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Li Wang and Chen Xu

microRNAs (miRNAs) are a class of small endogenous RNAs, 19–25 nucleotides in size, which play a role in the regulation of gene expression at transcriptional and post-transcriptional levels. Spermatogenesis is a complex process through which spermatogonial stem cells (SSCs) proliferate and differentiate into mature spermatozoa. A large number of miRNAs are abundantly expressed in spermatogenic cells. Growing evidence supports the essential role of miRNA regulation in normal spermatogenesis and male fertility and cumulative research has shown that this form of regulation contributes to the etiology of testicular germ cell tumors (TGCTs). In this review, we addressed recent advancements of miRNA expression profiles in testis and focused on the regulatory functions of miRNA in the process of SSC renewal, spermatogonial mitosis, spermatocyte meiosis, spermiogenesis, and the occurrence of TGCTs.

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Xingji You, Zixi Chen, Huina Zhao, Chen Xu, Weina Liu, Qianqian Sun, Ping He, Hang Gu and Xin Ni

Recent evidence suggests that uterine activation for labor is associated with inflammation within uterine tissues. Hydrogen sulfide (H2S) plays a critical role in inflammatory responses in various tissues. Our previous study has shown that human myometrium produces H2S via its generating enzymes cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS) during pregnancy. We therefore explored whether H2S plays a role in the maintenance of uterine quiescence during pregnancy. Human myometrial biopsies were obtained from pregnant women at term. Uterine smooth muscle cells (UMSCs) isolated from myometrial tissues were treated with various reagents including H2S. The protein expression of CSE, CBS and contraction-associated proteins (CAPs) including connexin 43, oxytocin receptor and prostaglandin F receptor determined by Western blot. The levels of cytokines were measured by ELISA. The results showed that CSE and CBS expression inversely correlated to the levels of CAPs and activated NF-κB in pregnant myometrial tissues. H2S inhibited the expression of CAPs, NF-κB activation and the production of interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα) in cultured USMCs. IL-1β treatment reversed H2S inhibition of CAPs. Knockdown of CSE and CBS prevented H2S suppression of inflammation. H2S modulation of inflammation is through KATP channels and phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways. H2S activation of PI3K and ERK signaling is dependent on KATP channels. Our data suggest that H2S suppresses the expression of CAPs via inhibition of inflammation in myometrium. Endogenous H2S is one of the key factors in maintenance of uterine quiescence during pregnancy.

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Chen Xu, Xingji You, Weina Liu, Qianqian Sun, Xiaoying Ding, Ying Huang and Xin Ni

Prostaglandin F2α (PGF2A) has multiple roles in the birth process in addition to its vital contractile role. Our previous study has demonstrated that PGF2A can modulate uterine activation proteins (UAPs) in cultured pregnant human myometrial smooth muscle cells (HMSMCs). The objective of this study was to define the signalling pathways responsible for PGF2A modulation of UAPs in myometrium. It was found that PGF2A stimulated the expression of (GJA1) connexin 43 (CX43), prostaglandin endoperoxide synthase 2 (PTGS2) and oxytocin receptor (OTR) in cultured HMSMCs. The inhibitors of phospholipase C (PLC) and protein kinase C (PKC) blocked PGF2A-stimulated expression of CX43. The inhibitors of ERK, P38 and NFκB also blocked the effect of PGF2A on CX43 expression, whereas PI3K and calcineurin/nuclear factor of activated T-cells (NFAT) pathway inhibitors did not reverse the effect of PGF2A on CX43. For PTGS2 and OTR, PLC, PI3K, P38 and calcineurin/NFAT signalling pathways were involved in PGF2A action, whereas PKC and NFκB signalling were not involved. In addition, PGF2A activated NFAT, PI3K, NFκB, ERK and P38 signalling pathways. Our data suggest that PGF2A stimulates CX43, PTGS2 and OTR through divergent signalling pathways.

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Kun Li, Yue Liu, Xiaoyu Xia, Li Wang, Meige Lu, Yanqin Hu and Chen Xu

Bactericidal/permeability-increasing protein (BPI) is a 455-residue (∼55 kDa) protein found mainly in the primary (azurophilic) granules of human neutrophils. BPI is an endogenous antibiotic protein that belongs to the family of mammalian lipopolysaccharide (LPS)-binding and lipid transport proteins. Its major function is to kill Gram-negative bacteria, thereby protecting the host from infection. In addition, BPI can inhibit angiogenesis, suppress LPS-mediated platelet activation, increase DNA synthesis, and activate ERK/Akt signaling. In this study, we found that Bpi was expressed in the testis and epididymis but not in the seminal vesicles, prostate, and solidification glands. BPI expression in the epididymis increased upon upregulation of testosterone, caused by injection of GNRH. In orchidectomized mice, BPI expression was significantly reduced, but its expression was restored to 30% of control levels in orchidectomized mice that received supplementary testosterone. The number of sperm fused per egg significantly decreased after incubation with anti-BPI antiserum. These results suggest that BPI may take part in the process of sperm–oocyte fusion and play a unique and significant role in reproduction.

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Yan Xu, Miao Liu, Yi-hua Gu, Xiao-feng Jia, Yong-Mei Chen, Michelle Santos, Ai-Zhen Wu, Xiao-dong Zhang, Hui-Juan Shi and Ching-Ling C Chen

With tetraspanning topology, members of the membrane-spanning four-domain subfamily A (MS4A) may facilitate signaling or ion channel functions in many tissues. In this study, we report the cloning of a full-length cDNA from rat testis, designated Ms4a14 (Sp3111), which encodes the MS4A protein with 1139 amino acid residues. In situ hybridization and immunohistochemical analyses indicate that Ms4a14 is predominantly expressed from round spermatids to spermatozoa at specific stages in the rat testis at both the mRNA and protein level. Immunofluorescence analysis revealed that MS4A14 (SP3111) is located in the acrosome and the midpiece of the flagellum in mature sperm. Previously, we explored and reported the involvement of MS4A14 in reproductive functions, using antibody blockage during IVF and a transgenic RNA interference method in a mouse model. Our results suggested that MS4A14 is involved in fertilization and zygote division. As MS4A14 protein exists in mammals, such as humans, cows, dogs, and rodents, MS4A14 may play a ubiquitous role in mammalian reproduction.

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Wei Wang, Xia Chen, Xinxiu Li, Li Wang, Haiyan Zhang, Yu He, Jingjing Wang, Yongyan Zhao, Baole Zhang and Yinxue Xu

FSH plays a critical role in granulosa cell (GC) proliferation and steroidogenesis through modulation by factors including bone morphogenetic proteins family, which belongs to transforming growth factor β (TGFB) superfamily. TGFBs are the key factors in maintaining cell growth and differentiation in ovaries. However, the interaction of FSH and TGFB on the GCs' proliferation and steroidogenesis remains to be elucidated. In this study, we have investigated the role of SMAD4, a core molecule mediating the intracellular TGFB/SMAD signal transduction pathway, in FSH-mediated proliferation and steroidogenesis of porcine GCs. In this study, SMAD4 was knocked down using interference RNA in porcine GCs. Our results showed that SMAD4-siRNA causes specific inhibition of SMAD4 mRNA and protein expression after transfection. Knockdown of SMAD4 significantly inhibited FSH-induced porcine GC proliferation and estradiol production and changed the expression of cyclin D2, CDK2, CDK4, CYP19a1, and CYP11a1. Thus, these observations establish an important role of SMAD4 in the regulation of the response of porcine GCs to FSH.

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Jingjing Guo, Hongyu Zhou, Zhijian Su, Bingbing Chen, Guimin Wang, Claire Q F Wang, Yunfei Xu and Ren-Shan Ge

The objective of this study was to purify cells in the Leydig cell lineage following regeneration after ethane dimethanesulfonate (EDS) treatment and compare their steroidogenic capacity. Regenerated progenitor (RPLCs), immature (RILCs), and adult Leydig cells (RALCs) were isolated from testes 21, 28 and 56 days after EDS treatment respectively. Production rates for androgens including androsterone and 5α-androstane-17β, 3α-diol (DIOL), testosterone and androstenedione were measured in RPLCs, RILCs and RALCs in media after 3-h in vitro culture with 100 ng/ml LH. Steady-state mRNA levels of steroidogenic enzymes and their activities were measured in freshly isolated cells. Compared to adult Leydig cells (ALCs) isolated from normal 90-day-old rat testes, which primarily produce testosterone (69.73%), RPLCs and RILCs primarily produced androsterone (70.21%) and DIOL (69.79%) respectively. Leydig cells isolated from testes 56 days post-EDS showed equivalent capacity of steroidogenesis to ALCs and primarily produced testosterone (72.90%). RPLCs had cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1 and 17α-hydroxylase but had almost no detectable 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities, while RILCs had increased 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities. Because RPLCs and RILCs had higher 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase activities they produced mainly 5α-reduced androgens. Real-time PCR confirmed the similar trends for the expressions of these steroidogenic enzymes. In conclusion, the purified RPLCs, RILCs and RALCs are similar to those of their counterparts during rat pubertal development.

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Meng-Ling Liu, Jing-Lei Wang, Jie Wei, Lin-Lin Xu, Mei Yu, Xiao-Mei Liu, Wen-Li Ruan and Jia-Xiang Chen

Tri-ortho-cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame retardants in industry and reported to have a deleterious effect on the male reproductive system in animals besides delayed neurotoxicity. Our preliminary results found that TOCP could disrupt the seminiferous epithelium in the testis and inhibit spermatogenesis, but the precise mechanism is yet to be elucidated. This study shows that TOCP inhibited viability of rat spermatogonial stem cells in a dose-dependent manner. TOCP could not lead to cell cycle arrest in the cells; the mRNA levels of p21, p27, p53, and cyclin D1 in the cells were also not affected by TOCP. Meanwhile, TOCP did not induce apoptosis of rat spermatogonial stem cells. After treatment with TOCP, however, both LC3-II and the ratio of LC3-II/LC3-I were markedly increased; autophagy proteins ATG5 and beclin 1 were also increased after treatment with TOCP, indicating that TOCP could induce autophagy in the cells. Ultrastructural observation under the transmission electron microscopy indicated that autophagic vesicles in the cytoplasm containing extensively degraded organelles such as mitochondria and endoplasmic reticulum increased significantly after the cells were treated with TOCP. In summary, we have shown that TOCP can inhibit viability of rat spermatogonial stem cells and induce autophagy of the cells, without affecting cell cycle and apoptosis.

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F Guo, B Yang, Z H Ju, X G Wang, C Qi, Y Zhang, C F Wang, H D Liu, M Y Feng, Y Chen, Y X Xu, J F Zhong and J M Huang

The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

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Shu-Fang Wang, Xi-Hua Chen, Bin He, De-Dong Yin, Hai-Jun Gao, Hao-Qi Zhao, Nan Nan, Shi-Ge Guo, Jian-Bing Liu, Bin Wu and Xiang-Bo Xu

Stress impacts the reproductive axis at the level of the hypothalamus and the pituitary gland, which exert an effect on the ovary. Menstruation is regulated by the hypothalamic–pituitary–ovary (HPO) axis. However, the role of stress in menstruation remains unclear. The objective of this study was to explore the role of stress in endometrial breakdown and shedding, using the pseudopregnant mouse menstrual-like model. Female mice were mated with vasectomized males and labeled day 0.5, upon observation of a vaginal seminal plug. On day 3.5, decidualization was induced in pseudopregnant mice using arachis oil. On day 5.5, pseudopregnant mice with artificial decidualization were placed in restraint tubes for 3 h. The findings indicated that acute restraint stress resulted in the disintegration of the endometrium. While corticosterone concentration in the serum increased significantly due to restraint stress, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and progesterone (P4) levels in the serum decreased significantly. An endometrial histology examination indicated that progesterone implants may rescue P4 decline caused by acute stress and block endometrium breakdown and shedding. In addition, mice were treated with metyrapone, an inhibitor of corticosterone synthesis, 1 h prior to being subjected to restraint stress. Interestingly, metyrapone not only inhibited stress-induced endometrium breakdown and shedding, but also prevented stress-induced reduction of P4, LH and FSH. Furthermore, real-time PCR and western blot showed that mRNA and protein expression of CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and steroidogenic acute regulatory protein (StAR), the two rate-limiting enzymes for progesterone synthesis in the ovary, decreased following acute stress. But metyrapone prevented the reduction of StAR expression induced by restraint stress. Overall, this study revealed that acute stress results in an increase in corticosterone, which may inhibit LH and FSH release in the serum and CYP11A1 and StAR expression in the ovary, which finally leads to the breakdown and shedding of the endometrium. These experimental findings, based on the mouse model, may enable further understanding of the effects of stress on menstruation regulation and determine the potential factors affecting stress-associated menstrual disorders.