Yanqing Geng

Sodium fluoride activates ERK and JNK via induction of oxidative stress to promote apoptosis and impairs ovarian function in rats

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.

Folate deficiency impairs decidualization and alters methylation patterns of the genome in mice

Existing evidence suggests that adverse pregnancy outcomes are closely related with dietary factors. Previous studies in mice have focused on the harm of folate deficiency (FD) on development of embryo, while the effect of low maternal folate levels on maternal intrauterine environment during early pregnancy remains unclear. Since our previous study found that FD treatment of mice causes no apparent defects in embryo implantation but is accompanied by female subfertility, we next chose to investigate a potential role of FD on molecular events after implantation. We observed that the decidual bulges began to be stunted on pregnancy day 6. The results of functional experiments in vivo and in vitro showed that FD inhibited the process of endometrial decidualization. It has been confirmed that DNA methylation participates in decidualization, and folate as a methyl donor could change the methylation patterns of genes. Thus, we hypothesized that FD impairs maternal endometrial decidualization by altering the methylation profiles of related genes. Reduced representation bisulphite sequencing was carried out to detect the methylation profiles of endometrium on pregnancy day 6-8, which is equivalent to the decidualization period in mice. The results confirmed that FD changes the methylation patterns of genome, and GO analysis of the differentially methylated regions revealed that the associated genes mainly participate in biological adhesion, biological regulation, cell proliferation, development, metabolism and signalling. In addition, we found some candidates for regulators of decidual transformation, such as Nr1h3 and Nr5a1. The data indicate that FD inhibits decidualization, possibly by altering methylation patterns of the genome in mice.

The Differential Expression of MicroRNAs Between Implantation Sites and Interimplantation Sites in Early Pregnancy in Mice and Their Potential Functions

Embryo implantation is a complex process that involves synchronized crosstalk between a receptive endometrium and a functional blastocyst. It can take place only during the window of implantation, a period when a series of changes in gene expression occur in the endometrium to accept the embryo. As modulators of gene expression, microRNAs (miRNAs) have been identified as regulators of embryo implantation. To better understand how miRNAs regulate implantation and the related molecular mechanisms, we compared the expression profiles of miRNAs and messenger RNAs between implantation sites (IMs) and inter-IMs in the endometrium of pregnant mice on day 5 by microarrays. The results showed that compared with inter-IMs, 30 miRNAs were upregulated and 42 miRNAs (>2-fold) were downregulated at the IMs. By combining the results of the microarray experiments, we found that 20 upregulated pathways and 14 downregulated pathways might be subject to miRNA regulation at IMs. We also found that some miRNAs and their targets may play a key role in implantation.

Mmu-microRNA-200a Overexpression Leads to Implantation Defect by Targeting Phosphatase and Tensin Homolog in Mouse Uterus

Successful mouse embryo implantation requires a receptive uterus and an activated blastocyst. A large number of genes, cytokines, and other factors are involved in the process. MicroRNAs (miRNAs) regulate the expression of many genes, and previous studies have investigated the relationship between miRNA expression and embryo implantation. In this study, we show that mmu-microRNA-200a (mmu-miR-200a) is expressed in a spatiatemporal manner during implantation in mouse uterus and found that phosphatase and tensin homolog (PTEN), SON, and programmed cell death 4 (Pdcd4) are the target genes of mmu-miR-200a by bioinformatics analysis. In vitro gain and loss of function experiments confirm that PTEN, a critical gene for cell proliferation and apoptosis, is the target gene of mmu-miR-200a. Our experiments also show that injection of the uterine horn with mmu-miR-200a lentivirus leads to a decreased implantation rate. Collectively, our results suggest that mmu-miR-200a affects embryo implantation by regulating PTEN protein expression. Thus, clarifying the physiological functions of uterine miRNAs will help to elucidate the embryo implantation process and may even contribute to curing infertility and inventing new contraceptives.

Folate Deficiency Could Restrain Decidual Angiogenesis in Pregnant Mice

The mechanism of birth defects induced by folate deficiency was focused on mainly in fetal development. Little is known about the effect of folate deficiency on the maternal uterus, especially on decidual angiogenesis after implantation which establishes vessel networks to support embryo development. The aim of this study was to investigate the effects of folate deficiency on decidual angiogenesis. Serum folate levels were measured by electrochemiluminescence. The status of decidual angiogenesis was examined by cluster designation 34 (CD34) immunohistochemistry and the expression of angiogenic factors, including vascular endothelial growth factor A (VEGFA), placental growth factor (PLGF), and VEGF receptor 2 (VEGFR2) were also tested. Serum levels of homocysteine (Hcy), follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), progesterone (P4), and estradiol (E2) were detected by Enzyme-linked immunosorbent assay. The folate-deficient mice had a lower folate level and a higher Hcy level. Folate deficiency restrained decidual angiogenesis with significant abnormalities in vascular density and the enlargement and elongation of the vascular sinus. It also showed a reduction in the expressions of VEGFA, VEGFR2, and PLGF. In addition, the serum levels of P4, E2, LH, and PRL were reduced in folate-deficient mice, and the expression of progesterone receptor (PR) and estrogen receptor α (ERα) were abnormal. These results indicated that folate deficiency could impaire decidual angiogenesis and it may be related to the vasculotoxic properties of Hcy and the imbalance of the reproductive hormone.

Altered β1,6-GlcNAc and bisecting GlcNAc-branched N-glycan on integrin β1 are associated with early spontaneous miscarriage in humans

STUDY QUESTION Do N-acetylglucosaminyltransferase (GnT-V) and N-acetylglucosaminyltransferase III (GnT-III) play an important role in early spontaneous miscarriage (ESM) in humans. SUMMARY ANSWER The dynamic balance between GnT-V and GnT-III expression in chorionic villi differed between early normal pregnancy and ESM and was associated with altered β1,6-N-acetylglucosamine (β1,6-GlcNAc) and bisecting N-acetylglucosamine (bis-GlcNAc) branched N-glycans on integrin β1. WHAT IS KNOWN ALREADY GnT-V contributes to metastasis, while GnT-III is recognized as a metastasis suppressor. It has been reported that GnT-V contributes to placentation in the early phase of pregnancy, possibly regulating trophoblast invasion. However, the expressions of GnT-V and GnT-III in ESM have not been reported. STUDY DESIGN, SIZE, DURATION Villous samples from 6 to 9 weeks of gestation were collected in the First Affiliated Hospital of Chongqing Medical University from May 2013 to September 2014 from 60 normal pregnant women undergoing elective termination of pregnancy and from 40 patients with a clinical diagnosis of ESM. PARTICIPANTS, MATERIALS, SETTING, METHODS Quantitative PCR and western blots were used to examine the GnT-V and GnT-III mRNA (Mgat5 and Mgat3) and protein expression, respectively, of chorionic villi in both the ESM group and the normal group from week 6 to week 9. We used immunofluorescence and immunohistochemistry to detect the location of GnT-V and GnT-III. Lectin fluorescence and histochemistry were used to test the location of β1,6-GlcNAc and bis-GlcNAc branching in the normal and ESM groups. To assess the functional capacity of GnT-V and GnT-III in the chorionic villi between the two groups, we used an enzyme-linked immunosorbent assay kit to measure the activity of these enzymes. Using co-precipitated integrin α5β1 followed by phytohaemagglutinin (PHA)-L and PHA-E blotting, we investigated whether GnT-V and GnT-III could modify the N-glycosylation profile in terms of the β1,6-GlcNAc and bis-GlcNAc structures in integrin α5β1 during the first trimester in both groups. MAIN RESULTS AND THE ROLE OF CHANCE In the normal group expression and activity of GnT-V and the concentration of its product, β1,6-GlcNAc were higher at week 9 than at weeks 6, 7 and 8 (P < 0.05). In contrast, the expression and activity of GnT-III and the concentration of its product, bis-GlcNAc were higher at week 6 than at weeks 7, 8 and 9 (P < 0.05). Compared with the normal group, the ESM group exhibited a lower expression of GnT-V and β1,6-GlcNAc (P < 0.05) and a higher expression of GnT-III and bis-GlcNAc (P < 0.05) with consistent changes in enzymatic activity. Immunofluorescence showed that GnT-V was located mainly in the cytoplasm of syncytiotrophoblasts (STBs) and chorionic villous cytotrophoblasts (CTBs), in both the ESM group and the normal group. β1,6-GlcNAc N-glycan was mainly located outside of the STB and CTB layer in normal villi and was expressed only rarely in the ESM villi. GnT-III was expressed primarily in the cytoplasm of STBs and expressed only very weakly in the CTBs of normal villi, whereas it was highly expressed in both the STBs and CTBs in the ESM group. bis-GlcNAc was primarily located outside of the STBs in the normal villi, whereas it was expressed much more abundantly outside of both the STBs and CTBs in the ESM group at each week of gestation. Moreover, decreased β1,6-GlcNAc-branched N-glycans and increased bis-GlcNAc-branched N-glycans on integrin β1 (P < 0.05) were observed in the ESM group. WIDER IMPLICATIONS OF THE FINDINGS Our findings provide a new insight for studying the mechanism of clinical ESM in humans and it might be valuable for the clinical diagnosis and treatment of ESM. LIMITATIONS, REASONS FOR CAUTION The study lacks experiments in vitro to disclose the precise mechanism by which GnT-V and GnT-III regulate ESM. In some cases, degradation of the tissues after the miscarriage event cannot be ruled out. STUDY FUNDING/COMPETING INTERESTS This study was supported by grants from the National Natural Science Foundation of China (31271546). The authors have no competing interests.

Exposure to benzo[a]pyrene impairs decidualization and decidual angiogenesis in mice during early pregnancy ☆

Benzo[a]pyrene (BaP) is a ubiquitous environmental persistent organic pollutant and a well-known endocrine disruptor. BaP exposure could alter the steroid balance in females. Endometrium decidualization and decidual angiogenesis are critical events for embryo implantation and pregnancy maintenance during early pregnancy and are modulated by steroids. However, the effect of BaP on decidualization is not clear. This study aimed to explore the effects of BaP on decidualization and decidual angiogenesis in pregnant mice. The result showed that the uteri in the BaP-treated groups were smaller and exhibited an uneven size compared with those in the control group. Artificial decidualization was detected in the uteri of the controls, but weakened decidualization response was observed in the BaP-treated groups. BaP significantly reduced the levels of estradiol, progesterone, and their cognate receptors ER and PR, respectively. The expression of several decidualization-related factors, including FOXO1, HoxA10, and BMP2, were altered after BaP treatment. BaP reduced the expression of cluster designation 34 (CD34), which indicated that the decidual angiogenesis was inhibited by BaP treatment. In addition, BaP induced the downregulation of vascular endothelial growth factor A. These data suggest that oral BaP ingestion compromised decidualization and decidual angiogenesis. Our results provide experimental data for the maternal reproductive toxicity of BaP during early pregnancy, which is very important for a comprehensive risk assessment of BaP on human reproductive health.

Mouse Endometrium Temporal and Spatial Expression mRNA and MicroRNA Associated With Embryo Implantation.

Embryo implantation is a dynamic physiological process involving morphological and molecular changes in the endometrium during the pre-receptivity, receptivity, and implantation phases. A comprehensive analysis of messenger RNA (mRNA) and microRNA (miRNA) profiles during implantation will likely provide new clues to elucidate the underlying mechanisms governing embryo implantation. We characterized the mRNA and miRNA transcriptomes using next generation sequencing (NGS) of the endometrium 1 day postcoitum (dpc) and 4dpc and the implantation site (IMS) and inter-implantation (IIM) site of the endometrium on 5dpc. Real-time quantitative polymerase chain reaction was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression. Statistical analysis of the data based on Gene Ontology (GO) group annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the genes with significant expression at the IIM site were primarily involved in glucose, protein, and lipoprotein metabolism to provide energy for embryo implantation, while the genes identified at the IMS were involved in RNA functions to produce proteins in support of embryo development and trophoblast invasion. Extracellular matrix (ECM)-receptor interactions between cells and the ECM was the most remarkable event during implantation. The miRNA-mRNA interaction network unraveled the regulatory relationship between miRNAs and mRNAs. Hub miRNAs (mmu-miR-96 and mmu-miR-200b) were identified to target B-cell lymphoma 2 (Bcl-2), Kruppel-like factor 13 (Klf13), and Progesterone receptor (PGR), which are associated with the preparation of the receptive condition or the maintenance of early pregnancy.

The homologous genes Vangl1 and Vangl2 are required for embryo implantation in the uterus of mice during early pregnancy

Vangl1 and Vangl2 are homologous genes belonging to the group of highly conserved planar cell polarity proteins. It has been shown that Vangl1 and Vangl2 are essential for embryonic development, cell adhesion, migration and polarity. We examined the expression of Vangl1 and Vangl2 in the uterus of mice during early pregnancy. They are upregulated in the endometrium of peri-implantation and reached the peak on D5. Vangl1 mRNA is widely distributed in the luminal epithelium, glandular epithelium and stromal cells in the endometrium, while its protein only appeared in the stromal cells. The localization of Vangl2 protein overlapped with its mRNA. In addition, expression of Vangl1 in the endometrium of pseudopregnant mice was lower than that of pregnant mice, whereas the level of Vangl2 was not significantly different, suggesting that expression of Vangl1 is induced by embryo. Further study showed that implantation would be suppressed after silencing expressions of Vangl1 and Vangl2 by uterine injection with antisense oligonucleotides. These findings suggest that Vangl1 and Vangl2 may play a key role in the embryo implantation of mice.

Expression of mmu-miR-96 in the endometrium during early pregnancy and its regulatory effects on stromal cell apoptosis via Bcl2

Decidualization of endometrial stromal cells is an important feature of implantation and pregnancy. The molecular mechanism underlying decidualization remains unclear, particularly regarding the microRNA (miRNA/miR) regulation of this process. The present study revealed the temporal and spatial distribution of mmu-miR-96 in the mouse uterus during early pregnancy by reverse transcription-quantitative polymerase chain reaction and in situ hybridization. In addition, primary stromal cells were isolated from the mouse uterus and used to explore the role of mmu-miR-96 in decidualization. The results demonstrated that mmu-miR-96 was highly expressed in stromal cells during pregnancy, and was upregulated at implantation sites. In addition, mmu-miR-96 was strongly expressed during decidualization, which indicates that it may serve a role in the decidualization of stromal cells. Based on existing reports, mmu-miR-96 participates in apoptosis; therefore the present study investigated its effects on the apoptosis of primary endometrial stromal cells. The results indicated that overexpression of mmu-miR-96 may induce apoptosis of stromal cells. In further studies regarding the underlying mechanism, the target genes of mmu-miR-96 were screened by bioinformatics analysis, and it was confirmed that B-cell lymphoma 2, an anti-apoptotic gene, was the target of mmu-miR-96, as determined using a reporter gene assay. In conclusion, the present study suggested that mmu-miR-96 participates in the decidualization of endometrial stromal cells in mice, thereby serving a key role in pregnancy.