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.

Comparative transcriptomic analysis reveals that TPX2 and AURXA are involved in porcine PCV2 infection.

Porcine circovirus type 2 (PCV2) has been a notorious killer for the pig industry, causing substantial economic losses worldwide. However, its pathogenesis is still poorly understood. Comparative transcriptomic analysis and weighted gene co-expression network analysis (WGCNA) were performed in different porcine tissues after PCV2 infection. Our comparative transcriptomic analysis obtained 40 key differentially expressed genes (DEGs), and our WGCNA identified 458 hub genes. Significantly, both TPX2 microtubule nucleation factor (TPX2) and Aurora kinase A (AURKA) are included in these key DEGs and hubs genes. Our gene ontology (GO) analysis indicated that the key DEGs and hub genes participated in cell cycle regulation and immune response. The expressive levels of TPX2 and AURKA went down in the spleen but up in the kidneys after infection with PCV2. We conclude that TPX2 and AURKA played an essential role in PCV2 infection.

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.

Corticosterone induced apoptosis of mouse oviduct epithelial cells independent of the TNF-α system.

It has been reported in recent studies that restraint stress on pregnant mice during the preimplantation stage elevated corticotrophin-releasing hormone (CRH) and glucocorticoid levels in the serum and oviducts; furthermore, CRH and corticosterone (CORT) impacted preimplantation embryos indirectly by triggering the apoptosis of oviductal epithelial cells (OECs) through activation of the Fas system. However, it remains unclear whether TNF-α signaling is involved in CRH- and/or glucocorticoid-induced apoptosis of OECs. In the present study, it was shown that culture with either CRH or CORT induced significant apoptosis of OECs. The culture of OECs with CRH augmented both FasL expression and TNF-α expression. However, culture with CORT increased FasL, but decreased TNF-α, expression significantly. Although knocking down/knocking out FasL expression in OECs significantly ameliorated the proapoptotic effects of both CRH and CORT, knocking down/knocking out TNF-α expression relieved only the proapoptotic effect of CRH but not that of CORT. Taken together, our results demonstrated that CRH-induced OEC apoptosis involved both Fas signaling and TNF-α signaling. Conversely, CORT-induced OEC apoptosis involved only the Fas, but not the TNF-α, signaling pathway. The data obtained are crucial for our understanding of the mechanisms by which various categories of stress imposed on pregnant females impair embryo development, as well as for the development of measures to protect the embryo from the adverse effects of stress.

Heat stress decreases egg production of laying hens by inducing apoptosis of follicular cells via activating the FasL/Fas and TNF-α systems.

Heat stress (HS) causes significant economic losses in the poultry industry every year. However, the mechanisms for the adverse effects of HS on avian follicular development are largely unknown. The aim of this study was to test whether HS induces apoptosis of follicular cells and impairs egg production by activating the FasL/Fas and tumor necrosis factor (TNF)-α systems. To this end, Hy-Line Brown laying hens, at 32 wk of age, were either exposed to HS of 35°C to 37°C or maintained at 24°C to 26°C (control) for 5 D. At the end of the HS period, follicle numbers, apoptosis, FasL/Fas and TNF-α activation, oxidative stress, and hormone secretion were examined in ovarian follicles. Egg production was observed daily during both the stressed (day S1-S5) and the poststress recovery (day R1-R15) periods. The results demonstrated that HS on hens significantly 1) decreased laying rates from day S3 to R6; 2) reduced numbers of large yellow and hierarchical follicles; 3) triggered apoptosis while increasing the expression of FasL, Fas, TNF-α, and TNF-receptor 1 in small and large yellow follicles; and 4) increased levels of oxidative stress, corticotrophin-releasing hormone, and corticosterone while decreasing the estradiol/progesterone ratio in follicular fluid in small and large yellow follicles. Taken together, the results suggested that hen HS impaired egg production by reducing the number of follicles through inducing apoptosis and that it triggered apoptosis in follicular cells by activating the FasL/Fas and TNF-α systems.

Glucocorticoids impair oocyte competence and trigger apoptosis of ovarian cells via activating the TNF-α system.

Mechanisms by which female stress and particularly glucocorticoids impair oocyte competence are largely unclear. Although one study demonstrated that glucocorticoids triggered apoptosis in ovarian cells and oocytes by activating the FasL/Fas system, other studies suggested that they might induce apoptosis through activating other signaling pathways as well. In this study, both in vivo and in vitro experiments were conducted to test the hypothesis that glucocorticoids might trigger apoptosis in oocytes and ovarian cells through activating the TNF-α system. The results showed that cortisol injection of female mice (1.) impaired oocyte developmental potential and mitochondrial membrane potential with increased oxidative stress; (2.) induced apoptosis in mural granulosa cells (MGCs) with increased oxidative stress in the ovary; and (3.) activated the TNF-α system in both ovaries and oocytes. Culture with corticosterone induced apoptosis and activated the TNF-α system in MGCs. Knockdown or knockout of TNF-α significantly ameliorated the pro-apoptotic effects of glucocorticoids on oocytes and MGCs. However, culture with corticosterone downregulated TNF-α expression significantly in oviductal epithelial cells. Together, the results demonstrated that glucocorticoids impaired oocyte competence and triggered apoptosis in ovarian cells through activating the TNF-α system and that the effect of glucocorticoids on TNF-α expression might vary between cell types.

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 and elevation of corticotrophin-releasing hormone in female mice impair oocyte competence through activation of the tumour necrosis factor α (TNF-α) system.

Studies have observed that restraint stress (RS) and the associated elevation in corticotrophin-releasing hormone (CRH) impair oocyte competence by triggering apoptosis of ovarian cells but the underlying mechanisms are largely unclear. Although one study demonstrated that RS and CRH elevation triggered apoptosis in ovarian cells and oocytes via activating Fas/FasL signalling, other studies suggested that RS might damage cells by activating other pathways as well as Fas signalling. The objective of this study was to test whether RS and CRH elevation impairs oocytes by activating tumour necrosis factor α (TNF-α) signalling. Our invivo experiments showed that RS applied during oocyte prematuration significantly increased expression of TNF-α and its receptor (TNFR1) while inducing apoptosis in both oocytes and mural granulosa cells (MGCs). Invitro treatment of MGCs with CRH significantly increased their apoptotic percentages and levels of TNF-α and TNFR1 expression. Invitro knockdown by interfering RNA, invivo knockout of the TNF-α gene or injection of TNF-α antagonist etanercept significantly relieved the adverse effects of RS and CRH on apoptosis of MGCs and/or the developmental potential and apoptosis of oocytes. The results suggest that RS and CRH elevation in females impair oocyte competence through activating TNF-α signalling and that a TNF-α antagonist might be adopted to ameliorate the adverse effects of psychological stress on oocytes.

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.

Restraint stress of male mice induces apoptosis in spermatozoa and spermatogenic cells: role of the FasL/Fas system†.

The mechanisms by which psychological stress impairs semen quality are largely unknown. By using a restraint-stressed mouse model, we studied the role of the FasL/Fas system in psychological stress-induced apoptosis of spermatozoa and spermatogenic cells. Male mice were restrained for 48 h before examination for sperm fertilizing potential and for apoptosis and FasL/Fas expression in spermatozoa, spermatogenetic cells/seminiferous tubules, and caudae epididymides. The results showed that the male restraint reduced motility, fertilization rates, and mitochondrial membrane potential while increasing apoptosis and Fas expression in spermatozoa. Restraint also facilitated apoptosis and FasL/Fas expression in spermatogenic cells/seminiferous tubules and caudae epididymides. The restraint-induced apoptosis in spermatozoa and spermatogenic cells was significantly ameliorated in gld mice that harbor a loss-of-function mutation in FasL. However, incubation with FasL did not affect sperm motility and apoptosis, while incubation with tumor necrosis factor (TNF)-α did. The epididymis of the gld mice produced significantly less TNF-α and TNF-related apoptosis-inducing ligand (TRAIL) than that of wild-type mice did after male restraint. Thus, the results confirmed that the FasL/Fas system played an important role in the psychological stress-induced apoptosis of spermatozoa and spermatogenic cells and that FasL triggered sperm apoptosis in epididymis dependently through promoting TNF-α and TRAIL secretion.

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.

Restraint-induced corticotrophin-releasing hormone elevation triggers apoptosis of ovarian cells and impairs oocyte competence via activation of the Fas/FasL system.

Mechanisms by which psychological stress damages oocytes are largely undetermined. Although a previous study showed that the stress-induced corticotrophin-releasing hormone (CRH) elevation impaired oocyte competence by triggering apoptosis of ovarian cells, how CRH causes apoptosis in ovarian cells and oocytes is unknown. In this study, we have examined the hypothesis that restraint stress (RS)-induced CRH elevation triggers apoptosis of ovarian cells and impairs oocyte competence through activating the Fas/FasL system. The results showed that RS of female mice impaired oocyte competence, enhanced expression of CRH and CRH receptor (CRH-R) in the ovary, and induced apoptosis while activating the Fas/FasL system in mural granulosa cells (MGCs) and oocytes. Injecting mice with CRH-R1 antagonist antalarmin significantly alleviated the adverse effect of RS on oocyte developmental potential. Treatment of cultured MGCs recapitulated the effects of CRH and antalarmin on apoptosis and Fas/FasL expression in MGCs. Silencing FasL gene by RNA interference in cultured MGCs further confirmed the involvement of the Fas/FasL system in the CRH triggered apoptosis of ovarian cells. It is concluded that the RS-induced CRH elevation triggers apoptosis of ovarian cells and impairs oocyte competence via activation of the Fas/FasL system.

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.

Mechanisms by which in vitro meiotic arrest and sexual maturity improve developmental potential of mouse oocytes.

To study the relationship between chromatin condensation, gene transcription and developmental competence during oocyte maturation and to explore the mechanisms by which meiotic arrest maintenance (MAM) and sexual maturity improve oocyte competence, we examined effects of MAM with roscovitine or db-cAMP on chromatin condensation, gene transcription and developmental potential of NSN or SN oocytes from prepubertal or adult mice. MAM with roscovitine improved the developmental competence and global gene transcription of prepubertal NSN (prep-NSN) and adult-SN oocytes while having no effect on those of prep-SN oocytes. MAM with db-cAMP facilitated neither development nor transcription in any type of oocytes. MAM with either roscovitine or db-cAMP promoted chromatin condensation of prep-NSN oocytes. MAM with roscovitine promoted gene transcription and chromatin condensation simultaneously through inhibiting cyclin-dependent kinase (CDK) 5 and 2, respectively. The results suggested that MAM with roscovitine improved oocyte competence by promoting gene transcription via inhibiting CDK5. Oocyte cytoplasmic maturation is correlated with gene transcription but not with chromatin condensation. The difference in developmental competence between prepubertal NSN and SN oocytes and between prepubertal and adult SN oocytes was because while the former had not, the latter had completed or acquired the ability for transcription of important genes.

Meiotic arrest with roscovitine and follicular fluid improves cytoplasmic maturation of porcine oocytes by promoting chromatin de-condensation and gene transcription.

The developmental capacity of in vitro matured oocytes is inferior to that of the in vivo matured ones due to insufficient cytoplasmic maturation. Although great efforts were made to accomplish better cytoplasmic maturation by meiotic arrest maintenance (MAM) before in vitro maturation (IVM), limited progress has been achieved in various species. This study showed that MAM of porcine oocytes was better achieved with roscovitine than with dibutyryl cyclic adenosine monophosphate (db-cAMP) or 3-isobutyl-1-methylxanthine. Oocyte developmental competence after IVM was significantly improved following MAM in 199 + FF medium (TCM-199 containing 10% porcine follicular fluid and 25 µM roscovitine) to a level even higher than that in control oocytes matured without pre-MAM. Observations on other markers further confirmed the positive effects of MAM in 199 + FF on oocyte cytoplasmic maturation. During MAM culture in 199 + FF, re-decondensation (RDC) of condensed chromatin occurred, and transcription of genes beneficial to cytoplasmic maturation was evident in some of the oocytes with surrounded nucleoli (SN). However, MAM with db-cAMP neither induced RDC nor improved oocyte developmental potential. Together, the results suggest that MAM in the presence of FF and roscovitine improved the developmental competence of porcine oocytes by promoting a pre-GVBD chromatin de-condensation and expression of beneficial genes.

Corticotrophin-releasing hormone and corticosterone impair development of preimplantation embryos by inducing oviductal cell apoptosis via activating the Fas system: an in vitro study.

STUDY QUESTION: What are the mechanisms by which corticotrophin-releasing hormone (CRH) and corticosterone impair the development of preimplantation embryos in the oviduct. SUMMARY ANSWER: CRH and corticosterone do not affect preimplantation embryos directly, but impair their development indirectly by triggering apoptosis of oviductal epithelial cells (OECs) through activation of the Fas system. WHAT IS KNOWN ALREADY: Studies report that stress impairs embryo development with facilitated secretion of CRH and glucocorticoids. Although an in vivo study demonstrated that preimplantation stress impaired embryo development in conjunction with oviductal apoptosis and activation of the Fas system, whether CRH or glucocorticoids damage embryos directly or indirectly by way of oviductal cells remains to be clarified. STUDY DESIGN, SIZE, DURATION: Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in Fas ligand in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female mice were used 8-10 weeks after birth. PARTICIPANTS/MATERIALS, SETTING, METHODS: While some female mice were killed 48 h after being injected with equine CG to collect oviducts and prepare OECs, others were killed to recover zygotes after mating with males following superovulation with eCG and hCG. The zygotes obtained were cultured with or without CRH or corticosterone (CRH/Cort) either in Chatot-Ziomek-Bavister (CZB) medium with or without OECs or in conditioned medium (CM) conditioned with OECs pretreated or not with CRH/Cort. Preimplantation development, levels of redox potential and apoptosis, and expression of CRH receptor 1 (CRHR1), glucocorticoid receptor (GR), Fas and 11ß-hydroxysteroid dehydrogenase (HSD) were observed in embryos recovered at different times of in vitro culture. After culture of OECs with or without CRH/Cort, levels of redox potential and apoptosis, mRNA and protein expression of growth factors, and protein expression of CRHR1, GR and Fas were examined in OECs and the level of FasL was measured in CM. The gld mice were used to confirm a role for the Fas system in triggering apoptosis of embryos and oviducts. MAIN RESULTS AND THE ROLE OF CHANCE: This study showed that blastocyst development was unaffected when mouse zygotes were cultured in CZB medium containing various concentrations of CRH/Cort but was impaired when embryos were cultured with CRH/Cort plus OECs or in CM conditioned with OECs pretreated with CRH/Cort (treatment CM). Culture in treatment-CM induced oxidative stress and apoptosis in embryos. Preimplantation embryos expressed GR and Fas at all stages and CRHR1 at the blastocyst stage only. Mouse 4-cell embryos and blastocysts expressed HSD2 but not HSD1. Culture of OECs with CRH/Cort increased their oxidative stress, apoptosis, CRHR1, Fas and FasL while decreasing their GR and growth factors. Blastocyst development in treatment-CM conditioned with OECs from gld mice harboring FasL mutations was superior to treatment-CM conditioned with wild-type mouse OECs. The results suggest that CRH/Cort impairs embryo development indirectly by inducing oviductal apoptosis via activating the Fas system. The insensitivity of preimplantation embryos to CRH and corticosterone is due to, respectively, a lack of CRHR and the exclusive expression of HSD2 that inactivate corticosterone. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Although significant, the conclusions were drawn from limited results obtained using mice and thus they need further verification in other species. For example, bovine embryos express both HSD1 and HSD2 at all the preimplantation stages whereas mouse preimplantation embryos express HSD2 exclusively without HSD1. WIDER IMPLICATIONS OF THE FINDINGS: The data are important for our understanding of the mechanisms by which stress affects female reproduction in both human and animals, as early stages of pregnancy are considered more vulnerable to stress than the late stages. STUDY FUNDING AND COMPETING INTEREST(S): This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (Nos. 31272444 and 30972096) and the Animal breeding improvement program of Shandong Province. All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Preimplantation maternal stress impairs embryo development by inducing oviductal apoptosis with activation of the Fas system.

STUDY QUESTION: What are the mechanisms by which the preimplantation restraint stress (PIRS) impairs embryo development and pregnancy outcome? SUMMARY ANSWER: PIRS impairs embryo development by triggering apoptosis in mouse oviducts and embryos,and this involves activation of the Fas system. WHAT IS KNOWN ALREADY: Although it is known that the early stages of pregnancy are more vulnerable than later stages to prenatalstress, studies on the effect of preimplantation stress on embryo developmentare limited. Furthermore, the mechanisms by which psychological stress impairs embryo development are largely unknown. These issues are worth exploring using the mouse PIRS models because restraint of mice is an efficient experimental procedure developed for studies of psychogenic stress. STUDY DESIGN, SIZE AND DURATION: Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in FasL in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female and male mice were used 8-10 weeks and 10-12 weeks after birth, respectively. Female mice showing vaginal plugs were paired by weight and randomly assigned to restraint treatments or as controls. For restraint treatment, an individual mouse was put in a micro-cage with food and water available. Control mice remained in their cages with food and water during the time treated females were stressed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Female mice were exposed to PIRS for 48 h starting from 16:00 on the day of vaginal plug detection. At the end of PIRS, levels of glucorticoids (GC), corticotropin-releasing hormone (CRH)and redox potential were measured in serum, while levels of GC, GC receptor (GR), CRH, CRH receptor (CRHR), Fas and Fas ligand (FasL) protein, mRNAs for brain derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1), oxidative stress (OS) and apoptosis were examined in oviducts. Preimplantation development and levels of GR, Fas, redox potential and apoptosis were observed in embryos recovered at different times after the initiation of PIRS. The gld mice were used to confirm a role for the Fas system in triggering apoptosis of embryos and oviducts. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to those in control mice, while the number of blastocysts/mouse (5.0 ± 0.7 versus 11.1 ± 0.5), cell number/blastocyst (49.1 ± 1.3 versus 61.5 ± 0.9), percentages of term pregnancy (37.5% versus 90.9%) and litter size (3.7 ± 0.1versus 9.6 ± 0.6) decreased, blood CRH (560 ± 23 versus 455 ± 37 pg/ml), cortisol (27.3 ± 3.4 versus 5 ± 0.5 ng/ml) and OS index (OSI: 2.8 versus 1.7) increased significantly (all P < 0.05) following PIRS. In the oviduct, while levels of CRH (1175 ± 85 versus 881 ± 33 pg/100 mg), cortisol (28.9 ± 1.7 versus14 ± 4 ng/g), CRHR (2.3 ± 0.3 versus 1.0 ± 0.0), FasL (1.31 ± 0.06 versus 1.08 ± 0.05 ng/g), Fas (1.42 ± 0.13 versus 1.0 ± 0.0) and apoptotic cells (19.1 ± 0.5% versus 8.4 ± 0.4%) increased, levels of GR proteins (0.67 ± 0.14 versus 1.0 ± 0.0) and Igf-1 (0.6 ± 0.09 versus 1.0 ± 0.0) and Bdnf (0.73 ± 0.03 versus 1.0 ± 0.0) mRNAs decreased significantly (all P < 0.05 versus control) after PIRS. Mouse embryos expressed GR and Fas at all stages of preimplantation development and embryo OS (GSH/GSSG ratio: 0.88 ± 0.03 versus 1.19 ± 0.13) and annexin-positive cells (blastocysts: 31.4 ± 3.8% versus 10.96 ± 3.4%) increased significantly (P < 0.05) following PIRS. Furthermore, the detrimental effects of PIRS on embryo development and oviductal apoptosis were much reduced in gld mice. Thus, PIRS triggered apoptosis in oviductal cells with activation of the Fas/FasL system. The apoptotic oviductal cells promoted embryo apoptosis with reduced production of IGF-1 and BDNF and increased production of FasL. LIMITATIONS, REASONS FOR CAUTION: Although important, the conclusions were drawn from limited results obtained using a single model in one species and thus they need further verification using other models and/or in other species. Furthermore, as differences in stressed samples were modest and sometimes not significant between gld and wild-type mice whereas differences between control and stressed samples were always present within gld mice, it is deduced that signaling pathways other than the Fas/FasL system might be involved as well in the PIRS-triggered apoptosis of oviducts and embryos. WIDER IMPLICATIONS OF THE FINDINGS: The data are important for studies on the mechanisms by which psychological stress affects female reproduction, as FasL expression has been observed in human oviduct epithelium. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (Nos. 31272444 and 30972096) and the Animal breeding improvement program of Shandong Province. All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

The relationship between apoptosis, chromatin configuration, histone modification and competence of oocytes: A study using the mouse ovary-holding stress model.

The epigenetic factors causing competence differences between SN (surrounded nucleolus) and NSN (non-surrounded nucleolus) oocytes, the significance for the increased histone acetylation and methylation in SN oocytes, and whether chromatin configuration or histone modification determines oocyte competence, are unclear. This study has addressed these issues by using the ovary-holding (OH) stress models where oocyte SN configuration was uncoupled from histone modifications and developmental potential. Prepubertal mouse ovaries containing high percentages of NSN oocytes were preserved at 37 or 39 °C for 1 or 2 h before examination for oocyte chromatin configuration, developmental competence, histone modification and apoptosis. Whereas 1-h OH at 37 °C caused a moderate apoptosis with increased oocyte competence, improved histone modification and a normal NSN-to-SN transition, harsher OH conditions induced a severe apoptosis with decreased oocyte competence, impaired histone modification and a pseudo (premature) NSN-to-SN transition. Observations on Fas/FasL expression and using the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations indicated that OH triggered oocyte apoptosis with activation of the Fas signaling. It was concluded that OH stress caused oocyte apoptosis with activation of the Fas/FasL system and that oocyte competence was more closely correlated with histone modification than with chromatin configuration.

Glucocorticoids impair oocyte developmental potential by triggering apoptosis of ovarian cells via activating the Fas system.

Previous studies indicate that stress damages oocytes with increased secretion of glucorticoids. However, although injection of female mice with cortisol decreased oocyte competence, exposure of mouse oocytes directly to physiological or stress-induced concentrations of glucorticoids did not affect oocyte maturation and embryo development. This study has explored the mechanisms by which glucocorticoids impair oocyte competence. Female mice were injected with cortisol and the effects of cortisol-injection on oocyte competence, ovarian cell apoptosis and Fas/FasL activation were observed. The results showed that cortisol-injection decreased (a) oocyte developmental potential, (b) the E2/P4 ratio in serum and ovaries, and (c) expression of insulin-like growth factor 1, brain-derived neurotrophic factor and glucocorticoid receptor in mural granulosa cells (MGCs), while increasing levels of (a) cortisol in serum and ovaries, (b) apoptosis in MGCs and cumulus cells (CCs), (c) FasL secretion in ovaries and during oocyte maturation in vitro, and (d) Fas in MGCs, CCs and oocytes. The detrimental effects of cortisol-injection on oocyte competence and apoptosis of MGCs and CCs were significantly relieved when the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations were observed. Together, the results suggested that glucocorticoids impair oocyte competence by triggering apoptosis of ovarian cells via activating the Fas system.

An essential role for the intra-oocyte MAPK activity in the NSN-to-SN transition of germinal vesicle chromatin configuration in porcine oocytes.

The mechanisms for the transition from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) chromatin configuration during oocyte growth/maturation are unclear. By manipulating enzyme activities and measuring important molecules using small-follicle pig oocytes with a high proportion of NSN configuration and an extended germinal vesicle stage in vitro, this study has the first time up-to-date established the essential role for intra-oocyte mitogen-activated protein kinase (MAPK) in the NSN-to-SN transition. Within the oocyte in 1-2 mm follicles, a cAMP decline activates MAPK, which prevents the NSN-to-SN transition by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) while inhibiting histone deacetylase (HDAC). In cumulus cells of 1-2 mm follicles, a lower level of estradiol and oocyte-derived paracrine factor (ODPF) reduces natriuretic peptide receptor 2 (NPR2) while enhancing FSH and cAMP actions. FSH elevates cAMP levels, which decreases NPR2 while activating MAPK. MAPK closes the gap junctions, which, together with the NPR2 decrease, reduces cyclic guanosine monophosphate (cGMP) delivery leading to the cAMP decline within oocytes. In 3-6 mm follicles, a higher level of estradiol and ODPF and a FSH shortage initiate a reversion of the above events leading to MAPK inactivation and NSN-to-SN transition within oocytes.