos were obtained here by the parthenogenetic activation of meiosis II oocytes and cultured in the PZM-5 medium with or without TMX. These embryos were evaluated by various methods. The expansion and hatching of blastocysts treated with TMX decreased by 21.73% and 16.71%, respectively, as compared with controls. In an analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation, the rate of cell proliferation was 44.33% lower as compared with expanded blastocysts of the control group. ROS and γH2AX levels were higher in the TMX group than in the control group. Real-time reverse-transcription polymerase chain reaction showed that Sod1 expression increased and the expression of Mnsod, Gpx1, Igta5, and Cox2 decreased. A CDK1 kinase assay revealed that maturation-promoting factor (MPF) activity diminished by 31.41% in expanding blastocysts. In conclusion, these results suggest that TMX inhibits blastocyst expansion and hatching by ROS-induced DNA damage checkpoint activation, which inhibits the activation of MPF and cell cycle progression in porcine blastocysts.

Cumulus cell-released tumor necrosis factor (TNF)-α promotes post-ovulatory aging of mouse oocytes.

Although previous studies indicated that cumulus cells (CCs) accelerate oocyte aging by releasing soluble factors, the factors have yet to be characterized. While demonstrating that CCs promoted oocyte aging by releasing soluble Fas ligand (sFasL), our recent study suggested that CCs might secrete other factors to mediate oocyte aging as well. This study tested whether CCs accelerate oocyte aging by secreting tumor necrosis factor (TNF)-α. The results showed that mouse CCs undergoing apoptosis released soluble TNF-α (sTNF-α) during in vitro aging. While ethanol activation rates were higher, the maturation-promoting factor (MPF) activity was lower significantly after culture of cumulus-denuded oocytes (DOs) in medium conditioned with CCs for 36 h than in medium conditioned for 24 h. Aging mouse oocytes expressed TNF-receptor 1. The CCs released equal amounts of sTNF-α and sFasL during aging in vitro, and the TNF-α-knockdown CCs secreted less sFasL than the control CCs did. Treatment of DOs in vitro with sTNF-α significantly accelerated their aging. The aging-promoting effect of sTNF-α was significantly reduced in TNF-α-knocked-down CCs and in CCs from the TNF-α-knockout mice. It is concluded that mouse CCs accelerate oocyte aging by secreting sTNF-α as well as sFasL.

Activity of MPF and expression of its related genes in mouse MI oocytes exposed to cadmium

Research has revealed that cadmium can disrupt ovarian function; however, few reports have focused on MI oocytes meiotic progression, especially the activity of maturation promoting factor (MPF) and its related genes (Cdk1, Ccnb1, and Cdc25b) expression. In this study, GV oocytes cultured in vitro for 0, 6, and 9 hours with five groups (control and doses of 0.05, 0.5, 2.5, and 5 μM Cd). At the same dose of cadmium but different exposure time: compared with 0h, Periodic changes in MPF activity were changed and continuously increased over time. The mRNA and protein expression of each MPF-related gene in different cadmium dose groups were changed compared with that of 0h. At the same exposure time but different dose of cadmium: compared with control group, MPF activity, mRNA and protein expressions of each MPF-related gene in all the cadmium exposure groups were increased at 9h after exposure. Cadmium maintains the high MPF activity in mouse MI oocytes during its meiotic process and disturbs the periodic change of MPF activity; meanwhile, cadmium exposure promotes the syntheses of MPF-related gene, which may be one of the molecular mechanisms for the maintenance of high MPF activity, and ultimately prevents the meiotic progression in oocytes.

Maintained MPF Level after Oocyte Vitrification Improves Embryonic Development after IVF, but not after Somatic Cell Nuclear Transfer.

Levels of maturation-promoting factor (MPF) in oocytes decline after vitrification, and this decline has been suggested as one of the main causes of low developmental competence resulting from cryoinjury. Here, we evaluated MPF activity in vitrified mouse eggs following treatment with caffeine, a known stimulator of MPF activity, and/or the proteasome inhibitor MG132. Collected MII oocytes were vitrified and divided into four groups: untreated, 10 mM caffeine (CA), 10 μM MG132 (MG), and 10 mM caffeine +10 μM MG132 (CA+MG). After warming, the MPF activity of oocytes and their blastocyst formation and implantation rates in the CA, MG, and CA+MG groups were much higher than those in the untreated group. However, the cell numbers in blastocysts did not differ among groups. Analysis of the effectiveness of caffeine and MG132 for improving somatic cell nuclear transfer (SCNT) technology using cryopreserved eggs showed that supplementation did not improve the blastocyst formation rate of cloned mouse eggs. These results suggest that maintaining MPF activity after cryopreservation may have a positive effect on further embryonic development, but is unable to fully overcome cryoinjury. Thus, intrinsic factors governing the developmental potential that diminish during oocyte cryopreservation should be explored.

The potential roles of c-Jun N-terminal kinase (JNK) during the maturation and aging of oocytes produced by a marine protostome worm.

Previous investigations have indicated that c-Jun N-terminal kinase (JNK) regulates the maturation and aging of oocytes produced by deuterostome animals. In order to assess the roles of this kinase in a protostome, oocytes of the marine nemertean worm Cerebratulus were stimulated to mature and subsequently aged before being probed with phospho-specific antibodies against active forms of JNK and maturation-promoting factor (MPF). Based on blots of maturing oocytes, a 40-kD putative JNK is normally activated during germinal vesicle breakdown (GVBD), which begins at 30 min post-stimulation with seawater, whereas treating immature oocytes with JNK inhibitors downregulates both the 40-kD JNK signal and GVBD, collectively suggesting a 40-kD JNK may facilitate oocyte maturation. Along with this JNK activity, mature oocytes also exhibit high levels of MPF at 2 h post-stimulation. However, by ~6-8 h post-GVBD, mature oocytes lose the 40-kD JNK signal, and at ~20-30 h of aging, an ~48-kD phospho-JNK band arises as oocytes deactivate MPF and begin to lyse during a necroptotic-like mode of death. Accordingly, JNK inhibitors reduce the aging-related 48-kD JNK phosphorylation while maintaining MPF activity and retarding oocyte degradation. Such findings suggest that a 48-kD JNK may help deactivate MPF and trigger death. Possible mechanisms by which JNK activation either together with, or independently of, protein neosynthesis might stimulate oocyte degradation are discussed.

Effects of CDK inhibitors on the maturation, transcription, and MPF activity of porcine oocytes

In mammals, cyclin-dependent kinases (CDKs) are involved in regulating both the cell cycle and transcription. Although CDK1 is known to act as the kinase subunit of maturation-promoting factor (MPF), the roles of the other CDKs in mammalian oocyte maturation are not yet understood. Here, we show that inhibition of various CDKs by small molecule inhibitors has different effects on the maturation and transcriptional activity of pig oocytes in vitro. Inhibition of CDK1 did not significantly affect cumulus cell expansion, but its kinase activity was necessary for germinal vesicle breakdown (GVBD). The inhibitions of CDK2, CDK4, or CDK6 had no effect on cumulus expansion or GVBD. The catalytic activity of CDK7 was crucial for GVBD but less important for cumulus expansion, whereas inhibition of CDK9 severely blocked both cumulus cell expansion and GVBD. CDK1, -2, -4, and -6 appeared to be dispensable for nuclear transcription, as their inhibitions did not affect nascent RNA production in oocytes. However, inhibition of CDK7 or CDK9 dramatically decreased the transcriptional activity in oocytes. Finally, we found that the GVBD arrest triggered by CDK9 inhibition was not due to altered MPF activity, but rather the inhibition of transcription. Overall, our results show that CDK7 and CDK9 are important for the nuclear maturation and transcriptional activity of pig oocytes.

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.