High glucose exposure of preimplantation embryos causes glucose intolerance and insulin resistance in F1 and F2 male offspring.

BACKGROUND: Although studies suggest that maternal high glucose (HG) increases offspring susceptibility to type 2 diabetes mellitus (T2DM), the underlying mechanisms are largely unclear. We studied whether glucose levels in oviducts are elevated when pregestational diabetic females get pregnant and whether the oviductal HG (OVHG) would act directly on embryos to increase offspring's T2DM susceptibility. METHODS: We established an in vivo model of OVHG by injecting female mice with streptozotocin (STZ) during the preimplantation period and an in vitro model of embryo culture with HG (ECHG) by culturing preimplantation embryos with HG, before examining glucose tolerance and insulin resistance (IR) in F1 and F2 offspring. FINDINGS: Injection of female mice with STZ induced a lasting significant glucose elevation in blood and oviduct fluid during the preimplantation period. The glucose tolerance test showed that both the STZ-induced OVHG and the ECHG caused glucose intolerance in F1 male and F1-sired F2 male offspring but had no effect on female offspring. Insulin tolerance test and the analysis for IR-related gene expression and glycogen contents in liver and muscle revealed significant IR in these male offspring. INTERPRETATION: This study provided evidence that HG can act directly on preimplantation embryos to increase offspring's T2DM susceptibility suggesting that the preimplantation period is a critical stage for transmission of mother's diabetes to offspring. FUND: This study was supported by grants from the China National Natural Science Foundation (Nos. 31772599, 32072738, 31702114, and 31902160), and the Natural Science Foundation of Shandong Province (Nos. ZR2022MC036, ZR2017BC025 and ZR2020QC102).

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.

Glucocorticoid Exposure of Preimplantation Embryos Increases Offspring Anxiety-Like Behavior by Upregulating miR-211-5p via Trpm1 Demethylation.

Most studies on mechanisms by which prenatal stress affects offspring behavior were conducted during late pregnancy using in vivo models; studies on the effect of preimplantation stress are rare. In vivo models do not allow accurate specification of the roles of different hormones and cells within the complicated living organism, and cannot verify whether hormones act directly on embryos or indirectly to alter progeny behavior. Furthermore, the number of anxiety-related miRNAs identified are limited. This study showed that both mouse embryculture with corticosterone (ECC) and maternal preimplantation restraint stress (PIRS) increased anxiety-like behavior (ALB) while decreasing hippocampal expression of glucocorticoid receptor (GR) and brain-derived neurotrophic factor (BDNF) in offspring. ECC/PIRS downregulated GR and BDNF expression by increasing miR-211-5p expression via promoter demethylation of its host gene Trpm1, and this epigenetic cell fate determination was exclusively perpetuated during development into mature hippocampus. Transfection with miR-211-5p mimic/inhibitor in cultured hippocampal cell lines confirmed that miR-211-5p downregulated Gr and Bdnf. Intrahippocampal injection of miR-211-5p agomir/antagomir validated that miR-211-5p dose-dependently increased ALB while decreasing hippocampal GR/BDNF expression. In conclusion, preimplantation exposure to glucocorticoids increased ALB by upregulating miR-211-5p via Trpm1 demethylation, and miR-211-5p may be used as therapeutic targets and biomarkers for anxiety-related diseases.

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.

A next-generation sequencing study on mechanisms by which restraint and social instability stresses of male mice alter offspring anxiety-like behavior.

Pathophysiological mechanisms for depression/anxiety are largely unknown. Evidence for transgenerational transmission of acquired epigenetic marks remains limited. We bred unstressed (US) female mice with adolescently restraint-stressed (RS), social instability-stressed (SI) or US males to produce RS, SI and control F1 offspring, respectively. Compared to controls, while paternal RS decreased anxiety-like behavior (ALB) in both female and male offspring, paternal SI increased ALB only in female offspring. Next-generation sequencing and bioinformatics using RS and SI female offspring identified 5 candidate anxiety-transmitting (CAT) genes; each showed a consistent pattern of DNA methylation from F0 spermatozoa through F1 blastocysts to fetal and adult hippocampi. Further analyses validated 4 CAT genes, demonstrated that paternal SI caused ALB differences between male and female offspring through modifying the CAT genes, and indicated a strong correlation between inflammation and ALB pathogenesis and an important function for intronic DNA methylation in regulating ALB-related genes. In conclusion, this study identified important CAT genes and suggested the possibility that stresses on males might alter offspring's ALB by modifying sperm DNA methylation.

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.

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.

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.

Parental life events cause behavioral difference among offspring: Adult pre-gestational restraint stress reduces anxiety across generations.

While effects of gestational, neonatal or adolescent stress on psychological alterations in progeny have been extensively studied, much less is known regarding the effects of adult pre-gestational life events on offspring behavior. Although full siblings often display behavioral differences, whether the different parental life events prior to different pregnancies contribute to these behavioral differences among siblings is worth studying. In this study, male and female adult mice were restrained for 60 days before mating with unstressed or stressed partners. F1 offspring were examined for anxiety or mated to generate F2. Both F1 females and males from restrained mothers and/or fathers showed significantly reduced anxiety and serum cortisol and increased mRNA levels of glucocorticoid receptor and brain-derived neurotrophic factor compared to control offspring from unstressed parents. Similar behavioral and molecular changes were also observed in F2 females and males. Although restraint of adolescent mice reduced anxiety in F1 of both sexes, social instability of them increased anxiety predominantly in F1 females. Thus, adult pre-gestational restraint reduced offspring's anxiety across generations; different stressors on parents may cause different phenotypes in offspring; individual behaviors can depend on adult life experiences of parents.

Stresses on Female Mice Impair Oocyte Developmental Potential: Effects of Stress Severity and Duration on Oocytes at the Growing Follicle Stage.

AIM: Although previous studies found that 1-time acute stress applied during follicle maturation impaired oocyte competence, it is unknown whether repeated chronic stress, which is known to cause animal behavioral adaptation, would damage oocytes when applied during follicle growth. METHODS AND RESULTS: In this study, female mice were exposed to repeated restraint stress (RRS) or unpredictable stress (UPS) for different days before equine chorionic gonadotropin injection to initiate oocyte prematuration development and to observe effects of different stressors on oocytes in the growing follicles. The results showed that although oocyte pre- and postimplantation development was unaffected when mice were exposed to RRS or UPS once a day for 4 days, development was impaired when mice were exposed to RRS for 8 or more days or to UPS twice a day for 4 days (4 × 2). The 4 × 2 UPS caused more oxidative stress in oocytes and severer apoptosis in antral follicles than did the 4-day RRS. The RRS mice were stressed consistently from days 1 to 23 of restraint, and the stress that a mouse had 4 × 2 UPS was severer than that from 4-day RRS. CONCLUSION: The results suggest that (1) the degree that a stress damages oocytes is the product of duration × severity of the stress; (2) RRS impaired oocyte developmental potential through cumulative effects on growing follicles; and (3) preantral follicles were not as sensitive to stress as antral follicles were.

Role of cytoskeleton in regulating fusion of nucleoli: a study using the activated mouse oocyte model.

Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca²âº-free CZB medium containing 10 mM SrCl2) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF.