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Benzo(a)pyrene [B(a)P] is an environmental endocrine disruptor with reproductive toxicity. The corpus luteum (CL) of the ovary plays an important role in embryo implantation and pregnancy maintenance. Our previous studies have shown that B(a)P exposure affects embryo implantation and endometrial decidualization in mouse, but its effects and mechanisms on CL function remain unclear. In this study, we explore the mechanism of ovarian toxicity of B(a)P using a pregnant mouse model and an in vitro model of human ovarian granulosa cells (GCs) KGN. Pregnant mice were gavaged with corn oil or 0.2 mg/kg.bw B(a)P from pregnant day 1 (D1) to D7, while KGN cells were treated with DMSO, 1.0IU/mL hCG, or 1.0IU/mL hCG plus benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a B(a)P metabolite. Our findings revealed that B(a)P exposure damaged embryo implantation and reduced estrogen and progesterone levels in early pregnant mice. Additionally, in vitro, BPDE impaired luteinization in KGN cells. We observed that B(a)P/BPDE promoted oxidative stress (OS) and inflammation, leading to apoptosis rather than pyroptosis in ovaries and luteinized KGN cells. This apoptotic response was mediated by the activation of inflammatory Caspase1 through the cleavage of BID. Furthermore, B(a)P/BPDE inhibited TRAF2 expression and suppressed NFκB signaling pathway activation. The administration of VX-765 to inhibit the Caspase1 activation, over-expression of TRAF2 using TRAF2-pcDNA3.1 (+) plasmid, and BetA-induced activation of NFκB signaling pathway successfully alleviated BPDE-induced apoptosis and cellular dysfunction in luteinized KGN cells. These findings were further confirmed in the KGN cell treated with H 2 O 2 and NAC. In conclusion, this study elucidated that B(a)P/BPDE induces apoptosis rather than pyroptosis in GCs via TRAF2-NFκB-Caspase1 during early pregnancy, and highlighting OS as the primary contributor to B(a)P/BPDE-induced ovarian toxicity. Our results unveil a novel role of TRAF2-NFκB-Caspase1 in B(a)P-induced apoptosis and broaden the understanding of mechanisms underlying unexplained luteal phase deficiency., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Benzo(a)pyrene (B(a)P) is a widespread persistent organic pollutant (POP) and a well-known endocrine disruptor. Exposure to BaP is known to disrupt the steroid balance and impair embryo implantation, but the mechanism under it remains unclear. The corpus luteum (CL), the primary source of progesterone during early pregnancy, plays a pivotal role in embryo implantation and pregnancy maintenance. The inappropriate luteal function may result in implantation failure and spontaneous abortions. Therefore, this study was conducted to assess the effects and potential mechanisms of B(a)P on the CL function. Our results showed that pregnant mice received B(a)P displayed impaired embryo implantation and dysfunction of ovarian CL. The estrogen and progesterone levels decreased by B(a)P. In vitro, exposure to BPDE, which is the metabolite of B(a)P, affected the luteinization of granular cell KK-1. Additionally, melatonin and its receptors, which are important for ovarian function and anti-oxidative damage, were affected by B(a)P or BPDE. B(a)P or BPDE-treated alone impaired antioxidant capacity of ovarian granulosa cells, caused an increasing of ROS and cell apoptosis, and disrupted the PI3K/AKT/GSK3β signaling pathway in vivo and in vitro. Co-treatment with melatonin alleviated B(a)P or BPDE-induced CL dysfunction by ameliorating oxidative stress, counteracting phosphorylation of PI3K/AKT/GSK3β signaling pathway, decreasing the apoptosis of the ovarian cells. Moreover, activation of the melatonin receptor by ramelteon in KK-1 cells exhibits an analogous protective effect as melatonin. In conclusion, our findings not only firstly clarify the potential mechanisms of BaP-induced CL dysfunction, but also extend the understanding about the ovarian protection of melatonin and its receptors against B(a)P exposure., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Women with hyperinsulinism and insulin resistance have reduced fertility, but the underlying mechanism is still poorly understood. Aberrant endometrial decidualization in early pregnancy was linked to pregnancy complications. In this study, we aimed to test whether elevated insulin levels compromise decidualization in early-stage pregnancy. C57BL/6J mice in high insulin-exposed group were given a subcutaneous injection of recombinant insulin at a concentration of 0.05 IU daily. During decidualization in early pregnancy, serum levels of insulin, E2, P4, LH, FSH and blood glucose were significantly altered in mice treated with high insulin levels. The number of embryo implantation sites and endometrial decidual markers BMP2, ER, PR was significantly decreased by high insulin levels in vivo. Artificial decidual induction in primary mouse endometrial stromal cells and immortal human endometrial stromal cells line were all compromised after treated with 100 nmol/L insulin levels. All these results on flow cytometry, transmission electron microscopy and western blotting of Bax, Bcl2, cleaved Caspase3, cleaved PARP proteins level showed that decidual cells apoptosis was significantly decreased. Mitochondrial transmembrane potential also significantly increased by the influence of high insulin levels. PI3K and p-Akt were much higher after insulin exposure and the compromised decidualization by high insulin treatment was rescued by PI3K/Akt inhibitor LY294002 both in vitro and in vivo. In conclusion, we demonstrated that elevated insulin levels could compromise mice decidualization in early-stage pregnancy and PI3K/p-Akt-regulated apoptosis was essential for this role. It provides a clue for future investigation on compromised reproduction in women with hyperinsulinemia.

Benzo(a)pyrene (BaP) is an endocrine-disrupting pollutant present in various aspects of daily life, and studies have demonstrated that BaP exerts reproductive toxicity. We previously showed that BaP damages endometrial morphology and decreases the number of implantation sites in early pregnant mice, but the mechanisms underlying these effects remain unclear. The endometrial function is crucial for implantation, which is associated with endometrial cell apoptosis. In this study, we focused on the effect of BaP on endometrial cell apoptosis and the role of WNT signaling during this process. Pregnant mice were gavaged with corn oil (control group) or 0.2 mg·kg -1 ·day -1 BaP (treatment group) from Days 1 to 6 of pregnancy. BaP impaired endometrial function by decreasing the expression of HOXA10 and BMP2, two markers of receptivity and decidualization. WNT5A and β-catenin were activated in the BaP group. BaP affected the expression of apoptosis-related proteins and inhibited the apoptosis of endometrial stromal cells. In vitro, human endometrial stromal cells (HESCs) were treated with different concentrations of BaP (dimethyl sulfoxide (DMSO); 5, 10 µM). WNT5A and β-catenin were also upregulated in the BaP treatment group. HESC apoptosis was restrained by BaP. Inhibiting WNT5A by SFRP5 partially restored the effect of BaP on apoptosis. In summary, these results suggested that BaP exposure during early pregnancy activates WNT5A/β-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function., (© 2018 Wiley Periodicals, Inc.)

Folate deficiency is a major risk factor of birth defects. Mechanistic studies on folate deficiency resulting in birth defects have mainly focused on fetal development. There have been few studies on folate deficiency from the point of view of the mother's uterus. In our previous study, we demonstrated that folate deficiency inhibits apoptosis of decidual cells, thereby restraining decidualization of the endometrium and impairing pregnancy. In this study, we further investigated the potential mechanism by which folate deficiency decreases endometrial apoptosis during decidualization. To investigate whether endometrium autophagy was inhibited under folate deficiency during decidualization, we performed real-time PCR for endometrial LC3 and P62 on day 6 (D6) to D8 of pregnancy in mice, and both were significantly changed compared to non-folate-deficient mice. Western blots showed that LC3-II and P62 were also changed in folate-deficient mice. Compared with control mice, a few punctuate LC3-II structures were detected in the folate deficiency group by immunofluorescence. Transmission electron micrographs of decidual cells on D8 showed that there were no evident autophagosomes in the folate deficiency group. In addition, apoptosis-related protein analysis by western blotting, TUNEL staining and flow cytometry showed that decreased endometrial apoptosis on D8 of pregnancy under folate deficiency was reversed after treatment with rapamycin, an autophagy inducer. ROS measurement showed that the endometrium ROS level was reduced by folate deficiency and that rapamycin reversed this effect on day 8 of pregnancy. All the results suggest that inhibiting endometrial autophagy may be implicated in the decreased endometrial apoptosis under folate deficiency during decidualization.

Studies have shown that metabolic syndrome is associated with reproductive problems. Women with metabolic syndrome, characterized by hyperinsulinemia, have common ovarian dysfunction, but the mechanism remains elusive. The aim of this research is to explore the effects of high levels of insulin on ovary function during early pregnancy. Mice in the high insulin-exposed group were given a subcutaneous injection of human recombinant insulin. After insulin treatment, changes in various hormones were tested using ELISA kits which showed hormones secreted by the ovary were significantly altered in the insulin group. TUNEL staining showed less TUNEL-positive cells in the insulin group. A significant decrease in Bax and an increase in Bcl2 in the ovary were found in the insulin group by immunohistochemical studies. Western blotting showed the expressions of apoptosis related proteins in the ovaries from the insulin group were obviously altered. In addition, expression of p-Akt proteins in the ovaries from the insulin group was significantly upregulated. Moreover, the Akt inhibitor LY294002 reversed the anti-apoptotic effects of high insulin in the ovary tissues in early pregnancy mice. All of these results showed that insulin impaired ovarian function during early pregnancy and ovarian apoptosis is imbalanced under the role of insulin. The PI3K/AKT signalling pathway might participate in this process., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The toxicity of sodium fluoride (NaF) to female fertility is currently recognized; however, the mechanisms are unclear. Previously, we reported a reduction in successful pregnancy rates, ovarian atrophy and dysfunction following exposure to NaF. The purpose of this study was to elucidate the underlying molecular mechanisms. Female Sprague-Dawley rats (10 rats/group) received 100 or 200mg/L NaF in their drinking water for 6 months or were assigned to an untreated control group. Apoptotic indices and oxidative stress indicators in blood and ovarian tissue were analyzed following sacrifice. The results confirmed the NaF-induced ovarian apoptosis, with concomitant activation of oxidative stress. Further investigations in ovarian granular cells showed that exposure to NaF activated extracellular regulated protein kinase (ERK) and c-Jun NH2 kinase (JNK), disrupting the ERK and JNK signaling pathways, while p38 and PI3K remained unchanged. These data demonstrated that oxidative stress may play a key role in NaF-induced ovarian dysfunction by activating the apoptotic ERK and JNK signaling pathways., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The Leydig cell tumor, derived from interstitial cells, is a rare neoplasm. In most cases, Leydig cell tumors are benign, however, if the tumor is malignant, no effective treatments are currently available. In this study, we aimed to evaluate the effects of icariin on the growth of the mouse Leydig tumor cell line MLTC-1 and to examine its underlying mechanism. Icariin caused a dose-dependent decrease in the viability of MLTC-1 cells, which coincided with an increase in cell apoptosis through regulation of the expression of Bcl-2/Bax and cytochrome c, activation of caspase-9 and -3. Moreover, the pro-apoptotic effect of icariin on MLTC-1 cells is related to piwil4, since icariin induced a decrease in piwil4 protein expression and piwil4 silencing significantly enhanced the cytotoxic effects of icariin in MLTC-1 cells. These findings suggest a novel anticancer effect of icariin in Leydig cell tumor through activation of the mitochondrial pathway and down-regulation of the expression of piwil4.

Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) is a 29 kDa mitochondrial precursor protein, which is proteolytically processed in mitochondria into a 23 kDa mature protein. It is released from the mitochondrial intermembrane space to cytosol after an apoptotic trigger. Smac/DIABLO acts as a dimer and it contributes to caspase activation by sequestering the inhibitor of apoptosis proteins (IAPs). In order to further investigate the mechanism of Smac/DIABLO action, we used the mature form of Smac/DIABLO as a bait and screened proteins that interact with mature Smac/DIABLO in human liver cDNA library using the yeast two-hybrid system. Forty-two colonies were obtained after 5.8x10(6) colonies were screened by nutrition limitation and X-galactosidase assay. After DNA sequence analysis and homology retrieval, one of the candidate proteins was identified as TRAF domain of the TNF receptor associated factor 3 (TRAF3). The interaction site between TRAF3 and Smac/DIABLO was identified by beta-galactosidase test. The interaction between TRAF3 and Smac/DIABLO via TRAF domain was identified in vivo by co-immunoprecipitation in HepG2 cells, and the direct interaction between TRAF3 and Smac/DIABLO in vitro was identified by GST-pull down assay. Co-expression of TRAF3 and mature Smac/DIABLO in 293 cells could enhance the Smac/DIABLO-mediated apoptosis. These results suggested that TRAF3 interacted with Smac/DIABLO via TRAF domain, leading to an increased proapoptotic effect of Smac/DIABLO in cytoplasm.

Aim: Recurrent miscarriage (RM) is associated with numerous clinical factors. However, some RM occurred without specific factors. It has been revealed that some molecules such as hormones, miRNAs, and transcription factors are involved in RM by regulating proliferation, apoptosis, etc. However, the mechanism of RM has yet to be identified clearly. Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that often act as sponges for miRNAs or binds to proteins involved in biological processes. However, the functional role of circRNAs in the uterine decidua of patients with early RM is still unclear. In this study, we aimed to investigate the mechanisms of circ-CYP24A1 in RM. Methods: The Dual-Luciferase Activity Assay was designed to analyze the bonding between circ-CYP24A1 and miR-224, and miR-224 and prolactin receptor (PRLR) mRNA 3′UTR. In situ hybridization (ISH) and immunohistochemistry (IHC) were used to observe the expression of circ-CYP24A1 and PRLR in the decidua. Rescue experiments were performed to investigate the regulating effects of circ-CYP24A1, miR-224, and PRLR. Western blotting was conducted to test the expression level of PRLR. The proliferation and apoptosis-related markers in Ishikawa cells were analyzed using CCK8, immunofluorescence staining, and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Results: In this study, based on the microarray analysis data, we identified a high level of circ-CYP24A1 and PRLR in the decidua of patients with early RM. Based on the bioinformatics prediction, the binding relationship between circ-CYP24A1 and miR-224, as well as miR-224 and PRLR, were verified. Functional experiments demonstrated that circ-CYP24A1 regulated proliferation and apoptosis by binding to and inhibiting miR-224, resulting in increased PRLR expression. Taken together, this study provides new insights into the mechanism of RM. Conclusion: In this study, we found that circ-CYP24A1 plays a role in RM by impairing the balance of cell proliferation and apoptosis by sponging miR-224, thereby regulating PRLR. [ABSTRACT FROM AUTHOR]

Embryo implantation is a complex process requiring reciprocal interactions between implantation-competent blastocysts and receptive uteri. Accumulating evidence from Digital Protein Expression Profiling indicates that DEK protein expression at implantation sites (ISs) was much higher than that at inter-implantation sites (IISs). In this study, we investigated the expression of DEK in mouse uterus by immunohistochemistry (IHC), Western blotting. We explored its function during decidualization of uterine stromal cells by inhibiting the expression of DEK. In further study of mechanism, the cell proliferation, apoptosis and DNA damage were detected after inhibiting DEK during decidualization of stromal cells. The results suggest that DEK participates in decidualization of stromal cells through mediating cell proliferation, apoptosis and DNA repair. [ABSTRACT FROM AUTHOR]