Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. METHODS: Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. RESULTS: The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. CONCLUSIONS: Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.

Immune and oxidative stress disorder in ovulation-dysfunction women revealed by single-cell transcriptome.

Introduction: Ovulation dysfunction is now a widespread cause of infertility around the world. Although the impact of immune cells in human reproduction has been widely investigated, systematic understanding of the changes of the immune atlas under female ovulation remain less understood. Methods: Here, we generated single cell transcriptomic profiles of 80,689 PBMCs in three representative statuses of ovulation dysfunction, i.e., polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI) and menopause (MENO), and identified totally 7 major cell types and 25 subsets of cells. Results: Our study revealed distinct cluster distributions of immune cells among individuals of ovulation disorders and health. In patients with ovulation dysfunction, we observed a significant reduction in populations of naïve CD8 T cells and effector memory CD4 T cells, whereas circulating NK cells and regulatory NK cells increased. Discussion: Our results highlight the significant contribution of cDC-mediated signaling pathways to the overall inflammatory response within ovulation disorders. Furthermore, our data demonstrated a significant upregulation of oxidative stress in patients with ovulation disorder. Overall, our study gave a deeper insight into the mechanism of PCOS, POI, and menopause, which may contribute to the better diagnosis and treatments of these ovulatory disorder.

Glucose promoting the early embryonic development by increasing the lipid synthesis at 2-cell stage.

The optimization of culture conditions is one of the main strategies to improve the embryo development competence in in vitro fertilization (IVF). Glucose is an important carbon source while also exists in the oviductal fluid in vivo, the effect of glucose in embryo development microenvironment is still unclear. Here we employed the LC-MS to detect and analyze the metabolites in the culture medium of different cleavage stages including 2-Cell, 4-Cell and 8-Cell mouse embryos, respectively. The effects of the external glucose were estimated by measuring the development rate at different glucose concentrations from 0 to 5 mmol/L, and the gene expression changes were detected to explore the potential mechanism after the addition of glucose in the media. Our results indicated the 2-Cell and 8-Cell stages had defined characteristic metabolites, while 4-Cell stage was the transition state. Global and contiguous metabolic characteristics showed the glycometabolism play a critical role at each early cleavage stages during the embryo development. The 8-Cell rates demonstrated the addition of glucose in culture media significantly improve the embryo competence, the highest rate was 87.33% using 3 mmol/L glucose in media, in contrast only 9.95% using the media without glucose. Meanwhile, the blocked embryos were mainly enriched at 2-Cell stage. Further transcriptome study found 3 mmol/L glucose in media remarkably upregulated the gene expression of lipid biosynthesis at 2-Cell stage, the increased lipid was confirmed by nile red staining. These data indicated the glucose may promote the development competence through increasing the lipid biosynthesis to overcoming the 2-Cell block. Our findings were helpful for the further optimization of IVF culture media, as well as the estimation of embryo quality using metabolites in the culture media.

Single-cell immune profiling reveals broad anti-inflammation response in bipolar disorder patients with quetiapine and valproate treatment.

Bipolar disorder (BD) is a common mental disorder characterized by manic and depressive episodes. Mood disorders have been associated with immune dysfunction. The combination of quetiapine and valproate has shown positive effects in treating BD, but the impact on immune dynamics remains less understood. Using single-cell RNA sequencing, we observed that B cells exhibited downregulation of inflammation-related genes, while pro-inflammatory mast and eosinophil cells decreased following treatment. Ribosomal peptide production genes were found to be reduced in both B and T cells after treatment. Additionally, our findings suggest that the combined therapy effectively alleviates inflammation by reducing myloid-mediated immune signaling pathways. This study provides valuable insights into the immune atlas and uncovers a potential mechanism for immune disorder alleviation in patients with BD treated with quetiapine and valproate.

Single-cell transcriptomics reveals variable trajectories of CSPCs in the progression of osteoarthritis.

Osteoarthritis (OA) is characterised by cartilage destruction; however, there are no specific drugs available for its treatment. Cartilage-derived stem/progenitor cells (CSPCs) are multipotent cells that play an essential role in cartilage renewal and may provide critical insights into the medical needs for OA treatment. However, alterations in cell function and fate of CSPCs during OA progression have seldom been analysed, especially at the single-cell level. Additionally, it has been reported that CSPCs can migrate to the cartilage injury area, although the mechanism of migration remains elusive. Thus, understanding the changing patterns of CSPCs in the pathological process of OA is important in the effort to develop stem cell therapy for OA. Here, we downloaded single-cell transcriptomic data of patients with OA from the Gene Expression Omnibus (GEO) database and performed unbiased clustering of the cells based on gene expression patterns using the Seurat package. Using common stem cell markers and chondrogenic transcription factors, we traced CSPCs throughout all stages of OA. We further explored the dynamics of CSPCs in OA progression and validated the single-cell RNA sequencing data in vitro using qPCR, immunofluorescence, and western blotting. Specifically, we primarily explored the heterogeneity of CSPCs at the single-cell level and found that it was closely associated with OA progression. Our results indicate significantly reduced chondrogenic differentiation capacity in CSPCs during the late stage of OA, while their proliferation capacity tended to increase. We also found that genes implicated in fibrosis, cell motility, and extracellular matrix remodelling were upregulated in CSPCs during the progression of OA. Our study revealed the dynamics of stem cells in OA progression and may inform the development of stem cell therapy for OA.

Decreased expression of SEMA4D in recurrent implantation failure induces reduction of trophoblast invasion and migration via the Met/PI3K/Akt pathway.

Recurrent implantation failure (RIF) associated with impaired endometrial receptivity and other factors. Disease-specific therapy has yet to be developed due to the lack of understanding of underlying mechanism(s). Herein we investigated the key factors of endometrial receptivity in RIF patients by transcriptomic sequencing. In vitro cellular model was used to delineate the molecular mechanism of key factors on proliferation, invasion and migration of trophoblast cells. SEMA4D was identified as the key factors of endometrial receptivity with significantly lower expression in the mid-secretory endometrium of RIF patients compared with those from normal fertile women. The binding of SEMA4D to its receptor Plexin-B1 on human trophoblast cells HTR-8/SVneo resulted in the activation of Met/PI3K/Akt signaling, which promotes trophoblast cell invasion and migration by enhancing MMP-2 expression. Moreover, the effect of SEMA4D on HTR-8/SVneo could be blocked by knocking down Met with specific siRNA or treating with LY294002. Collectively, our data indicate that decreased expression of SEMA4D in endometrium impair the process of trophoblast invasion and migration through Met/PI3K/Akt pathway, which provides insights into this essential physiological process in the development of RIF.

Pathological changes and oxidative stress of the HPG axis in hypothyroid rat.

Hypothyroidism is a common endocrine disease caused by a deficiency of thyroid hormones, which could affect the hypothalamus-pituitary-gonadal (HPG) axis and cause additional severe fertility problems. However, the pathogenesis of abnormal reproductive capacity caused by hypothyroidism and whether there are differences between females and males need more study. Here, we constructed a prolonged neonatal hypothyroid rat model using 6-propyl-2-thiouracil (PTU). H&E staining and RNA-sequencing were performed to detect histopathological and transcriptome changes. Our results indicated that the numbers of ventromedial hypothalamus nuclei were increased, and the number of pituitary chromophobes was sharply increased, whereas the proportion of pituitary acidophils and pituitary basophils were obviously reduced. The differentially expressed genes of the HPG axis organs were identified, and different tissues shared similar steroid hormone and oxidative stress-related terms in gene ontology analysis. Weighted gene co-expression network analysis (WGCNA) and differential expression analysis indicated oxidative stress, and apoptosis-related genes were more enriched in male hypothyroid pituitaries, whereas the serum levels of growth hormone, follicle-stimulating hormone, and luteinizing hormone that were detected by ELISA were also reduced more in male hypothyroid rats, suggesting that prolonged neonatal hypothyroidism may have a more significant impact on male pituitaries. Moreover, the multi-organ oxidative stress in hypothyroid rats was confirmed by the higher expression of oxidative stress-related genes, such as the Txnip. The increased level of oxidative stress may have contributed to the histopathological and transcriptome changes of HPG axis organs in the prolonged neonatal hypothyroidism rats, especially in male pituitaries.

Analysis of differences in the transcriptomic profiles of eutopic and ectopic endometriums in women with ovarian endometriosis.

BACKGROUND: Endometriosis is a common gynecological disease among women in their reproductive years. Although much effort has been made, the pathogenesis of this disease and the detailed differences between eutopic endometrial cells and ectopic endometrial cells are still unclear. METHODS: In this study, eutopic and ectopic endometrial cells were collected from patients with and without endometriosis and RNA sequencing was performed. The gene expression patterns and differentially expressed genes (DEGs) in eutopic and ectopic endometrial cells, as well as control endometrial cells, were analyzed using a weighted gene co-expression network analysis (WGCNA) and the DESeq2 package. The functions of significant genes were detected using Gene ontology (GO) enrichment analysis, and qRT-PCR validation was performed. RESULTS: The results indicated that eight gene modules were found among these three groups. They also indicated that the gene module, which is highly related to eutopic endometrial cells, was mainly enriched in cell adhesion, embryo implantation, etc., while the gene module related to ectopic endometrial cells was mainly enriched in cell migration, etc. The results of differential expression analysis were generally consistent with the WGCNA results through identified significant DEGs between different groups. These DEGs may play an important role in the occurrence of endometriosis, including the infertility associated gene ARNTL and PIWIL2, tissue remodeling gene MMP11, cell survival and migration gene FLT1, inflammatory response gene GNLY, the tumor suppressor genes PLCD1, etc. Further analysis suggested the function of adhesion is stronger in ectopic endometrial cells than in eutopic endometrial cells, while the ectopic endometrium may have a higher potential risk of malignant transformation than eutopic endometrium. CONCLUSIONS: Overall, these data provide a reference for understanding the pathogenesis of endometriosis and its relationship with malignant transformation.

Activation of Blood Vessel Development in Endometrial Stromal Cells In Vitro Cocultured with Human Peri-Implantation Embryos Revealed by Single-Cell RNA-Seq.

In humans, the maternal endometrium participates in the physical and physiological interaction with the blastocyst to begin implantation. A bidirectional crosstalk is critical for normal implantation and then a successful pregnancy. While several studies have used animal models or cell lines to study this step, little knowledge was acquired to address the role of endometrial cells in humans. Here, we analyzed single-cell sequencing data from a previous study including 24 non-coculture endometrial stromal cells (EmSCs) and 57 EmSCs after coculture with embryos. We further explored the transcriptomic changes in EmSCs and their interactions with trophoblast cells after coculture. Differentially expressed gene (DEG) analysis showed 1783 upregulated genes and 569 downregulated genes in the cocultured embryos. Weight gene coexpression network and gene ontology analysis of these DEGs showed a higher expression of RAMP1, LTBP1, and LRP1 in EmSCs after coculture, indicating the enrichment of biological processes in blood vessel development and female pregnancy. These data imply that EmSCs start blood vessel development at the implantation stage. Compared with endometrium data in vivo at the implantation window, key pathways including epithelial cell development and oxygen response were involved at this stage. Further analysis using CellphoneDB shed light on the interactions between EmSCs and embryonic trophoblasts, suggesting the important role of integrins and fibroblast growth factor pathways during implantation. Taken together, our work reveals the synchronization signaling and pathways happening at the implantation stage involving the acquisition of receptivity in EmSCs and the interaction between EmSCs and trophoblast cells.

Characterizing disease progression of nonalcoholic steatohepatitis in Leptin-deficient rats by integrated transcriptome analysis.

Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease threatening human health, yet no medicine is developed to treat this disease. In this study, we first discovered that Leptin mutant rats (LepΔI14/ΔI14) exhibit characteristic NASH phenotypes including steatosis, lymphocyte infiltration, and ballooning after postnatal week 16. We then examined NASH progression by performing an integrated analysis of hepatic transcriptome in Leptin-deficient rats from postnatal 4 to 48 weeks. Initially, simple steatosis in LepΔI14/ΔI14 rats were observed with increased expression of the genes encoding for rate-limiting enzymes in lipid metabolism such as acetyl-CoA carboxylase and fatty acid synthase. When NASH phenotypes became well developed at postnatal week 16, we found gene expression changes in insulin resistance, inflammation, reactive oxygen species and endoplasmic reticulum stress. As NASH phenotypes further progressed with age, we observed elevated expression of cytokines and chemokines including C-C motif chemokine ligand 2, tumor necrosis factor ɑ, interleukin-6, and interleukin-1ß together with activation of the c-Jun N-terminal kinase and nuclear factor-κB pathways. Histologically, livers in LepΔI14/ΔI14 rats exhibited increased cell infiltration of MPO+ neutrophils, CD8+ T cells, CD68+ hepatic macrophages, and CCR2+ inflammatory monocyte-derived macrophages associated with macrophage polarization from M2 to M1. Subsequent cross-species comparison of transcriptomes in human, rat, and mouse NASH models indicated that Leptin-deficient rats bear more similarities to human NASH patients than previously established mouse NASH models. Taken together, our study suggests that LepΔI14/ΔI14 rats are a valuable pre-clinical rodent model to evaluate NASH drug safety and efficacy.

Effect of Sperm Cryopreservation on miRNA Expression and Early Embryonic Development.

Although sperm preservation is a common means of personal fertility preservation, its effects on embryonic development potential need further investigation. The purpose of this study was to identify key microRNA (miRNA) in cryopreserved sperm and determine the changes of these miRNAs and their target genes during embryonic development using cryopreserved sperm. Moreover, the embryonic development potential of cryopreserved sperm was estimated in assisted reproductive technology (ART), where key miRNAs and target genes were validated in sperm and subsequent embryos. Clinical data of embryonic development from cryopreserved sperm indicated a significant decrease in fertilization rate in both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cases, as well as a reduction in blastocyst formation rate in ICSI cases. Meanwhile there was a significant increase in blocked embryo ratio of Day1, Day2, and Day3.5 embryos when frozen-thawed mouse sperm was used, compared with fresh mouse sperm, suggesting a potential negative effect of sperm cryopreservation on embryonic development. From frozen-thawed and fresh sperm in humans and mice, respectively, 21 and 95 differentially expressed miRNAs (DEmiRs) were detected. miR-148b-3p were downregulated in both human and mouse frozen-thawed sperm and were also decreased in embryos after fertilization using cryopreserved sperm. Target genes of miR-148b-3p, Pten, was identified in mouse embryos using quantitative real-time PCR (qRT-PCR) and Western blot (WB). In addition, common characters of cryopreservation of mouse oocytes compared with sperm were also detected; downregulation of miR-148b-3p was also confirmed in cryopreserved oocytes. In summary, our study suggested that cryopreservation of sperm could change the expression of miRNAs, especially the miR-148b-3p across humans and mice, and may further affect fertilization and embryo development by increasing the expression of Pten. Moreover, downregulation of miR-148b-3p induced by cryopreservation was conserved in mouse gametes.

Single-Cell Transcriptomic Analysis Reveals Mitochondrial Dynamics in Oocytes of Patients With Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS), characterized by polycystic ovarian morphology, ovarian follicular maturation arrest, and hormonal disorders, affects numerous women in the reproductive age worldwide. A recent study has found that mitochondria are likely to play an essential role in oocyte quality. However, it is still unclear whether oocyte development failure is associated with mitochondria in patients with PCOS. We analyzed the single-cell RNA sequencing data from the previous study, including data from 14 oocytes from 7 healthy fertile women and 20 oocytes from 9 patients with PCOS at the germinal vesicle (GV) stage, metaphase I (MI) stage, and metaphase II (MII) stage. We revealed the transcriptomic dynamics by weighted gene co-expression network analysis (WGCNA) and investigated the differences between stages using PCA and Deseq2 analyses to identify the differential expression genes (DEGs). Gene ontology (GO) was performed using clusterProfiler R package and Metascape. Our results indicated that specific gene modules were related to different stages of oocyte development using WGCNA. Functional enrichment analysis and gene co-expression network analysis found significant enrichment of the mitochondrial regulation genes at the GV stage. PCA (principal component analysis) and differential gene expression analysis suggested that GV was significantly different from the MI and MII stages between the two groups. Further analysis demonstrated that the upregulated differentially expressed genes at the GV stage of patients with PCOS mainly related to mitochondrial function, such as COX6B1, COX8A, COX4l1, and NDUFB9. Meanwhile, these genes tended to be activated at the MII stage in healthy cells, suggesting that some mitochondrial functions may be prematurely activated at the GV stage of PCOS oocytes, whereas this process occurs at the MII stage in healthy oocytes. Collectively, our study showed that aberrant mitochondrial function at the GV stage may contribute to a decline in oocyte quality of PCOS patients.

Roles of Exosomes from Mesenchymal Stem Cells in Treating Osteoarthritis.

Exosomes are small extracellular vesicles (EVs) with a diameter of 50-150 nm that play important roles in cell-to-cell communication through transportation of proteins, microRNAs, lncRNAs, and mRNAs. Some components, such as miRNAs, have been proven to be involved in inflammation regulation. Osteoarthritis (OA) is a progressive disease resulting in articular cartilage degeneration and subchondral bone deficiency. Complicated relationships between the breakdown of extracellular matrix and inflammation make it difficult to recover thoroughly. Current studies reported that exosomes secreted by mesenchymal stem cells (MSCs) can change disease evolution and protect the cartilage matrix in OA. In addition, exosomes obtained from human adipose derived stem cells downregulate inflammation and oxidative stress, which might mediate antisenescence in OA. The goal of this review is to describe and summarize the role of mesenchymal stem cell (MSC)-derived exosomes in OA, focusing on their potential mechanism and possible therapeutic strategies.

Potential impact of maternal vitamin D status on peripheral blood and endometrium cellular immunity in women with recurrent implantation failure.

PROBLEM: This study aims to evaluate the modulatory effects of vitamin D on peripheral blood and endometrial cellular immunity in women with recurrent implantation failure (RIF). METHOD OF STUDY: One hundred and fifty-four women with RIF were identified at a fertility center from January 2018 and March 2019. Blood and endometrium samples were collected during the mid-luteal phase before IVF treatment or pregnancy. The serum vitamin D status, NK cell cytotoxicity, Th1 cytokine production, and endometrial immune cells were detected before and after vitamin D supplementation. RESULTS: The NK cell cytotoxicity at an effector:target (E:T) ratio of 50:1 or 25:1 was significantly higher in vitamin D insufficiency group (VDI) than those in vitamin D normal group (VDN) (P < .05 each). The percentage of IFN-γ- or TNF-α-producing Th cells was significantly increased in VDI or vitamin D deficiency group (VDD) when compared with VDN (P < .05 each). The percentage of CD68+ macrophages on all endometrial cells in VDI and VDD was significantly higher than in VDN (P < .05 each), while no significant differences in the percentage of other endometrial immune cells among the three groups were observed. This dysregulation was significantly reduced with vitamin D supplementation. CONCLUSION: Our findings highlighted that vitamin D may have an important role in the regulation of not only systemic but also local immune response for optimization of maternal tolerance for implantation in women with RIF. Pre-conception optimization of vitamin D status should be considered in women with RIF.

Light-induced injury in mouse embryos revealed by single-cell RNA sequencing.

BACKGROUND: Light exposure is a common stress factor in in vitro manipulation of embryos in the reproductive center. Many studies have shown the deleterious effects of high-intensity light exposure in different animal embryos. However, no transcriptomic studies have explored the light-induced injury and response in preimplantation embryos. RESULTS: Here, we adopt different time-courses and illumination intensities to treat mouse embryos at the 2-cell stage and evaluate their effects on blastulation. Meanwhile, single-cell transcriptomes from the 2-cell to blastocyst stage were analyzed after high-intensity light exposure. These data show that cells at each embryonic stage can be categorized into different light conditions. Further analyses of differentially expressed genes and GO terms revealed the light-induced injury as well as the potential repair response after high-intensity lighting. Maternal-to-zygote transition is also affected by the failure to remove maternal RNAs and deactivate zygotic genome expression. CONCLUSION: Our work revealed an integrated response to high-intensity lighting, involving morphological changes, long-lasting injury effects, and intracellular damage repair mechanisms.

Mitochondria: the panacea to improve oocyte quality?

Oocyte quality is one of the most important factors involving in female reproduction. The number of compromised oocytes will increase with maternal age, while mitochondrial dysfunction has implicated in age-related poor oocyte. Together with the successful application of ooplasmic transfer (OT) and the critical role of mitochondria in the oocyte, functional mitochondria transfer may be a feasible strategy to improve oocyte quality. However, limitation on ethics and laws are strictly and optimal condition or methods to exert transferring need to be further explored. Therefore, the role of oocyte mitochondria and the effective molecular involving in oocyte quality will be hot topics in next few years. In this review, we summarize the potential mechanism of mitochondria in oocyte and embryo development and discuss the next step for mitochondrial transfer therapy.

Light-induced injury in mouse embryos revealed by single-cell RNA sequencing

BACKGROUND: Light exposure is a common stress factor in in vitro manipulation of embryos in the reproductive center. Many studies have shown the deleterious effects of high-intensity light exposure in different animal embryos. However, no transcriptomic studies have explored the light-induced injury and response in preimplantation embryos. RESULTS: Here, we adopt different time-courses and illumination intensities to treat mouse embryos at the 2-cell stage and evaluate their effects on blastulation. Meanwhile, single-cell transcriptomes from the 2-cell to blastocyst stage were analyzed after high-intensity light exposure. These data show that cells at each embryonic stage can be categorized into different light conditions. Further analyses of differentially expressed genes and GO terms revealed the light-induced injury as well as the potential repair response after high-intensity lighting. Maternal-to-zygote transition is also affected by the failure to remove maternal RNAs and deactivate zygotic genome expression. CONCLUSION: Our work revealed an integrated response to high-intensity lighting, involving morphological changes, long-lasting injury effects, and intracellular damage repair mechanisms.

Effects of dimethyl carbonate-induced autophagic activation on follicular development in the mouse ovary.

Dimethyl carbonate (DMC) is a widely used industrial chemical, which may be increasingly used in the future. However, its toxicity profile remains largely unknown. The present study was designed to investigate the effects of DMC exposure on the ovaries and the effect of autophagy activation on follicular development. Rats were randomly divided into a control group and low, medium and high dose DMC groups (all n=10). Histological analyses identified no marked differences in the rate of apoptosis between the control and low dose groups; however, marked apoptosis occurred in the medium and high dose groups. The expression of cleaved caspase-3 was significantly increased in the medium and high dose groups, which was consistent with changes observed in the expression of Bcl-2 and Bax. These results indicated that DMC exposure induces toxicity on ovarian function via the induction of apoptosis. The increased expression of the autophagy-related proteins light chain 3II, beclin-1 and p62 following exposure to DMC further indicated that autophagy was activated primarily in the granulosa cells of ovarian follicles in a dose-dependent manner. In addition, the changes in the expression of hypoxia inducible factor 1 α subunit (HIF-1α) and its target protein BCL2 interacting protein 3 (BNIP3) indicated that they may serve a role in the follicular development process induced by DMC. The results of the current study demonstrated that DMC exposure activated autophagy in the ovarian tissue. Furthermore, exposure to low doses of DMC may protect follicular development by activating the HIF-1α/BNIP3 signaling pathway. Taken together, these results indicate that exposure to medium and high doses of DMC induced follicular atresia by activating the apoptotic signaling pathway. This may be an important mechanism of regulating follicular development and ovarian function in mammals.

Effect of high-fat diet-induced obesity on the Akt/FoxO/Smad signaling pathway and the follicular development of the mouse ovary.

Obesity has a negative effect on ovarian functions, which is reported to increase the risk of infertility. The mechanism underlying obesity­induced infertility is not yet clear. The present study established a high­fat diet (HFD)­induced obesity mouse model to elucidate the mechanisms underlying the effect of HFD­induced obesity on follicular development in the mouse ovary. The 4­week­old female mice were fed with HFD or normal control (NC) diet for 15 or 20 weeks. Body mass index was used to demonstrate that the mice were obese following HFD treatment. The follicular development of the ovaries from the HFD group mice was retarded in a time­dependent manner, as demonstrated by morphological and histological examination of the ovaries. Further investigation via western blot analysis demonstrated that the activity of the transcription factor, forkhead box O3a (FoxO3a), was increased by HFD through downregulated FoxO3a phosphorylation, which may contribute to the inhibited development of ovarian follicles. To determine the regulatory mechanism of FoxO3 on the follicular development, the expression levels of FoxO3a target protein, Smad1/5/8, were also determined and there was significant decrease in phosphorylated Smad1/5/8 in the ovaries from the HFD group compared with the NC group, indicating that FoxO3a/Smad1/5/8 may be important in the regulation of follicular development. The expression levels of the upstream regulator of FoxO3a, Akt, were also examined and it was demonstrated that Akt phosphorylation was significantly reduced in the HFD group compared with the NC group, indicating that Akt/FoxO3a may be also involved in follicular development. Together, the experiments demonstrated that HFD­induced obesity affected the activity of the Akt/FoxO3a/Smad1/5/8 signaling pathway in a time­dependent manner during the follicular development of the mouse ovary, leading to abnormal follicular development. These findings may provide part of a theoretical basis for the clinical prevention and treatment of obesity-associated female infertility.