Role of miRNAs in glucose metabolism of mouse cumulus cells†.

It is known that the oocyte has a limited capacity to acquire and metabolize glucose, and it must rely on cumulus cells (CCs) to take up glucose and produce pyruvate for use to produce ATP through oxidative phosphorylation. We therefore propose that miRNAs might regulate glucose metabolism (GM) in CCs and might be used as markers for oocyte quality assessment. Here, mouse CC models with impaired glycolysis or pentose phosphate pathway (PPP) were established, and miRNAs targeting the key enzymes in glycolysis/PPP were predicted using the miRNA target prediction databases. Expression of the predicted miRNAs was compared between CCs with normal and impaired glycolysis/PPP to identify candidate miRNAs. Function of the candidate miRNAs was validated by transfecting CCs or cumulus-oocyte-complexes (COCs) with miRNA inhibitors and observing effects on glucose metabolites of CCs and on competence of oocytes. The results validated that miR-23b-3p, let-7b-5p, 34b-5p and 145a-5p inhibited glycolysis, and miR-24-3p, 3078-3p,183-5p and 7001-5p inhibited PPP of CCs. Our observation using a more physiologically relevant model (intact cultured COCs) further validated the four glycolysis-targeting miRNAs we identified. Furthermore, miR-let-7b-5p, 34b-5p and 145a-5p may also inhibit PPP, as they decreased the production of glucose-6-phosphate. In conclusion, miRNAs play critical roles in GM of CCs and may be used as markers for oocyte quality assessment. Summary sentence:  We identified and validated eight new miRNAs that inhibit glycolysis and/or pentose phosphate pathways in cumulus cells (CCs) suggesting that miRNAs play critical roles in glucose metabolism of CCs and may be used for oocyte quality markers.

Expression profiling and function analysis identified new genes regulating cumulus expansion and apoptosis.

Studies on the mechanisms behind cumulus expansion and cumulus cell (CC) apoptosis are essential for understanding the mechanisms for oocyte maturation. Genes expressed in CCs might be used as markers for competent oocytes and/or embryos. In this study, both in vitro (IVT) and in vivo (IVO) mouse oocyte models with significant difference in cumulus expansion and CC apoptosis were used to identify and validate new genes regulating cumulus expansion and CC apoptosis of mouse oocytes. We first performed mRNA sequencing and bioinformatic analysis using the IVT oocyte model to identify candidate genes. We then analyzed functions of the candidate genes by RNAi or gene overexpression to select the candidate cumulus expansion and CC apoptosis-regulating genes. Finally, we validated the cumulus expansion and CC apoptosis-regulating genes using the IVO oocyte model. The results showed that while Spp1, Sdc1, Ldlr, Ezr and Mmp2 promoted, Bmp2, Angpt2, Edn1, Itgb8, Cxcl10 and Agt inhibited cumulus expansion. Furthermore, Spp1, Sdc1 and Ldlr inhibited CC apoptosis. In conclusion, by using both IVT and IVO oocyte models, we have identified and validated a new group of cumulus expansion and/or apoptosis-regulating genes, which may be used for selection of quality oocytes/embryos and for elucidating the molecular mechanisms behind oocyte maturation.

Role and action mechanisms of tPA in CRH-induced apoptosis of mouse oviductal epithelial and mural granulosa cells.

Understanding how stress hormones induce apoptosis in oviductal epithelial cells (OECs) and mural granulosa cells (MGCs) can reveal the mechanisms by which female stress impairs embryonic development and oocyte competence. A recent study showed that tissue plasminogen activator (tPA) ameliorates corticosterone-induced apoptosis in MGCs and OECs by acting on its receptors low-density lipoprotein receptor-related protein 1 (LRP1) and Annexin A2 (ANXA2), respectively. However, whether tPA is involved in corticotropin-releasing hormone (CRH)-induced apoptosis and whether it uses the same or different receptors to inhibit apoptosis induced by different hormones in the same cell type remains unknown. This study showed that CRH triggered apoptosis in both OECs and MGCs and significantly downregulated tPA expression. Moreover, tPA inhibits CRH-induced apoptosis by acting on ANXA2 in both OECs and MGCs. While ANXA2 inhibits apoptosis via phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling, LRP1 reduces apoptosis via mitogen-activated protein kinase (MAPK) signaling. Thus, tPA used the same receptor to inhibit CRH-induced apoptosis in both OECs and MGCs, however used different receptors to inhibit corticosterone-induced apoptosis in MGCs and OECs. These data helps understand the mechanism by which female stress impairs embryo/oocyte competence and proapoptotic factors trigger apoptosis in different cell types.

Role of calcium-sensing receptor in regulating activation susceptibility of postovulatory aging mouse oocytes.

The mechanisms underlying postovulatory oocyte aging (POA) remain largely unknown. The expression of the calcium-sensing receptor (CaSR) in mouse oocytes and its role in POA need to be explored. Our objective was to observe CaSR expression and its role in the susceptibility to activating stimuli (STAS) in POA mouse oocytes. The results showed that, although none of the newly ovulated oocytes were activated, 40% and 94% of the oocytes recovered 19 and 25 h after human chorionic gonadotropin (hCG) injection were activated, respectively, after ethanol treatment. The level of the CaSR functional dimer protein in oocytes increased significantly from 13 to 25 h post hCG. Thus, the CaSR functional dimer level was positively correlated with the STAS of POA oocytes. Aging in vitro with a CaSR antagonist suppressed the elevation of STAS, and cytoplasmic calcium in oocytes recovered 19 h post hCG, whereas aging with a CaSR agonist increased STAS, and cytoplasmic calcium of oocytes recovered 13 h post hCG. Furthermore, the CaSR was more important than the Na-Ca2+ exchanger in regulating oocyte STAS, and T- and L-type calcium channels were inactive in aging oocytes. We conclude that the CaSR is involved in regulating STAS in POA mouse oocytes, and that it is more important than the other calcium channels tested in this connection.

Topoisomerase II dysfunction causes metaphase I arrest by activating Aurora B, SAC and MPF and prevents PB1 abscission in mouse oocytes†.

Oocyte aneuploidy is caused mainly by chromosome nondisjunction and/or unbalanced sister chromatid pre-division. Although studies in somatic cells have shown that topoisomerase II (TOP2) plays important roles in chromosome condensation and timely separation of centromeres, little is known about its role during oocyte meiosis. Furthermore, because VP-16, which is a TOP2 inhibitor and induces DNA double strand breaks, is often used for ovarian cancer chemotherapy, its effects on oocytes must be studied for ovarian cancer patients to recover ovarian function following chemotherapy. This study showed that inhibiting TOP2 with either ICRF-193 or VP-16 during meiosis I impaired chromatin condensation, chromosome alignment, TOP2α localization, and caused metaphase I (MI) arrest and first polar body (PB1) abscission failure. Inhibiting or neutralizing either spindle assembly checkpoint (SAC), Aurora B or maturation-promoting factor (MPF) significantly abolished the effect of ICRF-193 or VP-16 on MI arrest. Treatment with ICRF-193 or VP-16 significantly activated MPF and SAC but the effect disappeared when Aurora B was inhibited. Most of the oocytes matured in the presence of ICRF-193 or VP-16 were arrested at MI, and only 11-27% showed PB1 protrusion. Furthermore, most of the PB1 protrusions formed in the presence of ICRF-193 or VP-16 were retracted after further culture for 7 h. In conclusion, TOP2 dysfunction causes MI arrest by activating Aurora B, SAC, and MPF, and it prevents PB1 abscission by promoting chromatin bridges.

Invivo zearalenone exposure dose-dependently compromises mouse oocyte competence by impairing chromatin configuration and gene transcription.

Although invivo and invitro zearalenone (ZEN) exposure impaired oocyte quality, the mechanisms by which ZEN damages oocytes and the lowest observed effect level remain unclear. Furthermore, although it is known that premature chromatin condensation may occur in oocytes under proapoptotic conditions, whether ZEN exposure compromises oocyte competence by impairing gene transcription by causing premature chromatin condensation remains to be investigated. This study tested the toxic concentrations of invivo ZEN exposure that impair oocyte preimplantation developmental potential (PIDP) and the hypothesis that ZEN exposure compromises oocyte competence by increasing oxidative stress and changing chromatin configuration and the transcription of related genes. We found that invivo treatment of mice (Kunming strain, 8 weeks after birth) with 0.5-1mg kg-1 ZEN daily for 5 days, impaired the PIDP of mouse oocytes, increased oxidative stress, disturbed spindle assembly and chromosome segregation, caused premature chromatin condensation, impaired global gene transcription and disturbed the expression of genes related to oocyte competence, spindle assembly, redox potential and apoptosis. In conclusion, ZEN dose-dependently compromised the competence of mouse oocytes by causing oxidative stress and impairing chromatin configuration and gene transcription.

Meiotic block with roscovitine improves competence of porcine oocytes by fine-tuning activities of different cyclin-dependent kinases.

Successful use of oocytes from small follicles (SFs) is of great importance for animal embryo production and human in vitro fertilization with reduced hormone-related side effects. How in vitro meiotic arrest maintenance (MAM) increases the competence of oocytes is not clear. In this study, pig oocytes recovered from SF of 1-2 mm and medium-follicles (MF) of 3-6 mm in diameter from abattoir ovaries were treated by various MAM treatments to improve their competence. The results showed that 25 µM roscovitine or 1 mM db-cAMP efficiently blocked germinal vesicle breakdown in both SF and MF oocytes suggesting a similar cyclin-dependent kinase (CDK) 1 level between the two oocyte groups. MAM with 15- and 25-µM roscovitine alone or with 1-mM db-cAMP improved competence of SF and MF oocytes, respectively, with a promoted chromatin configuration transition from surrounded nucleoli (SN) to re-decondensation (RDC) pattern that supported substantial gene transcription. However, MAM with db-cAMP alone or with higher concentrations of roscovitine did not improve oocyte competence, could not support an SN-to-RDC transition, and/or evoked a premature chromatin condensation (PMC) that suppressed gene transcription. Both CDK2 and CDK5 contents were higher (p < .05) in MF than in SF oocytes. It is concluded that the competence of pig oocytes, particularly that of SF oocytes can be improved by MAM using a proper roscovitine concentration that promotes gene transcription by inhibiting CDK5 while letting CDK2 off to prevent PMC.

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.

Restraint stress of male mice triggers apoptosis in spermatozoa and spermatogenic cells via activating the TNF-α system.

Studies have indicated that psychological stress impairs human fertility and that various stressors can induce apoptosis of testicular cells. However, the mechanisms by which psychological stress on males reduces semen quality and stressors induce apoptosis in testicular cells are largely unclear. Using a psychological (restraint) stress mouse model, we tested whether male psychological stress triggers apoptosis of spermatozoa and spermatogenic cells through activating tumour necrosis factor (TNF)-α signalling. Wild-type or TNF-α-/- male mice were restrained for 48 h before examination for apoptosis and expression of TNF-α and TNF receptor 1 (TNFR1) in spermatozoa, epididymis, seminiferous tubules and spermatogenic cells. The results showed that male restraint significantly decreased fertilization rate and mitochondrial membrane potential, while increasing levels of malondialdehyde, active caspase-3, TNF-α and TNFR1 in spermatozoa. Male restraint also increased apoptosis and expression of TNF-α and TNFR1 in caudae epididymides, seminiferous tubules and spermatogenic cells. Sperm quality was also significantly impaired when spermatozoa were recovered 35 days after male restraint. The restraint-induced damage to spermatozoa, epididymis and seminiferous tubules was significantly ameliorated in TNF-α-/- mice. Furthermore, incubation with soluble TNF-α significantly reduced sperm motility and fertilizing potential. Taken together, the results demonstrated that male psychological stress induces apoptosis in spermatozoa and spermatogenic cells through activating the TNF-α system and that the stress-induced apoptosis in spermatogenic cells can be translated into impaired quality in future spermatozoa.

Glucose metabolism during in vitro maturation of mouse oocytes: An study using RNA interference.

In previous studies on glucose metabolism during in vitro maturation, intact cumulus-oocyte complexes (COCs) were treated with enzyme inhibitors/activators. Because inhibitors/activators may have non-specificity and/or toxicity, and culture of COCs cannot differentiate whether glucose metabolism of cumulus cells (CCs) or that of the oocyte supports oocyte maturation, results from the previous studies must be verified by silencing genes in either CCs or cumulus-denuded oocytes (DOs). In this study, RNAi was adopted to specify the effects of glucose metabolism in CCs or DOs on oocyte maturation. Although silencing either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or glucose-6-phosphate dehydrogenase (G6PD) genes in CCs significantly decreased competence of the cocultured DOs, silencing G6PD impaired competence to a greater extent. While silencing G6PD or GAPDH of CCs decreased glutathione and ATP contents of cocultured DOs to similar extents, silencing G6PD increased oxidative stress as well. Analysis on metabolite contents and oxidative stress index and culture of DOs in medium conditioned with gene-silenced CCs indicated that CCs supported oocyte maturation by releasing glucose metabolites. Silencing mitochondrial pyruvate carrier 1 or NADH dehydrogenase (ubiquintone) flavoprotein 1 of DOs significantly impaired their maturation. The results have unequivocally confirmed that CCs promote oocyte maturation by releasing glucose metabolites from both pentose phosphate pathway (PPP) and glycolysis. Pyruvate is transferred into DOs by mitochondrial pyruvate carrier (MPC) and utilized through mitochondrial electron transport to support maturation.

Role of calcium-sensing receptor in regulating spontaneous activation of postovulatory aging rat oocytes.

Mechanisms for postovulatory aging (POA) of oocytes and for spontaneous activation (SA) of rat oocytes are largely unknown. Expression of calcium-sensing receptor (CaSR) in rat oocytes and its role in POA remain unexplored. In this study, expression of CaSR in rat oocytes aging for different times was detected by immunofluorescence microscopy, and western blotting and the role of CaSR in POA was determined by observing the effects of regulating its activity on SA susceptibility and cytoplasmic calcium levels. The results showed that CaSR was expressed in rat oocytes. Oocytes recovered 19 h post human chorionic gonadotropin (hCG) injection were more susceptible to SA and expressed more functional CaSR than oocytes recovered 13 h after hCG injection, although both expressed the same level of total CaSR protein. Treatment with CaSR antagonist significantly suppressed cytoplasmic calcium elevation and SA of oocytes. Activation of Na-Ca2+ exchanger with NaCl inhibited SA to a greater extent than suppression of CaSR with NPS-2143, suggesting that calcium sources other than CaSR-controlled channels contributed to the elevation of cytoplasmic calcium. Treatment with T- or L-type calcium channel blockers significantly reduced SA. Suppression of all calcium channels tested reduced SA to minimum. It is concluded that the level of CaSR functional dimer protein, but not that of the total CaSR protein, was positively correlated with the SA susceptibility during POA of rat oocytes confirming that CaSR is involved in POA regulation. Blocking multiple calcium channels might be a better choice for efficient control of SA in rat oocytes.

Mechanisms for the species difference between mouse and pig oocytes in their sensitivity to glucorticoids.

Although in vitro exposure to physiological concentrations of glucorticoids did not affect maturation of mouse oocytes, it significantly inhibited nuclear maturation of pig oocytes. Studies on this species difference in oocyte sensitivity to glucocorticoids will contribute to our understanding of how stress/glucocorticoids affect oocytes. We showed that glucorticoid receptors (NR3C1) were expressed in both oocytes and cumulus cells (CCs) of both pigs and mice; however, while cortisol inhibition of oocyte maturation was overcome by NR3C1 inhibitor RU486 in pigs, it could not be relieved by RU486 in mice. The mRNA level of 11ß-hydroxysteroid dehydrogenase 1 (HSD11B1) was significantly higher than that of HSD11B2 in pig cumulus-oocyte complexes (COCs), whereas HSD11B2 was exclusively expressed in mouse COCs. Pig and mouse cumulus-denuded oocytes (DOs) expressed HSD11B2 predominantly and exclusively, respectively. In the presence of cortisol, although inhibiting HSD11B2 decreased maturation rates of COCs in both species, inhibiting HSD11B1 improved maturation of pig COCs while having no effect on mouse COCs. Cortisol-cortisone interconversion observation confirmed high HSD11B1 activities in pig oocytes but none in mouse oocytes, a higher HSD11B2 activity in mouse than in pig oocytes, and a rapid cortisol-cortisone interconversion in pig COCs catalyzed by HSD11B1 from CCs and HSD11B2 from DOs. In conclusion, the species difference in glucocorticoid sensitivity between pig and mouse oocytes is caused by their different contents/ratios of HSD11B1 and HSD11B2, which maintain different concentrations of active glucocorticoids. While cortisol inhibited pig oocytes by interacting with NR3C1, glucocorticoid suppression of mouse oocytes was apparently not mediated by NR3C1.

Postovulatory Aging of Mouse Oocytes Impairs Offspring Behavior by Causing Oxidative Stress and Damaging Mitochondria.

Information on long-term effects of postovulatory oocyte aging (POA) on offspring is limited. Whether POA affects offspring by causing oxidative stress (OS) and mitochondrial damage is unknown. Here, in vivo-aged (IVA) mouse oocytes were collected 9 h after ovulation, while in vitro-aged (ITA) oocytes were obtained by culturing freshly ovulated oocytes for 9 h in media with low, moderate, or high antioxidant potential. Oocytes were fertilized in vitro and blastocysts transferred to produce F1 offspring. F1 mice were mated with naturally bred mice to generate F2 offspring. Both IVA and the ITA groups in low antioxidant medium showed significantly increased anxiety-like behavior and impaired spatial and fear learning/memory and hippocampal expression of anxiolytic and learning/memory-beneficial genes in both male and female F1 offspring. Furthermore, the aging in both groups increased OS and impaired mitochondrial function in oocytes, blastocysts, and hippocampus of F1 offspring; however, it did not affect the behavior of F2 offspring. It is concluded that POA caused OS and damaged mitochondria in aged oocytes, leading to defects in anxiety-like behavior and learning/memory of F1 offspring. Thus, POA is a crucial factor that causes psychological problems in offspring, and antioxidant measures may be taken to ameliorate the detrimental effects of POA on offspring.

Role and action mechanisms of miR-149 and miR-31 in regulating function of pig cumulus cells and oocytes.

Understanding the mechanisms for oocyte maturation and optimizing the protocols for in vitro maturation (IVM) are greatly important for improving developmental potential of IVM oocytes. The miRNAs expressed in cumulus cells (CCs) play important roles in oocyte maturation and may be used as markers for selection of competent oocytes/embryos. Although a recent study from our group identified several new CCs-expressed miRNAs that regulate cumulus expansion (CE) and CC apoptosis (CCA) in mouse oocytes, validation of these findings and further investigation of mechanisms of action in other model species was essential before wider applications. By using both in vitro and in vivo pig oocyte models with significant differences in CE, CCA and developmental potential, the present study validated that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes. We demonstrated that miR-149 and miR-31 targeted SMAD family member 6 (SMAD6) and transforming growth factor ß2 (TGFB2), respectively, in the transforming growth factor-ß (TGF-ß) signaling. Furthermore, both miR-149 and miR-31 increased CE and decreased CCA via activating SMAD family member 2 (SMAD2) and increasing the expression of SMAD2 and SMAD family member 4. In conclusion, the present results show that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes by activating the TGF-ß signaling, suggesting that they might be used as markers for pig oocyte quality.

Control of spontaneous activation of rat oocytes by regulating plasma membrane Na+/Ca2+ exchanger activities.

Inhibiting oocyte spontaneous activation (SA) is essential for successful rat cloning by nuclear transfer (NT). This study tested the hypothesis that activities of the Na(+)/Ca(2+) exchanger (NCX) would decrease with oocyte aging and that SA of rat oocytes could be inhibited if the intraoocyte Ca(2+) rises were prevented by activating the NCX through increasing Na(+) concentrations in the culture medium. Elevating Na(+) levels in culture medium by supplementing NaCl inhibited SA of rat oocytes, while maintaining a constant level of maturation-promoting factor and mitogen-activated protein kinase activities. Experiments using the NCX inhibitor bepridil, the Na(+)/K(+)-ATPase inhibitor ouabain, and an assay for intraoocyte Ca(2+) concentrations showed that extracellular Na(+) inhibited rat oocyte SA by enhancing NCX activity and preventing intracellular Ca(2+) rises. Immunohistochemical quantification indicated that the density of NCX1 decreased significantly in aged oocytes that were prone to SA compared with that in freshly ovulated oocytes whose SA rates were low during in vitro culture. Cumulus cell NT showed that sham enucleation caused marked SA in freshly ovulated rat oocytes and that Na(+) supplementation prevented the manipulation-induced SA and improved the in vitro and in vivo development of rat somatic cell NT embryos. Taken together, the results have confirmed our hypothesis that the NCX is active in rat oocytes and its activity decreases with oocyte aging and that activating the NCX by increasing extracellular Na(+) inhibits SA of rat oocytes and improves the development of rat somatic cell NT embryos. These data are also important for understanding the mechanisms of oocyte aging.

Restraint stress impairs oocyte developmental potential in mice: role of CRH-induced apoptosis of ovarian cells.

This study examined the role of CRH-induced ovarian cell apoptosis in the restraint stress (RS)-induced impairment of oocyte competence. Oocyte percentages of apoptotic cumulus cells (CCs) did not differ between stressed and control mice before in vitro maturation (IVM) but became significantly higher in stressed mice after IVM without serum, growth factor, and hormone. The level of Bcl2 mRNA decreased significantly in mural granulosa cells (MGCs) and ovarian homogenates after RS. Whereas ovarian estradiol, testosterone, and IGF1 decreased, cortisol and progesterone increased significantly following RS. RS increased the level of CRH in serum, ovary, and oocyte while enhancing the expression of CRHR1 in CCs, MGCs, and thecal cells. RS down-regulated ovarian expression of glucocorticoid receptor and brain-derived neurotrophic factor. Furthermore, CRH supplementation to IVM medium impaired oocyte developmental potential while increasing apoptotic CCs, an effect that was completely overcome by addition of the CRHR1 antagonist antalarmin. Results suggest that RS impaired oocyte competence by increasing CRH but not glucocorticoids. Increased CRH initiated a latent apoptotic program in CCs and oocytes during their intraovarian development, which was executed later during IVM to impair oocyte competence. Thus, elevated CRH interacted with increased CRHR1 on thecal cells and MGCs, reducing the production of testosterone, estrogen, and IGF1 while increasing the level of progesterone. The imbalance between estrogen and progesterone and the decreased availability of growth factors triggered apoptosis of MGCs and facilitated CC expression of CRHR1, which interacted with the oocyte-derived CRH later during IVM to induce CC apoptosis and reduce oocyte competence.

Mechanisms by which a lack of germinal vesicle (GV) material causes oocyte meiotic defects: a study using oocytes manipulated to replace GV with primary spermatocyte nuclei.

Oocytes with germinal vesicles (GVs) replaced with somatic nuclei exhibit meiotic abnormalities. Although this suggests an exclusive role for GV material in meiosis, mechanisms by which a lack of GV material causes meiotic defects are unknown. Knowledge of these mechanisms will help us to understand meiotic control, nuclear-cytoplasmic interactions, and cellular reprogramming. This study showed that although oocytes with prometaphase I chromosomes replaced with primary spermatocyte nuclei (PSN) did not, oocytes with GV replaced with PSN (PSG oocytes) did display meiotic defects. Among the defects, insufficient chromosome condensation with chromosome bridges was associated with spindle abnormalities. Abnormal spindle migration, cortical nonpolarization, and the aberrant spindle caused randomly positioning of cleavage furrows, leading to large first polar bodies (PB1) and unequal allocation of chromosomes and mitogen-activated protein kinases (MAPK) between oocyte and PB1. Spindle assembly checkpoint was activated but did not stop the incorrect division. The unequal MAPK allocation resulted in differences in pronuclear formation and PB1 degeneration; oocytes receiving more MAPK were more capable of forming pronuclear rudiments, whereas PB1 receiving more MAPK degenerated sooner than those that received less. Because none of the PSG oocytes or the enucleated GV oocytes injected with sperm heads showed cortical polarization in spite of chromosome localization close to the oolemma and because the PSG oocytes receiving more MAPK could form only pronuclear rudiments and not normal pronuclei, we suggest that the GV material plays essential roles in polarization and pronuclear formation on top of those played by chromosomes or MAPK. In conclusion, using PSG oocytes as models, this study has revealed the primary pathways by which a lack of GV material cause meiotic defects, laying a foundation for future research on the role of GV material in oocyte meiotic control.

Role of tPA in Corticosterone-Induced Apoptosis of Mouse Mural Granulosa and Oviductal Epithelial Cells.

Although studies indicate that female stress-increased secretion of glucocorticoids impairs oocyte competence and embryo development, by inducing apoptosis of ovarian and oviductal cells, respectively, the mechanisms by which glucocorticoids induce apoptosis of ovarian and oviductal cells are largely unclear. Tissue plasminogen activator (tPA) has been involved in apoptosis of different cell types. However, while some studies indicate that tPA is proapoptotic, others demonstrate its antiapoptotic effects. This study has explored the role and action mechanisms of tPA in corticosterone-induced apoptosis of mouse mural granulosa cells (MGCs) and oviductal epithelial cells (OECs). The results demonstrate that culture with corticosterone significantly increased apoptosis, while decreasing levels of tPA (Plat) mRNA and tPA protein in both MGCs and OECs. Culture with tPA ameliorated corticosterone-induced apoptosis of MGCs and OECs. Furthermore, while tPA protected MGCs from corticosterone-induced apoptosis by interacting with low-density lipoprotein receptor-related protein 1 (LRP1), it protected OECs from the apoptosis by acting on Annexin 2 (ANXA2). In conclusion, tPA is antiapoptotic in both MGCs and OECs, and it protects MGCs and OECs from corticosterone-induced apoptosis by interacting with LRP1 and ANXA2, respectively, suggesting that tPA may use different receptors to inhibit apoptosis in different cell types.

Combined inhibitory effects of pyruvate and low temperature on postovulatory aging of mouse oocytes.

This study tested the hypothesis that oocyte aging could be prevented for a longer time by reducing the culture temperature while supplementing the culture medium with more pyruvate. Newly ovulated mouse oocytes were cultured at various temperatures for various times in HCZB medium (Kimura and Yanagimachi, Biol Reprod 1995; 52:709-720) containing various concentrations of pyruvate before examining for aging parameters and developmental potential. The increase in susceptibility to activating stimuli was efficiently prevented when oocytes were cultured in HCZB with 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 96 h, and 5°C for 48 h. Satisfactory blastocyst development of both parthenotes and fertilized zygotes was achieved after oocyte culture in HCZB containing 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 36 h, and 5°C for 24 h. Transfer of two-cell embryos or blastocysts showed no difference between newly ovulated control oocytes and oocytes cultured at 15°C for 36 h in either term pregnancy, live young per pregnant recipient, live young/transferred embryos, or birth weight of young. Oocytes with impaired developmental potential after culture at 15°C for 96 h and at 5°C for 48 h showed unrecoverable decreases in the content of glutathione, the glutathione/oxidized glutathione ratio, the BCL2 content, and in the numbers of oocytes with normal spindles and cortical granule distribution, suggesting induction of oxidative stress, which caused oocyte apoptosis and cytoskeleton alterations by downregulating BCL2. Because oocytes cultured at 15°C for 36 h were activated or fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 42 h without impairing their developmental potential.

Restraint stress of female mice during oocyte development facilitates oocyte postovulatory aging.

Studies suggest that psychological stress on women can impair their reproduction and that postovulatory oocyte aging (POA) might increase the risk of early pregnancy loss and affect offspring's reproductive fitness and longevity. However, whether psychological stress during oocyte development would facilitate POA is unknown but worth exploring to understand the mechanisms by which psychological stress and POA damage oocytes. This study observed effects of female restraint stress during oocyte development (FRSOD) on oocyte resistance to POA. Female mice were restrained for 48 h before superovulation, and they were sacrificed at different intervals after ovulation to recover aging oocytes for analyzing their early and late aged characteristics. The effects of FRSOD on aging oocytes included: (1) increasing their susceptibility to activation stimulus with elevated cytoplasmic calcium; (2) impairing their developmental potential with downregulated expression of development-beneficial genes; (3) facilitating degeneration, cytoplasmic fragmentation and apoptosis; (4) worsening the disorganization of cortical granules and spindle/chromosomes; and (5) impairing redox potential with increased oxidative stress. In conclusion, FRSOD impairs oocyte resistance to POA, so that stressed oocytes become aged significantly quicker than unstressed controls. Thus, couples wishing to achieve pregnancy should take steps to avoid not only fertilization of aged oocytes but also pregestational stressful life events.