Adenomyosis is a finding that is associated with dysmenorrhea and heavy menstrual bleeding, associated with PI3K/AKT signaling overactivity. To investigate the effect of metformin on the growth of eutopic endometrial stromal cells (ESCs) from patients with adenomyosis and to explore the involvement of AMP-activated protein kinase (AMPK) and PI3K/AKT pathways. Primary cultures of human ESCs were derived from normal endometrium (normal endometrial stromal cells (N-ESCs)) and adenomyotic eutopic endometrium (adenomyotic endometrial stroma cells (A-ESCs)). Expression of AMPK was determined using immunocytochemistry and western blot analysis. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays were used to determine the effects of metformin and compound C on ESCs and also to detect growth and proliferation of ESCs. AMPK and PI3K/AKT signaling was determined by western blotting. A-ECSs exhibited greater AMPK expression than N-ESCs. Metformin inhibited proliferation of ESCs in a concentration-dependent manner. The IC50 was 2.45 mmol/l for A-ESCs and 7.87 mmol/l for N-ESCs. Metformin increased AMPK activation levels (p-AMPK/AMPK) by 2.0±0.3-fold in A-ESCs, 2.3-fold in A-ESCs from the secretory phase, and 1.6-fold in the proliferation phase. The average reduction ratio of 17β-estradiol on A-ESCs was 2.1±0.8-fold in proliferative phase and 2.5±0.5-fold in secretory phase relative to the equivalent groups not treated with 17β-estradiol. The inhibitory effects of metformin on AKT activation (p-AKT/AKT) were more pronounced in A-ESCs from the secretory phase (3.2-fold inhibition vs control) than in those from the proliferation phase (2.3-fold inhibition vs control). Compound C, a selective AMPK inhibitor, abolished the effects of metformin on cell growth and PI3K/AKT signaling. Metformin inhibits cell growth via AMPK activation and subsequent inhibition of PI3K/AKT signaling in A-ESCs, particularly during the secretory phase, suggesting a greater effect of metformin on A-ESCs from secretory phase.
Jing Xue, Hui Zhang, Wei Liu, Ming Liu, Min Shi, Zeqing Wen and Changzhong Li
Xin Huang, Chang Liu, Cuifang Hao, Qianqing Tang, Riming Liu, Shaoxia Lin, Luping Zhang and Wei Yan
Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women and is characterised by polycystic ovaries, hyperandrogenism and chronic anovulation. Although the clinical and biochemical signs of PCOS are typically heterogeneous, abnormal folliculogenesis is considered a common characteristic of PCOS. Our aim is to identify the altered miRNA and mRNA expression profiles in the cumulus cells of PCOS patients to investigate their molecular function in the aetiology and pathophysiology of PCOS. In this study, the miRNA expression profiles of the cumulus cell samples isolated from five PCOS and five control patients were determined by an miRNA microarray. At the same time, the altered mRNA profiles of the same cumulus cell samples were also identified by a cDNA microarray. From the microarray data, 17 miRNAs and 1263 mRNAs showed significantly different expression in the PCOS cumulus cells. The differentially expressed miRNA-509-3p and its potential target gene (MAP3K8) were identified from the miRNA and mRNA microarrays respectively. The expression of miRNA-509-3p was up-regulated and MAP3K8 was down-regulated in the PCOS cumulus cells. The direct interaction between miRNA-509-3p and MAP3K8 was confirmed by a luciferase activity assay in KGN cells. In addition, miRNA-509-3p mimics or inhibitor transfection tests in KGN cells further confirmed that miRNA-509-3p improved oestradiol (E2) secretion by inhibiting the expression of MAP3K8. These results help to characterise the pathogenesis of anovulation in PCOS, especially the regulation of E2 production.
Wenqian Xiong, Ling Zhang, Lan Yu, Wei Xie, Yicun Man, Yao Xiong, Hengwei Liu and Yi Liu
Endometriosis is an estrogen-dependent disease that involves the adhesion, invasion, and angiogenesis of endometrial tissues outside of the uterine cavity. We hypothesized that a link exists between estrogen and beta-catenin (β-catenin) signaling in the pathogenesis of endometriosis. Human endometrial stromal cells (HESCs) were separated from eutopic endometrial tissues that were obtained from patients with endometriosis. β-catenin expression and cells invasiveness ability were up-regulated by 17β-estradiol (E2) in an estrogen receptor (ESR)-dependent manner, whereas β-catenin siRNA abrogated this phenomenon. Moreover, co-immunoprecipitation and dual immunofluorescence studies confirmed ESR1, β-catenin, and lymphoid enhancer factor 1/T cell factor 3 co-localization in the nucleus in HESCs after E2 treatment. To determine the role of β-catenin signaling in the implantation of ectopic endometrium, we xenotransplanted eutopic endometrium from endometriosis patients into ovariectomized severe combined immunodeficiency mice. The implantation of the endometrium was suppressed by β-catenin siRNA. Collectively, studies regarding β-catenin signaling are critical for improving our understanding of the pathogenesis of estrogen-induced endometriosis, which can translate into the development of treatments and therapeutic strategies for endometriosis.
Fei Qu, Xiaoqian Ying, Wei Guo, Qiangsu Guo, Guowu Chen, Yue Liu and Zhide Ding
Sperm motility is essential for male reproduction or natural fertilization. The cyclic AMP (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway is generally recognized as one of the significant signaling pathways in the regulation of mammalian spermatozoan motility. Since Zn-α2-glycoprotein (ZAG) activity in mammalian adipose tissue is mediated via the β3-adrenoreceptor, with upregulation of the cAMP pathway, we hypothesize that ZAG may play the same role in sperm motility regulation, a new factor of regulation of sperm motility. Therefore, the gene encoding human ZAG was cloned and polyclonal antibodies were generated, and then laser scanning confocal microscopy and flow cytometry were employed to identify this protein in human spermatozoa. The results showed that ZAG protein was mostly localized on the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum of spermatozoa. Furthermore, using computer-assisted sperm analysis, we found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90- or 120-min incubation (P<0.05 and P<0.01 respectively), together with the decreasing of intracellular cAMP and PKA levels. In conclusion, these data suggest that ZAG is present in human spermatozoa and may be involved in the regulation of sperm motility via the cAMP/PKA signaling pathway.
Guixiang Ji, Lifeng Yan, Wei Liu, Cong Huang, Aihua Gu and Xinru Wang
The DNA double-strand breaks (DSBs) repair pathway plays a critical role in repairing double-strand breaks, and genetic variants in DSBs repair pathway genes are potential risk factors for various diseases. To test the hypothesis that polymorphisms in DSBs genes are associated with susceptibility to male infertility, we examined 11 single nucleotide polymorphisms in eight key DSBs genes (XRCC3, XRCC2, BRCA2, RAG1, XRCC5, LIG4, XRCC4 and ATM) in 580 infertility cases and 580 controls from a Chinese population-based case–control study (NJMU Infertility Study). Genotypes were determined using the OpenArray platform, and sperm DNA fragmentation was detected using the TUNEL assay. The adjusted odds ratio (OR) and 95% CI were estimated using logistic regression. The results indicate that LIG4 rs1805388 (Ex2+54C>T, Thr9Ile) T allele could increase the susceptibility to male infertility (adjusted OR=2.78; 95% CI, 1.77–4.36 for TT genotype; and adjusted OR=1.58; 95% CI, 1.77–4.36 for TC genotype respectively). In addition, the homozygous variant genotype GG of RAG1 rs2227973 (A>G, K820R) was associated with a significantly increased risk of male infertility (adjusted OR, 1.44; 95% CI, 1.01–2.04). Moreover, linear regression analysis revealed that carriers of LIG4 rs1805388 or RAG1 rs2227973 variants had a significantly higher level of sperm DNA fragmentation and that T allele carriers of LIG4 rs1805388 also had a lower level of sperm concentration when compared with common homozygous genotype carriers. This study demonstrates, for the first time, to our knowledge, that functional variants of RAG1 rs2227973 and LIG4 rs1805388 are associated with susceptibility to male infertility.
Shui-Kei Poon, Wai-Kin So, Xiaobin Yu, Lin Liu and Wei Ge
Inhibin and activin are closely related disulphide-linked dimers that belong to the transforming growth factor β superfamily. Although inhibin has been extensively studied in mammals, the information about its existence and function in lower vertebrates is very scarce. Using zebrafish as a model, the present study demonstrated that the inhibin-specific α subunit (inha) was predominantly expressed in the gonads and no transcript could be detected in other tissues including the pituitary and brain. In the ovary, the expression of inha was restricted to the somatic follicle cells surrounding the oocyte, together with the β subunits (inhbaa and inhbb). This was further supported by the absence of its expression in the ovulated unfertilized eggs. During folliculogenesis, inha expression in the follicles slightly but steadily increased from primary growth to the mid-vitellogenic stage; however, its expression surged dramatically at the full-grown stage. Interestingly, the expression level of inha decreased significantly in the follicles whose oocytes were undergoing spontaneous maturation or germinal vesicle breakdown. When tested on cultured ovarian fragments, both goldfish pituitary extract and forskolin significantly stimulated inha expression. Further experiments showed that recombinant zebrafish FSH but not LH significantly increased inha expression in the same assay system. When tested in vitro, human inhibin A exhibited a slight but significant inhibitory effect on 17α, 20β-dihydroxyprogesterone-induced oocyte maturation after 4 h incubation. The stimulation of inha expression by FSH and the potential inhibition of FSH by inhibin suggest a possible existence of a negative feedback loop between the pituitary and ovary in the zebrafish.
Lina Wang, Zhiliang Xu, Muhammad Babar Khawar, Chao Liu and Wei Li
Meiosis is a specialized process that produces haploid gametes from diploid cells by a single round of DNA replication followed by two successive cell divisions. It contains many special events, such as programmed DNA double-strand break (DSB) formation, homologous recombination, crossover formation and resolution. These events are associated with dynamically regulated chromosomal structures, the dynamic transcriptional regulation and chromatin remodeling are mainly modulated by histone modifications, termed ‘histone codes’. The purpose of this review is to summarize the histone codes that are required for meiosis during spermatogenesis and oogenesis, involving meiosis resumption, meiotic asymmetric division and other cellular processes. We not only systematically review the functional roles of histone codes in meiosis but also discuss future trends and perspectives in this field.
Bingteng Xie, Heng Zhang, Renyue Wei, Qiannan Li, Xiaogang Weng, Qingran Kong and Zhonghua Liu
Aberrant epigenetic reprogramming is the main obstacle to the development of somatic cell nuclear transfer (SCNT) embryos and the generation of induced pluripotent stem (iPS) cells, which results in the low reprogramming efficiencies of SCNT and iPS. Histone H3 lysine 27 trimethylation (H3K27me3), as a repressive epigenetic mark, plays important roles in mammalian development and iPS induction. However, the reprogramming of H3K27me3 in pig remains elusive. In this study, we showed that H3K27me3 levels in porcine early cloned embryos were higher than that in IVF embryos. Then GSK126 and GSK-J4, two small molecule inhibitors of H3K27me3 methylase (EZH2) and demethylases (UTX/JMJD3), were used to regulate the H3K27me3 level. The results showed that H3K27me3 level was reduced in cloned embryos after treatment of PEF with 0.75 μM GSK126 for 48 h, incubation of one-cell reconstructed oocytes with 0.1 μM GSK126 and injection of antibody for EZH2 into oocyte. Meanwhile, the development of the cloned embryos was significantly improved after these treatments. On the contrary, GSK-J4 treatment increased the H3K27me3 level in cloned embryos and decreased the cloned embryonic development. Furthermore, iPS efficiency was both increased after reducing the H3K27me3 level in donor cells and in early reprogramming phase. In summary, our results suggest that H3K27me3 acts as an epigenetic barrier in SCNT and iPS reprogramming, and reduction of H3K27me3 level in donor cells and in early reprogramming phase can enhance both porcine SCNT and iPS efficiency.
Chao Wei, Xia Li, Pengfei Zhang, Yu Zhang, Tong Liu, Shaoshuai Jiang, Fei Han and Yunhai Zhang
Partially reprogrammed induced pluripotent stem cells (PiPSCs) have great potential for investigating reprogramming mechanisms and represent an alternative potential material for making genetically modified animals and regenerative medicine. To date, PiPSCs have scarcely been reported in detail when compared with mice and humans. In this study, we obtained PiPSCs from porcine adipose-derived stem cells (pADSCs) by ectopic expression of human transcription factors (OCT4, SOX2, c-MYC, and KLF4) in feeder-free condition. The morphology and proliferation activity of porcine PiPSCs (pPiPSCs) were similar to those of porcine fully reprogrammed iPSCs (pFiPSCs); furthermore, pPiPSCs expressed higher levels of the typical surface molecules (CD29) found in pADSCs. However, pPiPSCs were negative for key proteins (NANOG) connected with stemness and possessed lower differentiation ability in vivo and in vitro. When differentiation-inhibiting factors were withdrawn, pPiPSCs-derived cells (pPiPSC-DCs) showed similar features to pADSCs in many aspects, including proliferation, differentiation, and immunosuppression. When both types of cells were used to produce cloned embryos, we found that the blastocyst formation rate of 19DC (one of the pPiPSC-DC cell lines)-derived cloned embryos was obviously higher than that of others. The total cell number of 19DC-derived blastocysts was significantly higher than the 30DC (one pFiPSC-DC cell line)-derived blastocysts. In all, through limited differentiation ability, the proliferation activity of pPiPSCs is similar to that of pFiPSCs, and pPiPSCs can retain several of the features of pADSCs, which are beneficial to cell therapy. Furthermore, the differentiation of pPiPSCs is more favorable for producing high-quality reconstructed embryos.
Free Chinese abstract: A Chinese translation of this abstract is freely available at http://www.reproduction-online.org/content/149/5/485/suppl/DC2.
Jingmei Hou, Shi Yang, Hao Yang, Yang Liu, Yun Liu, Yanan Hai, Zheng Chen, Ying Guo, Yuehua Gong, Wei-Qiang Gao, Zheng Li and Zuping He
Infertility is a major and largely incurable disease caused by disruption and loss of germ cells. It affects 10–15% of couples, and male factor accounts for half of the cases. To obtain human male germ cells ‘especially functional spermatids’ is essential for treating male infertility. Currently, much progress has been made on generating male germ cells, including spermatogonia, spermatocytes, and spermatids, from various types of stem cells. These germ cells can also be used in investigation of the pathology of male infertility. In this review, we focused on advances on obtaining male differentiated germ cells from different kinds of stem cells, with an emphasis on the embryonic stem (ES) cells, the induced pluripotent stem (iPS) cells, and spermatogonial stem cells (SSCs). We illustrated the generation of male differentiated germ cells from ES cells, iPS cells and SSCs, and we summarized the phenotype for these stem cells, spermatocytes and spermatids. Moreover, we address the differentiation potentials of ES cells, iPS cells and SSCs. We also highlight the advantages, disadvantages and concerns on derivation of the differentiated male germ cells from several types of stem cells. The ability of generating mature and functional male gametes from stem cells could enable us to understand the precise etiology of male infertility and offer an invaluable source of autologous male gametes for treating male infertility of azoospermia patients.