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Oxidative stress-induced granulosa cell (GCs) injury is believed to be a common trigger for follicular atresia. Emerging evidence indicates that excessive autophagy occurs in mammalian cells with oxidative damage. N-acetyl-5-methoxytrypamine (melatonin) has been shown to prevent GCs from oxidative injury, although the exact mechanism remains to be elucidated. Here, we first demonstrated that the suppression of autophagy through the JNK/BCL-2/BECN1 signaling is engaged in melatonin-mediated GCs protection against oxidative damage. Melatonin inhibited the loss of GCs viability, formation of GFP-MAP1LC3B puncta, accumulation of MAP1LC3B-II blots, degradation of SQSTM1 and the expression of BECN1, which was correlated with impaired activation of JNK during oxidative stress. On the other hand, blocking of autophagy and/or JNK also reduced the level of H₂O₂-induced GCs death, but failed to further restore GCs viability in the presence of melatonin. Particularly, the suppression of autophagy provided no additional protective effects when GCs were pretreated with JNK inhibitor and/or melatonin. Importantly, we found that the enhanced interaction between BCL-2 and BECN1 might be a responsive mechanism for autophagy suppression via the melatonin/JNK pathway. Moreover, blocking the downstream antioxidant system of melatonin using specific inhibitors further confirmed a direct role of melatonin/JNK/autophagy axis in preserving GCs survival without scavenging reactive oxygen species (ROS). Taken together, our findings uncover a novel function of melatonin in preventing GCs from oxidative damage by targeting JNK-mediated autophagy, which might contribute to develop therapeutic strategies for patients with ovulation failure-related disorders.

Human placental villi are surfaced by a multinucleated and terminally differentiated epithelium, the syncytiotrophoblast, with a subjacent layer of mononucleated cytotrophoblasts that can divide and fuse to replenish the syncytiotrophoblast. The objectives of this study were i) to develop an approach to definitively identify and distinguish cytotrophoblasts from the syncytiotrophoblast, ii) to unambiguously determine the relative susceptibility of villous cytotrophoblasts and syncytiotrophoblast to constitutive and stress-induced apoptosis mediated by caspases, and iii) to understand the progression of apoptosis in villous trophoblasts. Confocal microscopy with co-staining for E-cadherin and DNA allowed us to clearly distinguish the syncytiotrophoblast from cytotrophoblasts and identified that many cytotrophoblasts are deeply interdigitated into the syncytiotrophoblast. Staining for specific markers of caspase-mediated apoptosis indicate that apoptosis occurs readily in cytotrophoblasts but is remarkably inhibited in the syncytiotrophoblast. To determine if an apoptotic cell or cell fragment was from a cytotrophoblast or syncytiotrophoblast, we found co-staining with E-cadherin along with a marker for apoptosis was essential: in the absence of E-cadherin staining, apoptotic cytotrophoblasts would easily be mistaken as representing localized regions of apoptosis in the syncytiotrophoblast. Regions with perivillous fibrin-containing fibrinoid contain the remnants of trophoblast apoptosis, and we propose this apoptosis occurs only after physical isolation of a region of the syncytium from the main body of the syncytium. We propose models for the progression of apoptosis in villous cytotrophoblasts and for why caspase-mediated apoptosis does not occur within the syncytium of placental villi.

Polycystic ovary syndrome, a common condition characterized by endocrine dysfunction, menstrual irregularity, anovulation and polycystic ovaries, affects 5–7% of reproductive-age women. RAB5B, which is identified by a genome-wide association study as a risk locus for this syndrome, encodes a small GTPase involved in control of receptor internalization and early endosome fusion. We found that RAB5A mRNA levels in luteinized granulosa cells of obese patients with polycystic ovary syndrome were lower than in those of obese women without the syndrome. RAB5A regulated follicle-stimulating hormone (FSH)-mediated translocation of the FSH receptor (FSHR) from the membrane to the cytoplasm and the subsequent FSH–FSHR signaling pathway. We showed that RAB5A negatively regulated aromatase expression and estradiol synthesis in human granulosa cells in association with changes in FSHR levels by way of the cAMP/PKA/CREB pathway. The regulation of FSHR by RAB5A may have been associated with two transcription factors, USF1 and USF2. In conclusion, RAB5A gene was abnormally expressed in luteinized granulosa cells of obese patients with polycystic ovary syndrome, which may help explain high FSHR levels found in this syndrome.

Our previous study has demonstrated that bicarbonate in the uterine fluid plays an indispensable role in sperm capacitation. However, the cellular mechanisms underlying the formation of bicarbonate-rich uterine fluid and the regulatory mechanism remained largely unknown. In this study, the expression profiles of bicarbonate transport/production proteins, the cystic fibrosis transmembrane conductance regulator (CFTR), SLC26A6, carbonic anhydrase 2 (CAR2, CA2) and CAR12 (CA12), throughout the estrous cycle, were examined in the mouse uterus by western blot. The results showed that the maximum expression levels of the proteins examined were observed at estrus. Luminal surface pH measurements showed that the resting uterine surface pH at estrus was significantly higher than that at diestrus, which could be reduced significantly by CFTR blocker, diphenylamine-2,2′-dicarboxylic acid, SLC26A6 inhibitor, 4′,4′-diisothiocyanostilbene-2′,2′-disulfonic acid, and CA inhibitor, acetazolamide. In ovariectomized mice and primary culture of endometrial epithelial cells, estrogen could upregulate CFTR, SLC26A6, CAR2, and CAR12 expression with a corresponding increase in the bicarbonate-dependent short-circuit current (I _sc) and endometrial surface pH. The present results have demonstrated dynamic changes in uterine bicarbonate secretion and expression of the proteins involved in bicarbonate secretion during the estrous cycle and suggested a novel role of estrogen in regulating uterine bicarbonate transport, which may be important for successful reproduction.

Recent studies have demonstrated that embryonic stem cells (ESCs) have an underdeveloped innate immune system, but the biological implications of this finding are poorly understood. In this study, we compared the responses of mouse ESCs (mESCs) and mESC differentiated fibroblasts (mESC-FBs) to tumor necrosis factor α (TNFα) and interferons (IFNs). Our data revealed that TNFα, IFNα, IFNβ, or IFNγ alone do not cause apparent effects on mESCs and mESC-FBs, but the combination of TNFα and IFNγ (TNFα/IFNγ) showed toxicity to mESC-FBs as indicated by cell cycle inhibition and reduced cell viability, correlating with the expression of inducible nitric oxide synthase (iNOS). However, none of these effects were observed in mESCs that were treated with TNFα/IFNγ. Furthermore, mESC-FBs, but not mESCs, are vulnerable to cytotoxicity resulting from lipopolysaccharide (LPS)-activated macrophages. The insensitivity of mESCs to cytotoxicity in all cases is correlated with their lack of responses to TNFα and IFNγ. Similar to mESCs, human ESCs (hESCs) and iPSCs (hiPSCs) do not respond to TNFα and are not susceptible to the cytotoxicity of TNFα, IFNβ, or IFNγ alone or in combination that significantly affects human foreskin fibroblast (hFBs) and Hela cells. However, unlike mESCs, hESCs and hiPSCs can respond to IFNγ, but this does not cause significant cytotoxicity in hESCs and hiPSCs. Our findings in both mouse and human PSCs together support the hypothesis that attenuated innate immune responses could be a protective mechanism that limits immunologic cytotoxicity resulting from inflammatory and immune responses.

Some studies have demonstrated that the implantation rate of fresh transfer cycles is lower in the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol than in the GnRH agonist (GnRH-a) protocol during in vitro fertilization (IVF). This effect may be related to endometrial receptivity. However, the mechanisms are unclear. Here, endometrial tissues obtained from the mid-secretory phase of patients treated with GnRH-a or GnRH-ant protocols and from patients on their natural cycle were assessed. Endometrial expression of B-type creatine kinase (CKB), which plays important roles in the implantation phase, was significantly reduced in the GnRH-ant group. At the same time, expression of the endometrial receptivity marker HOXA10 was considerably reduced in the GnRH-ant group. GnRH-ant exposure in endometrial epithelial cells (EECs) in vitro decreased CKB expression and ATP generation and blocked polymerization of actin. Furthermore, in vitro GnRH-ant-exposed Ishikawa cells showed enhanced F-actin depolymerization, and these effects were rescued by CKB overexpression. Similar effects were observed after CKB knockdown, and these effects were rescued by CKB overexpression. Moreover, cell migration was decreased in CKB-knockdown Ishikawa cells compared with that in control cells, and this effect was also rescued by CKB overexpression. Overall, these findings showed that GnRH-ant affected CKB expression in EECs, resulting in cytoskeletal damage and migration failure. These results provide insight into the roles and molecular mechanisms of GnRH-ant treatment in the endometrium.

With tetraspanning topology, members of the membrane-spanning four-domain subfamily A (MS4A) may facilitate signaling or ion channel functions in many tissues. In this study, we report the cloning of a full-length cDNA from rat testis, designated Ms4a14 (Sp3111), which encodes the MS4A protein with 1139 amino acid residues. In situ hybridization and immunohistochemical analyses indicate that Ms4a14 is predominantly expressed from round spermatids to spermatozoa at specific stages in the rat testis at both the mRNA and protein level. Immunofluorescence analysis revealed that MS4A14 (SP3111) is located in the acrosome and the midpiece of the flagellum in mature sperm. Previously, we explored and reported the involvement of MS4A14 in reproductive functions, using antibody blockage during IVF and a transgenic RNA interference method in a mouse model. Our results suggested that MS4A14 is involved in fertilization and zygote division. As MS4A14 protein exists in mammals, such as humans, cows, dogs, and rodents, MS4A14 may play a ubiquitous role in mammalian reproduction.

The porcine female reproductive tract undergoes estrogen receptor (ER) α-dependent development after birth (postnatal day=PND 0), the course of which can determine adult uterine function. Uterotrophic effects of relaxin (RLX) in the porcine neonate are age specific and may involve ER activation. Here, objectives were to determine effects of RLX and estrogen administered from birth on uterine and cervical growth and expression of ERα, vascular endothelial growth factor (VEGF), and the RLX receptor (RXFP1). On PND 0, gilts were treated with the antiestrogen ICI 182 780 (ICI) or vehicle alone and, 2 h later, were given estradiol-17β (E) or porcine RLX for 2 days. Neither RLX nor E affected uterine wet weight or protein content on PND 2. However, RLX, but not E, increased cervical wet weight and protein content when compared with controls. Pretreatment with ICI did not inhibit RLX-stimulated cervical growth. Uterine and cervical ERα increased in response to RLX, but not E. Both RLX and E increased VEGF in the uterus and cervix on PND 2. Pretreatment with ICI increased VEGF in both tissues and increased RLX-induced cervical VEGF. In the uterus E, but not RLX, increased RXFP1 mRNA. In the cervix, E increased RXFP1 gene expression whereas RLX decreased it. Results indicate that the neonatal uterus and cervix are sensitive to E and RLX and that growth responses to RLX in these tissues differ by PND 2. Effects of RLX on uterine and cervical ERα and VEGF expression may be important for neonatal reproductive tract development.

In cloned animals where somatic cell nuclei and oocytes are from the same or closely related species, the mitochondrial DNA (mtDNA) of the oocyte is dominantly inherited. However, in nuclear transfer (NT) embryos where nuclear donor and oocyte are from two distantly related species, the distribution of the mtDNA species is not known. Here we determined the levels of macaque and rabbit mtDNAs in macaque embryos reprogrammed by rabbit oocytes. Quantification using a real-time PCR method showed that both macaque and rabbit mtDNAs coexist in NT embryos at all preimplantation stages, with maternal mtDNA being dominant. Single NT embryos at the 1-cell stage immediately after fusion contained 2.6 × 10⁴ copies of macaque mtDNA and 1.3 × 10⁶ copies of rabbit mtDNA. Copy numbers of both mtDNA species did not change significantly from the 1-cell to the morula stages. In the single blastocyst, however, the number of rabbit mtDNA increased dramatically while macaque mtDNA decreased. The ratio of nuclear donor mtDNA to oocyte mtDNA dropped sharply from 2% at the 1-cell stage to 0.011% at the blastocyst stage. These results suggest that maternal mtDNA replicates after the morula stage.

Indian hedgehog (Ihh) signaling regulates endometrial receptivity and is an indispensable mediator of embryonic implantation. Hedgehog signaling is known to regulate autophagy, and aberrant regulation of autophagy is critically implicated in the pathogenesis of endometriosis and adenomyosis. However, potential dysregulation of Ihh signaling and its role in autophagy modulation in these diseases remain obscure. In this study, we found that components of Ihh signaling were significantly decreased, whereas the autophagy marker protein, LC3BII, was significantly increased in endometrial tissues of women with endometriosis or adenomyosis. Inhibition of Ihh signaling with the small-molecule inhibitor GANT61 or Gli1 silencing in primary endometrial stromal cells increased autophagic activity, as measured by LC3 turnover assay and tandem mCherry-eGFP-LC3B fluorescence microscopy. Furthermore, we observed that GANT61 treatment significantly attenuated hydrogen peroxide-induced cell death, whereas disruption of autophagy with chloroquine diminished this effect. Collectively, these findings reveal that Ihh signaling is suppressed in endometrial tissues of patients with endometriosis or adenomyosis. This abnormal decrease may contribute to endometrial autophagy activation, which may promote aberrant survival of endometrial cells in ectopic sites in these two gynecological diseases.