Ti3C2 nanosheet-induced autophagy derails ovarian functions.

BACKGROUND: Two-dimensional ultrathin Ti3C2 (MXene) nanosheets have gained significant attention in various biomedical applications. Although previous studies have described the accumulation and associated damage of Ti3C2 nanosheets in the testes and placenta. However, it is currently unclear whether Ti3C2 nanosheets can be translocated to the ovaries and cause ovarian damage, thereby impairing ovarian functions. RESULTS: We established a mouse model with different doses (1.25, 2.5, and 5 mg/kg bw/d) of Ti3C2 nanosheets injected intravenously for three days. We demonstrated that Ti3C2 nanosheets can enter the ovaries and were internalized by granulosa cells, leading to a decrease in the number of primary, secondary and antral follicles. Furthermore, the decrease in follicles is closely associated with higher levels of FSH and LH, as well as increased level of E2 and P4, and decreased level of T in mouse ovary. In further studies, we found that exposure toTi3C2 nanosheets increased the levels of Beclin1, ATG5, and the ratio of LC3II/Ι, leading to autophagy activation. Additionally, the level of P62 increased, resulting in autophagic flux blockade. Ti3C2 nanosheets can activate autophagy through the PI3K/AKT/mTOR signaling pathway, with oxidative stress playing an important role in this process. Therefore, we chose the ovarian granulosa cell line (KGN cells) for in vitro validation of the impact of autophagy on the hormone secretion capability. The inhibition of autophagy initiation by 3-Methyladenine (3-MA) promoted smooth autophagic flow, thereby partially reduced the secretion of estradiol and progesterone by KGN cells; Whereas blocking autophagic flux by Rapamycin (RAPA) further exacerbated the secretion of estradiol and progesterone in cells. CONCLUSION: Ti3C2 nanosheet-induced increased secretion of hormones in the ovary is mediated through the activation of autophagy and impairment of autophagic flux, which disrupts normal follicular development. These results imply that autophagy dysfunction may be one of the underlying mechanisms of Ti3C2-induced damage to ovarian granulosa cells. Our findings further reveal the mechanism of female reproductive toxicity induced by Ti3C2 nanosheets.

Uterine deficiency of Dnmt3b impairs decidualization and causes consequent embryo implantation defects.

Uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects. Recent advances in molecular technologies have allowed the unprecedented mapping of epigenetic modifications during embryo implantation. DNA methyltransferase 3a (DNMT3A) and DNMT3B are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. It was reported that conditional knockout of Dnmt3a in the uterus does not markedly affect endometrial function during embryo implantation, but the tissue-specific functions of Dnmt3b in the endometrium during embryo implantation remain poorly understood to investigate the role of Dnmt3b during peri-implantation period. Here, we generated Dnmt3b conditional knockout (Dnmt3bd/d) female mice using progesterone receptor-Cre mice and examined the role of Dnmt3b during embryo implantation. Dnmt3bd/d female mice exhibited compromised fertility, which was associated with defective decidualization, but not endometrial receptivity. Furthermore, results showed loss of Dnmt3b did not lead to altered genomic methylation patterns of the decidual endometrium during early pregnancy. Transcriptome sequencing analysis of uteri from day 6 pregnant mice identified phosphoglycerate kinase 1 (Pgk1) as one of the most variable genes in Dnmt3bd/d decidual endometrium. Potential roles of PGK1 in the decidualization process during early pregnancy were confirmed. Lastly, the compromised decidualization upon the downregulation of Dnmt3b could be reversed by overexpression of Pgk1. Collectively, our findings indicate that uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects.

Decidual derived exosomal miR-99a-5p targets Ppp2r5a to inhibit trophoblast invasion in response to CeO2NPs exposure.

BACKGROUND: The biological effects of cerium dioxide nanoparticles (CeO2NPs), a novel material in the biomedical field, have attracted widespread attention. Our previous study confirmed that exposure to CeO2NPs during pregnancy led to abnormal trophoblast invasion during early placental development, thereby impairing placental development. The potential mechanisms may be related to low-quality decidualization triggered by CeO2NPs exposure, such as an imbalance in trophoblast invasion regulators secreted by decidual cells. However, the intermediate link mediating the "dialogue" between decidual cells and trophoblasts during this process remains unclear. As an important connection between cells, exosomes participate in the "dialogue" between endometrial cells and trophoblasts. Exosomes transfer bioactive microRNA into target cells, which can target and regulate the level of mRNA in target cells. RESULTS: Here, we constructed a mice primary uterine stromal cell-induced decidualization model in vitro, and detected the effect of CeO2NPs exposure on the expression of decidual-derived exosomal miRNAs by high-throughput sequencing. Bioinformatics analysis and dual-luciferase reporter assays were performed to identify target genes of the screened key miRNAs in regulating trophoblast invasion. Finally, the role of the screened miRNAs and their target genes in regulating trophoblast (HTR-8/SVneo cells) invasion was confirmed. The results showed that CeO2NPs exposure inhibited trophoblast invasion by promoting miR-99a-5p expression in decidual-derived exosomes, and Ppp2r5a is a potential target gene for miR-99a-5p to inhibit trophoblast invasion. CONCLUSIONS: This study revealed the molecular mechanism by which CeO2NPs exposure inhibits trophoblast invasion from the perspective of decidual derived exosomal miRNAs. These results will provide an experimental basis for screening potential therapeutic targets for the negative biological effects of CeO2NPs exposure and new ideas for studying the mechanism of damage to trophoblast cells at the decidual-foetal interface by harmful environmental or occupational factors.

Exposure to Benzo(a)pyrene promotes proliferation and inhibits differentiation of stromal cells in mice during decidualization.

The environmental pollutant Benzo(a)pyrene (BaP) has an adverse effect on the reproductive performance of mammals. We previously showed that BaP treatment during early pregnancy damages endometrial morphology and impairs embryo implantation. Endometrial decidualization at the implantation site (IS) after embryo implantation is crucial for pregnancy maintenance and placental development. The balance between proliferation and differentiation in endometrial stromal cells (ESCs) is a crucial event of decidualization, which is regulated by the cell cycle. Here, we report that abnormal decidualization caused by BaP is associated with cell cycle disturbance of stromal cells. The mice in the treatment group were gavaged with 0.2 mg/kg/day BaP from day 1-8 of pregnancy, while those in control were gavaged with corn oil in parallel. BaP damaged the decidualization of ESCs and reduced the number of polyploid cells. Meanwhile, BaP up-regulated the expression of Ki67 and PCNA, affecting the differentiation of stromal cells. The cell cycle progression analysis during decidualization in vivo and in vitro showed that BaP induced polyploid cells deficiency with enhanced expressions of CyclinA(E)/CDK2, CyclinD/CDK4 and CyclinB/CDK1, which promote the transformation of cells from G1 to S phase and simultaneously activate the G2/M phase. The above results indicated that BaP exposure accelerates cell cycle progression, promotes ESC proliferation, inhibits differentiation, and impedes proper decidualization and polyploidy development. Thus, the imbalance of ESC proliferation and differentiation would be an important mechanism for BaP-induced defective decidualization.

Rictor/mTORC2 is involved in endometrial receptivity by regulating epithelial remodeling.

Successful embryo implantation requires well-functioning endometrial luminal epithelial cells to establish uterine receptivity. Inadequate uterine receptivity is responsible for approximately two thirds of implantation failures in humans. However, the regulatory mechanism governing this functional process remains largely unexplored. A previous study revealed that the expression of Rictor, the main member of mTORC2, in mouse epithelial cells is increased on the fourth day of gestation (D4). Here, we provide the first report of the involvement of Rictor in the regulation of endometrial receptivity. Rictor was conditionally ablated in the mouse endometrium using a progesterone receptor cre (PRcre ) mouse model. Loss of Rictor altered polarity remodeling and the Na+ channel protein of endometrial cells by mediating Rac-1/PAK1(pPAK1)/ERM(pERM) and Sgk1/pSgk1 signaling, respectively, ultimately resulting in impaired fertility. In the endometrium of women with infertility, the expression of Rictor was changed, along with the morphological transformation and Na+ channel protein of epithelial cells. Our findings demonstrate that Rictor is crucial for the establishment of uterine receptivity in both mice and humans. The present study may help improve the molecular regulatory network of endometrial receptivity and provide new diagnostic and treatment strategies for infertility.

Maternal exposure to CeO2NPs derails placental development through trophoblast dysfunction mediated by excessive autophagy activation.

BACKGROUND: The increasing use of cerium dioxide nanoparticles (CeO2NPs) in biomedical field has attracted substantial attention about their potential risks to human health. Recent studies have shown that nanoparticles can induce placental dysfunction and even fetal abortion, but a more detailed mechanism of nanoparticles affecting placental development remains elusive. RESULTS: Here, we constructed a mouse exposure model with different doses of CeO2NPs (2.5, 4, 5, 7.5, and 10 mg kg-1 day-1, average particle size 3-5 nm), finding that intravenous exposure to pregnant mice with CeO2NPs could cause abnormal placental development. Deposited nanoparticles were able to be observed in the placental trophoblast at doses of 5 and 7.5 mg kg-1 day-1. Diving into molecular mechanisms indicated that CeO2NPs exposure could lead to autophagy activation in placental trophoblast. At the cellular level, exposure to CeO2NPs inhibited the migration and invasion of HTR-8/SVneo and activated the autophagy through mammalian target of rapamycin complex1 (mTORC1) signaling pathway. Furthermore, inhibition of autophagy initiation by 3-Methyladenine (3-MA) partially restored the function of HTR-8/SVneo, while blocking autophagic flow by Chloroquine (CQ) aggravated the functional damage. CONCLUSIONS: Maternal exposure to CeO2NPs impairs placental development through trophoblast dysfunction mediated by excessive autophagy activation. These results suggested that autophagy dysfunction may be a potential mechanism for the impairment of trophoblast by CeO2NPs exposure. As above, our findings provide insights into the toxicity mechanism to the reproductive system induced by rare-earth nanoparticles exposure.

Exposure to two-dimensional ultrathin Ti3C2 (MXene) nanosheets during early pregnancy impairs neurodevelopment of offspring in mice.

BACKGROUND: Two-dimensional ultrathin Ti3C2 (MXene) nanosheets have been extensively explored for various biomedical applications. However, safety issues and the effects of Ti3C2 on human health remain poorly understood. RESULTS: To explore the influence on foetal or offspring after exposure to Ti3C2 nanosheets, we established a mouse model exposed to different doses of Ti3C2 nanosheets during early pregnancy in this study. We found that Ti3C2 nanosheets had negligible effect on the reproductive ability of maternal mice, including average pregnancy days, number of new-borns, and neonatal weight, etc. Unexpectedly, abnormal neurobehavior and pathological changes in the cerebral hippocampus and cortex in adult offspring were observed following Ti3C2 nanosheet treatment. In further studies, it was found that Ti3C2 exposure led to developmental and functional defects in the placenta, including reduced area of labyrinth, disordered secretion of placental hormones, and metabolic function derailment. The long-chain unsaturated fatty acids were significantly higher in the placenta after Ti3C2 exposure, especially docosahexaenoic acid (DHA) and linoleic acid. The metabolic pathway analysis showed that biosynthesis of unsaturated fatty acids was upregulated while linoleic acid metabolism was downregulated. CONCLUSIONS: These developmental and functional defects, particularly metabolic function derailment in placenta may be the cause for the neuropathology in the offspring. This is the first report about the effects of Ti3C2 nanosheet exposure on pregnancy and offspring. The data provides a better understanding of Ti3C2 nanosheets safety. It is suggested that future studies should pay more attention to the long-term effects of nanomaterials exposure, including the health of offspring in adulthood, rather than only focus on short-term effects, such as pregnancy outcomes. Metabolomics could provide clues for finding the prevention targets of the biological negative effect of Ti3C2 nanosheets.

AMPK/mTOR downregulated autophagy enhances aberrant endometrial decidualization in folate-deficient pregnant mice.

Existing evidence suggests that adverse pregnancy outcomes are closely related to dietary factors. Folate plays an important role in neural tube formation and fetal growth, folate deficiency is a major risk factor of birth defects. Our early studies showed that folate deficiency could impair enddecidualization, however, the mechanism is still unclear. Dysfunctional autophagy is associated with many diseases. Here, we aimed to evaluate the adverse effect of folate deficiency on endometrial decidualization, with a particular focus on endometrial cell autophagy. Mice were fed with no folate diet in vivo and the mouse endometrial stromal cell was cultured in a folate-free medium in vitro. The decrease of the number of endometrial autophagosomes and the protein expressions of autophagy in the folate-deficient group indicated that autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation were inhibited. Autophagic flux examination using mCherry-GFP-LC3 transfection showed that the fusion of autophagosomes with lysosomes was inhibited by folate deficiency. Autophagy inducer rapamycin could reverse the impairment of folate deficiency on endometrial decidualization. Moreover, folate deficiency could reduce autophagy by disrupting AMPK/mTOR signaling, resulting in aberrant endometrial decidualization and adverse pregnancy outcomes. Further co-immunoprecipitation examination showed that decidual marker protein Hoxa10 could interact with autophagic marker protein Cathepsin L, and the interaction was notably reduced by folate deficiency. In conclusion, AMPK/mTOR downregulated autophagy was essential for aberrant endometrial decidualization in early pregnant mice, which could result in adverse pregnancy outcomes. This provided some new clues for understanding the causal mechanisms of birth defects induced by folate deficiency.

Exposure to N-monoacetyl-p-phenylenediamine impaired ovarian function in mice.

p-Phenylenediamine (PPD) is the main constituent of permanent hair dye and is also widely used in the photographic and rubber industries. PPD and its metabolites have been shown to increase the risk of cancer (especially ovarian cancer); however, their effect on female reproduction is unclear. We investigated the effects of the PPD metabolite N-monoacetyl-PPD (MAPPD) on mouse blastocyst development and ovarian function. Sixty 8-week-old female Kunming mice were administered at 0-, 100-, and 300-mg/kg/day MPPD by gavage for 28 days. KGN (human ovarian granulosa cells) were treated with MAPPD at concentrations of 0, 50, 100, and 300 µg/ml for 48 h. The number of abnormal blastocysts increased on gestation day 3.5 in all treatment groups. Compared with the control group, in MAPPD exposed group, the number of antral follicles decreased, the levels of E2 and P4 decreased in ovarian tissue, the serum levels of E2 , P4 , luteinizing hormone (LH), and T decreased, and follicle-stimulating hormone (FSH) increased. The expression of FSH receptor (FSHR) and LH receptor (LHR) was significantly downregulated, and the level of oxidative stress was significantly increased. In KGN cells, the level of reactive oxygen species increased in a dose-dependent manner, and the mRNA levels of FSHR, LHR, and aromatase increased. These results suggest that MAPPD inhibits FSH- and LH-induced aromatase activity by causing oxidative stress, which decrease hormone levels, leading to abnormal follicle development. Meanwhile, MAPPD exposure could affect early embryonic development abnormalities by affecting the quality of ovum.

Exposure to ethylparaben and propylparaben interfere with embryo implantation by compromising endometrial decidualization in early pregnant mice.

Ethylparaben (EtP) and propylparaben (PrP) are common preservatives and well-known endocrine-disrupting chemicals. Studies have demonstrated that they can reduce female fertility, but the underlying mechanism, especially that on embryo implantation, is still poorly understood. Endometrial decidualization is a critical event for embryo implantation. In this study, we aimed to explore the effects of EtP/PrP on endometrial decidualization. Pregnant mice were dosed daily by oral gavage with EtP at 0, 400, 800 and 1600 mg/kg or with PrP at 0, 625, 1250 and 2500 mg/kg from Day 1 of pregnancy until sacrifice. The results showed that the rate of pregnant mice with impaired embryo implantation, whose number of implantation sites was less than 7, was significantly increased after exposure to 1600 mg/kg EtP or 2500 mg/kg PrP. Further study found that the expression of endometrial decidualization markers HOXA10, MMP9 and PR was significantly downregulated in 1600 mg/kg EtP group and 2500 mg/kg PrP group. Notably, serum oestrogen and progesterone levels were significantly increased, whereas the expression of uterine oestrogen receptor and progesterone receptor was decreased following 1600 mg/kg EtP or 2500 mg/kg PrP exposure. In the breeding test, fewer offspring were found after females were exposed to 1600 mg/kg EtP or 2500 mg/kg PrP in early pregnancy. This demonstrated that exposure to EtP/PrP interfered with embryo implantation by compromising endometrial decidualization in early-stage pregnant mice. Disorders of reproductive hormones and hormone receptor signals could be responsible for impaired decidualization. This study broadened the understanding on the biological safety of EtP and PrP.

Melatonin alleviates benzo(a)pyrene-induced ovarian corpus luteum dysfunction by suppressing excessive oxidative stress and apoptosis.

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/GSK3ß 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/GSK3ß 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.

Benzo(a)pyrene inhibits endometrial cell apoptosis in early pregnant mice via the WNT5A pathway.

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 ß-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 ß-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/ß-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function.

Autophagy regulates abnormal placentation induced by folate deficiency in mice.

Folate deficiency has been linked to a wide range of pregnancy disorders. Most research about folate-deficiency has focused on the embryo itself, little attention has been paid to possible effects on the placenta. According to our results, the morphology of the placenta, endocrine function, and the expression of genes involved in placental differentiation were all abnormal in folate-deficient mice on days 10, 12, and 14 of pregnancy. Similar results were found in human placenta explants cultured in folate-deficient medium. Autophagy is an inducible catabolic process activated by external nutrients starvation. Here we explored further, whether autophagy was involved in the abnormal placentation caused by folate-deficiency. The aberrant number of autophagosomes measured by transmission electron microscopy and the deviant expression of autophagy-related markers showed a disordered autophagy in placentas under conditions of folate-deficiency in vivo and in vitro dual-fluorescence mRFP-eGFP-LC3 analysis indicated enhanced autophagy was detected in HTR8/SVneo cells incubated in folate-deficient medium. Importantly, the placentation impairment in mice and human placenta explants could be recovered by inhibiting placental autophagy using 3-MA. In addition, the apoptosis and invasive capability of HTR8/SVneo cells were obviously suppressed by folate deficiency but notably elevated by 3-MA. These data suggest that folate deficiency can impair placentation and autophagy is a key factor in this. However, the signal pathway by which folate deficiency causes aberrant autophagy needs to be explored further.

Folate deficiency inhibits the PCP pathway and alters genomic methylation levels during embryonic development.

Folate deficiency results in abnormal embryonic development, but the underlying mechanisms remain to be comprehensively investigated. Mutation of Vangl genes belonging to the planar cell polarity (PCP) pathway is associated with abnormal embryonic development, but the effect of folate deficiency on the PCP pathway is unclear. In this study, we found that folate deficiency inhibited Vangl gene expression and Vangl protein binding to the ligand Dvl. As a methyl donor, folate can chemically alter the DNA methylation levels of genomic sequences. Here, reduced representation bisulfite sequencing (RRBS) was employed to detect the methylation profiles of mouse embryos. The results confirmed that folate deficiency affected the genomic methylation levels of mouse embryos, which resulted in down-regulation of key genes involved in embryonic development. Gene ontology (GO) analysis suggested that the genes located in the differentially methylated regions (DMRs) are primarily involved in biological regulation, cellular processes, development, metabolism, and signaling pathways. The data revealed that folate deficiency inhibits the PCP pathway and alters genomic methylation profiles, which may be the underlying mechanisms through which folate deficiency impairs embryonic development.

Expression and function of Pdcd4 in mouse endometrium during early pregnancy.

Embryo implantation is a complex process involving synchronised crosstalk between a receptive endometrium and functional blastocysts. Apoptosis plays an important role in this process as well as in the maintenance of pregnancy. In this study, we analysed the expression pattern of programmed cell death 4 (Pdcd4), a gene associated with apoptosis in the mouse endometrium, during early pregnancy and pseudopregnancy by real-time quantitative polymerase chain reaction, in situ hybridisation, Western blotting and immunohistochemistry. The results showed that Pdcd4 was increased along with days of pregnancy and significantly reduced at implantation sites (IS) from day 5 of pregnancy (D5). The level of Pdcd4 at IS was substantially lower than that at interimplantation sites (IIS) on D6 and D7. In addition, Pdcd4 expression in the endometrium was reduced in response to artificially induced decidualisation in vivo and in vitro Downregulation of Pdcd4 gene expression in cultured primary stromal cells promoted decidualisation, while upregulation inhibited the decidualisation process by increasing apoptosis. These results demonstrate that Pdcd4 is involved in stromal cell decidualisation by mediating apoptosis and therefore plays a role in embryo implantation in mice.

A palladium-platinum bimetal nanodendritic melamine network for signal amplification in voltammetric sensing of DNA.

A sandwich-type electrochemical DNA sensor is described for the detection of oligonucleotides typical for MECP2 gene mutations. Palladium nanoparticles (PdNPs) and platinum nanoparticles (PtNPs) were used to synthesize flower-like PdPt nanodendrites (NDs) by a one-pot method. The PdPt NDs possess a high specific surface area and excellent catalytic capabilities. They served as the carrier for the signal DNA probe (SP) and simultaneously catalyze the reduction of hydrogen peroxide (H2O2). The PdPt NDs were modified with melamine, and this results in the formation of a PdPt-melamine network through stable interactions between the PdPt NDs and the three amino groups of each melamine molecule. The network exhibits excellent catalytic ability in enhancing the current signal response in the voltammetric detection of MECP2 gene mutation, best measured at -0.4 V vs. SCE and using H2O2 as the electrochemical probe. In addition, gold nanoflowers were electrodeposited on the electrode interface in order to accelerate electron transfer and to capture the capture probe. The sensor is stable and can detect MECP2 gene mutations in the 1 fmol·L-1 to 1 nmol·L-1 concentration range, with a 0.33 fmol·L-1 lower detection limit at an S/N ratio of 3. Graphical abstract Schematic presentation of electrodes for the determination of the X-linked gene methyl-CpG-binding protein 2 (MECP2). The sensor is based on the electrooxidation of added H2O2 by using the melamine modified palladium platinum bimetal nanodendrites as network signal amplification strategy. This versatile platform expands studies on the detection of monogenic disease.

The regulation of high insulin levels on ovary apoptosis in early pregnant mice.

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.

Stomatin-like protein 2 is involved in endometrial stromal cell proliferation and differentiation during decidualization in mice and humans.

The molecular mechanisms underlying endometrial stromal cell proliferation and differentiation (decidualization) are still not fully understood. This study revealed that increased Slp-2 expression is a significant factor modulating endometrial stromal cell proliferation and decidualization in both mice and humans. Our results showed a significant difference in the mRNA and protein levels between the implantation site and inter-implantation site on day 5 and day 6 of pregnancy in mice (all P < 0.05). Strong Slp-2 immunostaining was mainly localized within the decidual zone of mice through the post-implantation period. Mice with artificially induced deciduoma showed significantly higher expression of Slp-2 compared with uninduced controls (P < 0.005). Human stromal cells in the middle and late-secretory phases demonstrated significantly (all P < 0.05) upregulated SLP-2, compared with cells in the proliferative phase and early secretory phases. Further analyses of the SLP-2 gene knocked down revealed a significant (P < 0.005) repression of both the decidualization marker gene's expression (decidual/trophoblast prolactin-related protein in mice, insulin-like growth factor binding protein and prolactin in human) and the cell proliferation in in vitro-induced decidualized primary endometrial stromal cells in mice and humans.

Silencing nc886, a Non-Coding RNA, Induces Apoptosis of Human Endometrial Cancer Cells-1A In Vitro.

BACKGROUND The role that nc886, a non-coding microRNA, plays in human endometrial cancer is unknown. The present study aimed to describe the functional role of nc886 in human endometrial cancer-1A (HEC-1A) cell line, which may provide another target for human endometrial cancer treatment. MATERIAL AND METHODS The expression levels of nv886 in normal human endometrial tissue and the early phase and late phase of human endometrial cancer tissues were determined and compared by fluorescence in situ hybridization (FISH). Small interference RNA (siRNA) was used to inhibit nc886, and cell proliferation was evaluated with the MTT test. mRNA levels of PKR, NF-κB, vascular endothelial growth factor (VEGF), and caspase-3 were determined against glyceraldehyde 3-phosphate dehydrogenase (GAPDH between the HEC-1A control group and the silenced group (nc886 silenced with siRNA) by real-time reverse transcription polymerase chain reaction (RT-PCR). The protein levels of PKR (total and phosphorylated form), NF-κB, VEGF, and caspase-3 were determined against GAPDH by Western blotting, and cell apoptosis was determined by flow cytometry. RESULTS Our results indicated that a higher level of nc886 was expressed in the late phase of human endometrial cancer tissue, less than in the early phase but still higher than in normal human endometrial tissue. After nc886 was silenced, protein levels of p-PKR (phosphorylated PKR) and caspase-3 were increased, whereas NF-κB and VEGF were decreased. CONCLUSIONS The rate of apoptosis in the silenced group was increased and the rate of cell proliferation was slower in comparison to the control.

nm23 regulates decidualization through the PI3K-Akt-mTOR signaling pathways in mice and humans.

STUDY QUESTION: Does nm23 have functional significance in decidualization in mice and humans? SUMMARY ANSWER: nm23 affects decidualization via the phosphoinositide 3 kinase/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathways in mouse endometrial stromal cells (ESCs; mESCs) and human ESCs. WHAT IS KNOWN ALREADY: The function of nm23 in suppressing metastasis has been demonstrated in a variety of cancer types. nm23 also participates in the control of DNA replication and cell proliferation and differentiation. STUDY DESIGN, SIZE AND DURATION: We first analyzed the expression profile of nm23 in mice during early pregnancy (n = 6/group), pseudopregnancy (n = 6/group) and artificial decidualization (n = 6/group) and in humans during the menstrual cycle phases and the first trimester. We then used primary cultured mESCs and a human ESC line, T-HESC, to explore the hormonal regulation of nm23 and the roles of nm23 in in vitro decidualization, and as a possible mediator of downstream PI3K-Akt-mTOR signaling pathways. PARTICIPANTS/MATERIALS, SETTINGS AND METHODS: We evaluated the dynamic expression of nm23 in mice and humans using immunohistochemistry, western blot and real-time quantitative RT-PCR (RT-qPCR). Regulation of nm23 by steroid hormones was investigated in isolated primary mESCs and T-HESCs by western blot. The effect of nm23 knockdown (using siRNA) on ESC proliferation was analyzed by 5-ethynyl-2'-deoxyuridine staining (EdU) and proliferating cell nuclear antigen protein (PCNA) expression. The influence of nm23 expression on the differentiation of ESCs was determined by RT-qPCR using the mouse differentiation markers decidual/trophoblast PRL-related protein (dtprp, also named prl8a2) and prolactin family 3 subfamily c member 1 (prl3c1) and the human differentiation markers insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). The effects of nm23 siRNA (si-nm23) and the PI3K inhibitor LY294002 on the downstream effects of nm23 on the PI3K-Akt-mTOR signaling pathway were estimated by western blot. MAIN RESULTS AND THE ROLE OF CHANCE: NM23-M1 was specifically expressed in the decidual zone during early pregnancy and in artificially induced deciduoma, and NM23-H1 was strongly expressed in human first trimester decidua. The expression of nm23 was upregulated by oestradiol and progesterone (P < 0.05 versus control) in vitro in mESCs and T-HESC, and this was inhibited by their respective receptor antagonists, ICI 182,780 and RU486. Mouse and human nm23 knockdown decreased ESC proliferation and differentiation (P < 0.05 versus control). The PI3K-Akt-mTOR signaling pathways were downstream mediators of nm23 in mESCs and T-HESCs decidualization. LIMITATIONS AND REASONS FOR CAUTION: Whether the nm23 regulates decidualization via the activation of AMPK, RAS, PKA, STAT3 or other signaling molecules remains to be determined. The role of nm23 in decidualization was tested in vitro only. WIDER IMPLICATIONS OF THE FINDINGS: Results demonstrate that nm23 plays a vital role in decidualization in mice and humans and that nm23 gene expression is hormonally regulated. The downregulation of nm23 in decidua during the first trimester may be associated with infertility in women. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Natural Science Foundation of China (grant nos. 81370731, 31571551 and 31571190), the Science and Technology Project of Chongqing Education Committee (KJ130309), open funding by the Chongqing Institute for Family Planning (1201) and the Excellent Young Scholars of Chongqing Medical University (CQYQ201302). The authors have no conflicts of interest to declare.