Chunlan Long

5-Aza-2′-deoxycytidine Leads to Reduced Embryo Implantation and Reduced Expression of DNA Methyltransferases and Essential Endometrial Genes

Background The DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) incorporates into DNA and decreases DNA methylation, sparking interest in its use as a potential therapeutic agent. We aimed to determine the effects of maternal 5-aza-CdR treatment on embryo implantation in the mouse and to evaluate whether these effects are associated with decreased levels of DNA methyltransferases (Dnmts) and three genes (estrogen receptor α [Esr1], progesterone receptor [Pgr], and homeobox A10 [Hoxa10]) that are vital for control of endometrial changes during implantation. Methods and Principal Findings Mice treated with 5-aza-CdR had a dose-dependent decrease in number of implantation sites, with defected endometrial decidualization and stromal cell proliferation. Western blot analysis on pseudo-pregnant day 3 (PD3) showed that 0.1 mg/kg 5-aza-CdR significantly repressed Dnmt3a protein level, and 0.5 mg/kg 5-aza-CdR significantly repressed Dnmt1, Dnmt3a, and Dnmt3b protein levels in the endometrium. On PD5, mice showed significantly decreased Dnmt3a protein level with 0.1 mg/kg 5-aza-CdR, and significantly decreased Dnmt1 and Dnmt3a with 0.5 mg/kg 5-aza-CdR. Immunohistochemical staining showed that 5-aza-CdR repressed DNMT expression in a cell type–specific fashion within the uterus, including decreased expression of Dnmt1 in luminal and/or glandular epithelium and of Dnmt3a and Dnmt3b in stroma. Furthermore, the 5′ flanking regions of the Esr1, Pgr, and Hoxa10 were hypomethylated on PD5. Interestingly, the higher (0.5 mg/kg) dose of 5-aza-CdR decreased protein expression of Esr1, Pgr, and Hoxa10 in the endometrium on PD5 in both methylation-dependent and methylation-independent manners. Conclusions The effects of 5-aza-CdR on embryo implantation in mice were associated with altered expression of endometrial Dnmts and genes controlling endometrial changes, suggesting that altered gene methylation, and not cytotoxicity alone, contributes to implantation defects induced by 5-aza-CdR.

The Mechanism of Environmental Endocrine Disruptors (DEHP) Induces Epigenetic Transgenerational Inheritance of Cryptorchidism

Discussion on the role of DEHP in the critical period of gonadal development in pregnant rats (F0), studied the evolution of F1-F4 generation of inter-generational inheritance of cryptorchidism and the alteration of DNA methylation levels in testis. Pregnant SD rats were randomly divided into two groups: normal control group and DEHP experimental group. From pregnancy 7d to 19d, experimental group was sustained to gavage DEHP 750mg/kg bw/day, observed the incidence of cryptorchidism in offspring and examined the pregnancy rate of female rats through mating experiments. Continuous recording the rat’s weight and AGD value, after maturation (PND80) recording testis and epididymis’ size and weight, detected the sperm number and quality. Subsequently, we examined the evolution morphological changes of testicular tissue for 4 generation rats by HE staining and Western Blot. Completed the MeDIP-sequencing analysis of 6 samples (F1 generation, F4 generation and Control). DEHP successfully induced cryptorchidism occurrence in offspring during pregnancy. The incidence of cryptorchidism in F1 was 30%, in F2 was 12.5%, and there was no cryptorchidism coming up in F3 and F4. Mating experiment shows conception rate 50% in F1, F2 generation was 75%, the F3 and F4 generation were 100%. HE staining showed that the seminiferous epithelium of F1 generation was atrophy and with a few spermatogenic cell, F2 generation had improved, F3 and F4 generation were tend to be normal. The DNA methyltransferase expression was up-regulated with the increase of generations by Real Time-PCR, immunohistochemistry and Western Blot. MeDIP-seq Data Analysis Results show many differentially methylated DNA sequences between F1 and F4. DEHP damage male reproductive function in rats, affect expression of DNA methyltransferase enzyme, which in turn leads to genomic imprinting methylation pattern changes and passed on to the next generation, so that the offspring of male reproductive system critical role in the development of imprinted genes imbalances, and eventually lead to producing offspring cryptorchidism. This may be an important mechanism of reproductive system damage.

Expression of DNA methyltransferases in the mouse uterus during early pregnancy and susceptibility to dietary folate deficiency

We have characterized the uterine expression of DNA methyltransferases (DNMTs) during early pregnancy in mice and determined whether a folate-deficient diet (FDD) can affect DNMTs in this context. Within endometrial cells, expressions of DNMT (cytosine-5) 1 (Dnmt1), Dnmt3a, and Dnmt3b were significantly elevated during the prereceptive phase of pregnancy but generally returned to baseline levels during receptive and postimplantation periods. As such, the transcription of DNMT genes is temporally regulated during early pregnancy. When comparisons were made between implantation sites (IS) and inter-IS on day 5 of pregnancy, lower levels of Dnmt3a were detected at IS. Comparisons between IS and inter-IS did not reveal significant expression differences for other DNMT genes. When tissue sections were examined, DNMT3A was specifically lower in the stroma of IS. Reduced DNMT1 and DNMT3B levels were also observed in the luminal and glandular epithelia of IS, whereas no obvious differences in the stroma were detected. In pseudo-pregnant mice subjected to a FDD, levels of Dnmt1 and Dnmt3a (but not Dnmt3b) were significantly upregulated in endometrial tissues, as compared with controls. When tissues from these folate-deficient mice were examined, DNMT1 levels were elevated in both the luminal and glandular epithelia, whereas DNMT3A was upregulated in the luminal epithelium and the stroma. A slight increase in DNMT3B levels was detected in the glandular epithelium. These results indicate that DNMTs may regulate the transcription of endometrial genes associated with embryo implantation and that levels of DNMTs are affected by dietary folate in mice.

Effect of folate deficiency on promoter methylation and gene expression of Esr1, Cdh1 and Pgr, and its influence on endometrial receptivity and embryo implantation

BACKGROUND Folate, one of the B vitamins, provides the one-carbon units required for methylation. Folate deficiency has been associated with many pathologies. However, much less is known about the effect of it on human reproduction, especially on implantation. The establishment of uterine receptivity is crucial for successful embryo implantation. Gene expression can be influenced by both heredity and epigenetics such as DNA methylation. However, it is not known whether the methylation and expression of genes related to uterine receptivity can be affected by folate levels. To explore whether folate deficiency affected the epigenetic regulation of genes related to uterine receptivity, and their influence on implantation, we investigated the methylation and expression of cadherin 1 (Cdh1), progesterone receptor (Pgr) and estrogen receptor 1 (Esr1) genes during implantation and the implantation efficiency using a folate-deficient pregnant mouse model. METHODS Serum folate levels of pregnant mice were measured using the electro-chemiluminescence immunoassay. The methylation status of Cdh1, Pgr and Esr1 promoter regions was determined by methylation-specific PCR and bisulfite sequencing. The expression of Cdh1, Pgr and Esr1 in the implantation-site endometrium was examined by real-time PCR, western blot and immunohistochemistry. The number and the morphology of pinopodes, important morphological markers of endometrial receptivity, were examined using scanning electron microscopy. The number of implantation sites demarcated by distinct blue bands was recorded. RESULTS Serum folate levels of the folate-deficient group were lower (5.42 ± 1.35 ng/ml, n= 42) than those of the normal group (24.13 ± 4 .26 ng/ml, n= 37; P = 0.003). Here we show that the methylation status and mRNA levels of Esr1 were decreased (P= 0.021, P= 0.045, respectively), while the Cdh1 and Pgr expression levels were slightly but not significantly elevated and the methylation status did not vary in the folate-deficient mice compared with the wild type. Neither the number nor morphology of pinopodes was affected by folate deficiency. Furthermore, folate deficiency did not affect the number of implantation sites in mice. CONCLUSIONS This study demonstrates for the first time that, unlike the effects on Esr1, folate deficiency in mice does not influence the methylation and expression of Pgr and Cdh1, two genes shown to be essential for uterine receptivity and embryo implantation. Embryo implantation in mice appears to be unaffected by a deficiency in folate, suggesting that abnormalities in a pregnancy caused by folate deficiency start to develop after implantation.

Urban fine particulate matter exposure causes male reproductive injury through destroying blood-testis barrier (BTB) integrity.

Blood-testis barrier (BTB) provides a suitable microenvironment for germ cells that is required for spermatogenesis. Exposure to particulate matter (PM) is recognized to occasion male reproductive impairment, but the mechanism of which remains unclear. Male Sprague-Dawley (SD) rats were used to establish animal models with PM2.5 exposure concentration of 0, 10, and 20mg/kg.b.w. once a day for four weeks. Success rate of mating, sperm quality, epididymal morphology, expressions of spermatogenesis markers, superoxide dismutases (SOD) activity and expression in testicular tissues, and expressions of BTB junction proteins were detected. In addition, in vitro experiments were also performed. After PM2.5 treatment, reactive oxygen species (ROS) production and apoptosis of Sertoli cells were analyzed. Our results indicated that after PM2.5 exposure male rats presented inferior uberty and sperm quality, with decreased expressions of spermatogenesis markers, escalated SOD activity and expression levels, and reduced expressions of tight junction, adherens junction, and gap junction proteins in testicular tissues. Meantime, PM2.5-treated Sertoli cells displayed increased SOD production and apoptosis. PM2.5 exposure engenders male reproductive function injury through breaking BTB integrity.

MiR-200a is involved in proliferation and apoptosis in the human endometrial adenocarcinoma cell line HEC-1B by targeting the tumor suppressor PTEN.

Abnormal cell proliferation is a main driver of tumor formation and development, which involves the deletion, mutation, and downregulation of tumor suppressor genes. One study recently demonstrated that miR-200a plays an oncogenic role by inhibiting phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression. In the human endometrial adenocarcinoma cell line HEC-1B, suppression of miR-200a expression inhibited cell proliferation and promoted apoptosis, whereas its over-expression had no effect on proliferation and apoptosis. Furthermore, inhibition or over-expression of miR-200a increased or reduced the expression of PTEN, respectively, with no change in PTEN mRNA levels. These effects were achieved by directly targeting miR-200a to the 3′ untranslated region of the PTEN mRNA to inhibit its translation. Taken together, we propose that in HEC-1B cells, miR-200a functions as an oncogene, affecting proliferation and apoptosis by regulating the expression of the tumor suppressor PTEN at the translational level.

Mmu-miR-193 Is Involved in Embryo Implantation in Mouse Uterus by Regulating GRB7 Gene Expression

Embryo implantation is a complicated process involving a series of endometrial changes that depend on differential gene expression. MicroRNAs (miRNAs) are important for regulation of gene expression. Previous studies have shown that miRNAs may participate in the regulation of gene expression during embryo implantation. To explore the role of endometrial miRNAs in early murine pregnancy, we used microarrays to investigate whether miRNAs were differentially expressed in the mouse endometrium on pregnancy day 4 (D4) and day 6 (D6). The results demonstrated that 17 miRNAs were upregulated and 18 were downregulated (>2-fold) in D6 endometria compared to D4. We identified that mmu-miR-193 exhibited the highest upregulation on D6, and the upregulation of mmu-miR-193 before embryo implantation could reduce the embryo implantation rate. Further, we demonstrated that mmu-miR-193 influenced embryo implantation by regulating growth factor receptor-bound protein 7 expression. In summary, our study suggests that mmu-miR-193 plays an important role in embryo implantation.

Human umbilical cord mesenchymal stem cell conditioned medium attenuates renal fibrosis by reducing inflammation and epithelial-to-mesenchymal transition via the TLR4/NF-κB signaling pathway in vivo and in vitro

BackgroundRenal fibrosis is characterized by infiltration of interstitial inflammatory cells and release of inflammatory mediators, activation and proliferation of fibroblasts, and deposition of excessive extracellular matrix (ECM). The aim of this study was to evaluate the effect of human umbilical cord-derived mesenchymal stem cell (hucMSC) conditioned medium (CM) on renal tubulointerstitial inflammation and fibrosis.MethodsRenal interstitial fibrosis was prepared in vivo using the unilateral ureteral obstruction (UUO). Rats were divided randomly into Sham group, Sham group with CM, UUO group, and UUO group with CM. The effect of hucMSC-CM on kidney injury induced by UUO was assessed by detecting kidney histopathology, serum creatinine (SCr), and blood urea nitrogen (BUN). The levels of TNF-α, IL-6, and IL-1β in serum and kidney tissues were detected by ELISA. The expression of proteins associated with fibrosis and renal inflammation was investigated using immunohistochemical staining and western blotting. The effects of hucMSC-CM on the TGF-β1-induced epithelial–mesenchymal transition (EMT) process and on inflammation in NRK-52E cells were investigated by immunofluorescent staining, ELISA, and western blotting.ResultshucMSC-CM reduced extracellular matrix deposition and inflammatory cell infiltration as well as release of inflammatory factors in UUO-induced renal fibrosis. Furthermore, hucMSC-CM markedly attenuated the EMT process and proinflammatory cytokines in rats with UUO and TGF-β1-induced NRK-52E cells. hucMSC-CM also inhibited the TLR4/NF-κB signaling pathway in vivo and in vitro.ConclusionsOur results suggest that hucMSC-CM has protective effects against UUO-induced renal fibrosis and that hucMSC-CM exhibits its anti-inflammatory effects through inhibiting TLR4/NF-κB signaling pathway activation.

Di-(2-ethylhexyl) phthalate (DEHP)-induced testicular toxicity through Nrf2-mediated Notch1 signaling pathway in Sprague-Dawley rats

Di‐(2‐ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor widely used in China that is harmful to the male reproductive system. Many studies have shown that DEHP causes testicular toxicity through oxidative stress, but the specific mechanism is unknown. Because the Notch pathway is a key mechanism for regulating cell growth and proliferation, we investigated whether Notch is involved in DEHP‐induced testicular toxicity and whether vitamins E and C could rescue testicular impairment in Sprague–Dawley (SD) rats. Compared with the control group, we found that DEHP exposure induced testicular toxicity through oxidative stress injury, and it decreased the testosterone level (P < .01) and upregulated nuclear factor‐erythroid 2 related factor (Nrf2) expression (P < .01). Therefore, because oxidative stress might be the initiating factor of DEHP‐induced testicular toxicity, treatment with the antioxidant vitamins E and C activated the Notch1 signaling pathway in the testis and in Leydig cells. Treatment with vitamins E and C normalized the oxidative stress state after DEHP exposure and restored testicular development to be similar to the control group. In summary, antioxidant vitamins E and C may be used to treat DEHP‐induced testicular toxicity.

Spermatogenesis dysfunction induced by PM2.5 from automobile exhaust via the ROS-mediated MAPK signaling pathway

Long-term exposure to particulate matter 2.5 (PM2.5) from automobile exhaust impairs spermatogenesis through oxidative stress injury, but the underlying mechanism is unknown. To investigate the toxic mechanism of PM2.5-induced spermatogenesis impairment, we focused on the MAPK signaling pathway. We also examined the effects of treatment with vitamins C and E on spermatogenic function. Male SD rats were divided randomly into three groups: control (0.9% sterilized saline), PM2.5 exposure (20 mg/kg.b.w.), and PM2.5 exposure (20 mg/kg.b.w.) with vitamin intervention (vitamin C, 100 mg/kg.b.w.; vitamin E, 50 mg/kg.b.w.). Male rats showed a marked decline in fertility and decreased sperm quality after PM2.5 exposure. The expression of SOD and Nrf2 was significantly decreased, and that of MDA was increased markedly. The expression of blood-testis barrier-associated proteins, such as ZO-1, occludin, connexin 43, and β-catenin, was significantly decreased, the Bcl-2/Bax ratio was downregulated, and the cleaved caspase-3 level was increased. Phosphorylation of MAPKs, including ERKs, JNKs, and p38, was upregulated. Treatment with vitamins C and E reversed the damage induced by PM2.5 exposure. These results suggest that PM2.5 from automobile exhaust disrupted spermatogenesis via ROS-mediated MAPK pathways, and that a combined vitamin C and E intervention effectively mitigated toxicity in the male reproductive system.