Spermatogenesis is a continuous and productive process supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs), which arise from undifferentiated precursors known as gonocytes and are strictly controlled in a special ‘niche’ microenvironment in the seminiferous tubules. Sertoli cells, the only somatic cell type in the tubules, directly interact with SSCs to control their proliferation and differentiation through the secretion of specific factors. Spermatocyte meiosis is another key step of spermatogenesis, which is regulated by Sertoli cells on the luminal side of the blood–testis barrier through paracrine signaling. In this review, we mainly focus on the role of Sertoli cells in the regulation of SSC self-renewal and spermatocyte meiosis, with particular emphasis on paracrine and endocrine-mediated signaling pathways. Sertoli cell growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), as well as Sertoli cell transcription factors, such as ETS variant 5 (ERM; also known as ETV5), nociceptin, neuregulin 1 (NRG1), and androgen receptor (AR), have been identified as the most important upstream factors that regulate SSC self-renewal and spermatocyte meiosis. Other transcription factors and signaling pathways (GDNF–RET–GFRA1 signaling, FGF2–MAP2K1 signaling, CXCL12–CXCR4 signaling, CCL9–CCR1 signaling, FSH–nociceptin/OPRL1, retinoic acid/FSH–NRG/ERBB4, and AR/RB–ARID4A/ARID4B) are also addressed.
Su-Ren Chen and Yi-Xun Liu
Yu-Qian Wang, Aalia Batool, Su-Ren Chen and Yi-Xun Liu
Reduced contractility of the testicular peritubular myoid (PTM) cells may contribute to human male subfertility or infertility. Transcription factor GATA4 in Sertoli and Leydig cells is essential for murine spermatogenesis, but limited attention has been paid to the potential role of GATA4 in PTM cells. In primary cultures of mouse PTM cells, siRNA knockdown of GATA4 increased the contractile activity, while GATA4 overexpression significantly attenuated the contractility of PTM cells using a collagen gel contraction assay. Using RNA sequencing and qRT-PCR, we identified a set of genes that exhibited opposite expressional alternation between Gata4 siRNA vs nontargeting siRNA-treated PTM cells and Gata4 adenovirus vs control adenovirus-treated PTM cells. Notably, ion channels, smooth muscle function, cytokines and chemokines, cytoskeleton, adhesion and extracellular matrix were the top four enriched pathways, as revealed by cluster analysis. Natriuretic peptide type B (NPPB) content was significantly upregulated by GATA4 overexpression in both PTM cells and their culture supernatant. More importantly, the addition of 100 μM NPPB could abolish the promoting effect of Gata4 silencing on PTM cell contraction. Taken together, we suggest that the inhibitory action of GATA4 on PTM cell contraction is mediated at least partly by regulating genes belonging to smooth muscle contraction pathway (e.g. Nppb).
Li-Juan Xiao, Jin-Xiang Yuan, Xin-Xin Song, Yin-Chuan Li, Zhao-Yuan Hu and Yi-Xun Liu
Stanniocalcin-1 (STC-1) is a recently discovered polypeptide hormone, while stanniocalcin-2 (STC-2) is a subsequently identified homologue of stanniocalcin-1. Although previous studies have shown that both STC-1 and -2 are involved in various physiological processes, such as ion transport, reproduction and development, their expression in the uterus and roles in implantation and early pregnancy are unclear. Here we have investigated the expression and regulation of both STC-1 and STC-2 in rat uterus during early pregnancy under various physiological conditions. We show that only basal levels of STC-1 and STC-2 mRNA were detected in the uterus from day one (D1) to day five (D5) of pregnancy. STC-2 immunostaining was gradually increased in the glandular epithelium from day two (D2), with a peak occurring on D5. High levels of both STC-1 and STC-2 mRNA were observed in the stoma cells at the implantation site on day six (D6) of pregnancy, whereas their immunostaining signals were also significant in the luminal epithelium. Basal levels of both STC-1 and STC-2 mRNA and STC-1 immunostaining were detected in the uterus with delayed implantation. After the delayed implantation was terminated by estrogen treatment, both STC-1 and STC-2 mRNA signals were significantly induced in the stroma underlying the luminal epithelium at the implantation site, and STC-2 immunostaining was also observed in the luminal epithelium surrounding the implanting blastocyst. Embryo transfer experiments further confirmed that STC-1 and STC-2 expression at the implantation sites was induced by the implanting blastocyst. Both STC-1 mRNA and immunostaining were seen in the decidualized cells from day seven (D7) to day nine (D9) of pregnancy. STC-2 mRNA was also found in the whole decidua from D7 to D9 of pregnancy; STC-2 protein, however, was strictly localized to the primary deciduas on D7 and D8, with a weak expression in the whole deciduas on D9. Consistent with the normal pregnancy process, strong STC-1 and STC-2 mRNA signals were detected in the decidualized cells under artificial decidualization, whereas only basal levels of STC-1 mRNA and immunostaining were observed in the control horn. These data suggest, for the first time, that STC-1 together with STC-2 may play important roles in the processes of implantation and decidualization in the rat.
Li-Juan Xiao, Jin-Xiang Yuan, Yin-Chuan Li, Rui Wang, Zhao-Yuan Hu and Yi-Xun Liu
The extracellular Ca2+-sensing receptor (CaR) is a member of the superfamily of G protein-coupled receptors (GPCRs). It is an important mediator of a wide range of Ca2+-dependent physiological responses in various tissues. In reproductive tissues it has been reported to play a significant role in promoting or maintaining placentation. Meanwhile, another Ca2+ regulated gene stanniocalcin-1 (STC-1) has been documented to be involved in decidualization and uterine remodelling. The phenomenon that CaR mediates STC-1’s transcription responding to extracellular calcium in fish urges us to suppose that CaR, like STC-1, may also play a role in implantation and decidualization. To resolve this conjecture, we have examined the expression and hormonal regulation of the CaR gene in rat uterus during peri-implantation period.
CaR mRNA was expressed at a moderate level in the luminal epithelium of the early stage of pregnancy (from day 1 to day 3). From day 2–3 it began to be expressed more strongly in the stromal cells immediately underneath the luminal epithelium, but decreased to a basal level on day 4. From day 6 to day 9 continuously, both CaR mRNA and protein were highly expressed in the primary decidua. Expression of CaR mRNA and protein in these cells was also observed when a delayed implantation was terminated by estrogen treatment to allow the embryo implantation. In contrast, only basal level expression of the molecules was detected in the cells of animals subjected to a normal-delayed implantation or the pseudopregnant condition.
Embryo transplantation experiment confirmed that CaR expression at the implantation site was induced by the implanting blastocyst. Consistent with the normal pregnant process, CaR mRNA and protein in the cells were also induced by an artificial decidualization procedure. Further experiments demonstrated that treatment of the ovariectomized rat with estrogen or/and progesterone stimulated a high level expression of CaR mRNA in the uterine epithelial and glandular epithelium. In conclusion, CaR was specifically induced during the processes of implantation and subsequent decidualization and may play a role in these processes.
Kien C Luu, Gui Ying Nie, Anne Hampton, Guo-Qiang Fu, Yi-Xun Liu and Lois A Salamonsen
The endometrium is hostile to embryo implantation except during the ‘window of receptivity’. A change in endometrial gene expression is required for the development of receptivity. Calbindin-d9k (CaBP-d9k) and calbindin-d28k (CaBP-d28k) are proteins possessing EF-hand motifs which have high affinity for Ca2+ ions. Previously, it has been demonstrated that, in mouse endometrium, the expression of both calbindins is highly regulated during implantation and that both proteins play critical but functionally redundant roles at implantation. This study was the first to determine the expression of these two calbindins in the human and rhesus monkey endometrium. Initial RT-PCR analysis demonstrated that CaBP-d28k but not CaBP-d9k mRNA expression is detectable in the endometrium of both species. Western blot analysis confirmed the presence of immuno-reactive CaBP-d28k protein in the primate endometrium. Furthermore, the endometrial expression pattern of CaBP-d28k mRNA and protein was examined by Northern blot analysis and immunohistochemistry respectively in both species across the menstrual cycle and during early pregnancy. Semi-quantitative statistical analysis of the immunohistochemistry results revealed that, in the human, CaBP-d28k protein expression was maximal in luminal and glandular epithelium during the mid-secretory phase, coinciding with the time when the endometrium is receptive to embryo implantation. Expression in rhesus monkey showed a similar trend. These results suggest that, in the primate endometrium, only CaBP-d28k is expressed and that the specific regulation of this calbindin is potentially important for the establishment of uterine receptivity.
Qiao-Song Zheng, Xiao-Na Wang, Qing Wen, Yan Zhang, Su-Ren Chen, Jun Zhang, Xi-Xia Li, Ri-Na Sha, Zhao-Yuan Hu, Fei Gao and Yi-Xun Liu
Spermatogenesis is a complex process involving the regulation of multiple cell types. As the only somatic cell type in the seminiferous tubules, Sertoli cells are essential for spermatogenesis throughout the spermatogenic cycle. The Wilms tumor gene, Wt1, is specifically expressed in the Sertoli cells of the mouse testes. In this study, we demonstrated that Wt1 is required for germ cell differentiation in the developing mouse testes. At 10 days post partum, Wt1-deficient testes exhibited clear meiotic arrest and undifferentiated spermatogonia accumulation in the seminiferous tubules. In addition, the expression of claudin11, a marker and indispensable component of Sertoli cell integrity, was impaired in Wt1 −/flox; Cre-ER TM testes. This observation was confirmed in in vitro testis cultures. However, the basal membrane of the seminiferous tubules in Wt1-deficient testes was not affected. Based on these findings, we propose that Sertoli cells' status is affected in Wt1-deficient mice, resulting in spermatogenesis failure.
Cheng Jin, Yan Zhang, Zhi-Peng Wang, Xiu-Xia Wang, Tie-Cheng Sun, Xiao-Yu Li, Ji-Xin Tang, Jin-Mei Cheng, Jian Li, Su-Ren Chen, Shou-Long Deng and Yi-Xun Liu
Spermatogenesis is crucial for male fertility and is therefore tightly controlled by a variety of epigenetic regulators. However, the function of enhancer of zeste homolog 2 (EZH2) in spermatogenesis and the molecular mechanisms underlying its activity remain poorly defined. Here, we demonstrate that deleting EZH2 promoted spermatogonial differentiation and apoptosis. EZH2 is expressed in spermatogonia, spermatocytes and round and elongated spermatids from stage 9 to 11 but not in leptotene and zygotene spermatocytes. Knocking down Ezh2 in vitro using a lentivirus impaired self-renewal in spermatogonial stem cells (SSCs), and the conditional knockout of Ezh2 in spermatogonial progenitors promoted precocious spermatogonial differentiation. EZH2 functions to balance self-renewal and differentiation in spermatogonia by suppressing NEUROG3 and KIT via a direct interaction that is independent of its histone methyltransferase activity. Moreover, deleting Ezh2 enhanced the activation of CASP3 in spermatids, resulting in reduced spermatozoa production. Collectively, these data demonstrate that EZH2 plays a nonclassical role in the regulation of spermatogonial differentiation and apoptosis in murine spermatogenesis.