Cyclooxygenase (COX), a rate-limiting enzyme that produces prostaglandins (PGs) from arachidonic acid, exists in two isoforms, COX-1 and COX-2. PGE2 synthase (PGES) is a terminal prostanoid synthase and can enzymatically convert the cyclooxygenase product PGH2 to PGE2, including two isoforms: microsomal PGES (mPGES) and cytosolic PGES (cPGES). cPGES is predominantly linked with COX-1 to promote the immediate response. mPGES is preferentially coupled with the inducible COX-2 to promote delayed PGE2 generation. COX-2-deficient female mice are infertile with abnormalities in ovulation, fertilization, implantation and decidualization. The aim of this study was to examine immunohistochemically the expression pattern of COX-1, COX-2, mPGES and cPGES proteins in the endometrium of the rhesus monkey during the menstrual cycle. COX-1 immunostaining was mainly localized in the luminal epithelium and glandular epithelium near the lumen, and detected in all the stages during the menstrual cycle. COX-2 immunostaining was mainly localized in the luminal and glandular epithelium, and strongly shown during the mid-luteal phase (days 16 and 20) of the menstrual cycle. There was a strong cPGES immunostaining in the luminal and glandular epithelium on days 12, 16, 20 and 25 of the menstrual cycle. mPGES immunostaining was strongly detected in the glandular epithelium on days 20 and 25 of the menstrual cycle. These data suggest that the coupling of cPGES and COX-1 in the luminal epithelium may be responsible for the synthesis of PGE2 in monkey endometrium, and the coupling of mPGES and COX-2 in the glandular epithelium may be of importance for preparing the receptive endometrium.
Tong Sun, Shi-Jie Li, Hong-Lu Diao, Chun-Bo Teng, Hong-Bin Wang and Zeng-Ming Yang
Chun-Bo Teng, Hong-Lu Diao, Hong Ma, Jing Cong, Hao Yu, Xing-Hong Ma, Li-Bin Xu and Zeng-Ming Yang
Signal transducer and activator of transcription 3 (Stat3), a member of the Stat family, is specifically activated during mouse embryo implantation. The aim of this study was to investigate the expression, activation and regulation of Stat3 in rat uterus during early pregnancy, pseudopregnancy, delayed implantation and artificial decidualization. Stat3 mRNA was highly expressed in the luminal epithelium on day 5 and in the luminal epithelium and underlying stromal cells at implantation sites on day 6 of pregnancy. There was a strong level of Stat3 protein expression and phosphorylation in the stromal cells near the lumen and in the luminal epithelium on day 5 of pregnancy, which was similar to day 5 of pseudopregnancy. In the afternoon of day 6, the strong level of Stat3 phosphorylation was detected only in the luminal epithelium. Stat3 was highly expressed and activated in the decidual cells from days 7 to 9 of pregnancy and under artificial decidualization in the present study. Our results suggest that the strong level of Stat3 activation in the luminal epithelium and underlying stromal cells during the pre-implantation period may be important for establishing uterine receptivity as in mice, and the high level of Stat3 expression and activation in decidual cells may play a role during decidualization.
Xing Su, Yi Hu, Ying Li, Jing-Li Cao, Xue-Qin Wang, Xu Ma and Hong-Fei Xia
Although the relationship between polymorphisms in microRNAs (miRNAs) and recurrent pregnancy loss (RPL) has been studied, there is very little data available in the literature. In the present study, we scanned 55 potentially functional polymorphisms in the miRNA coding region in Chinese women with unexplained RPL (URPL; no. 2011-10). The rs6505162 C>A in the MIR423 coding region was found to be significantly associated with the occurrence of human URPL. The rare A allele contributed to an increase in the expression of mature MIR423. C to A substitution in the polymorphism rs6505162 in pre-MIR423 repressed cell proliferation and migratory capacity. Further investigations showed that MIR 423 could inversely regulate the expression of proliferation-associated 2 group 4 (PA2G4) by binding the 3′-UTR of PA2G4. Dual-luciferase assay indicated that the A allele in the polymorphism rs6505162 could more effectively suppress the expression of PA2G4 than the C allele could. Collectively, the present data suggest that rs6505162 C>A in pre-MIR423 may contribute to the genetic predisposition to RPL by disrupting the production of mature MIR42 3 and its target gene, which consequently interferes with MIR423 functioning.
Tian-Hong Zhu, Shao-Jie Ding, Tian-Tian Li, Li-Bo Zhu, Xiu-Feng Huang and Xin-Mei Zhang
Endometriosis is an estrogen-dependent disease. Previous research has shown that abnormal enzymes associated with estrogen (E2) metabolism and an increased number of mast cells (MCs) in endometriomas are implicated in the pathogenesis of endometriosis. However, it remains unclear how MCs mediate the role of E2 in endometriosis. Accordingly, we investigated whether E2 was associated with the number of MCs, and the rate of degranulation, in local ovarian endometriomas, as well as the role of E2 on MCs during the pathogenesis of endometriosis. Using enzyme-linked immunosorbent assay and immunohistochemistry, we found that concentrations of E2, and the number and activity of MCs, were significantly higher in ovarian endometriomas than in controls, and that these parameters were correlated with the severity of endometriosis-associated dysmenorrhea. By measuring the release of hexosaminidase, we found that the rate of RBL2H3 cell degranulation increased after E2 treatment. Furthermore, activation of RBL2H3 cells by E2 was found to trigger the release of biologically active nerve growth factor, which promotes neurite outgrowth in PC12 cells and also sensitizes dorsal root ganglion cells via upregulation of Nav1.8 and transient receptor potential cation channel (subfamily V member 1) expression levels. When treated with E2, endometriotic cells could promote RBL2H3 cell recruitment by upregulating expression levels of stem cell factor, transforming growth factor-β and monocyte chemoattractant protein-1; these observations were not evident with control endometrial cells. Thus, elevated E2 concentrations may be a key factor for degranulation and recruitment of MCs in ovarian endometriomas, which play a key role in endometriosis-associated dysmenorrhea.
Kai Zhu, Shang Li, Jiansheng Liu, Yan Hong, Zi-Jiang Chen and Yanzhi Du
Polycystic ovary syndrome, a common condition characterized by endocrine dysfunction, menstrual irregularity, anovulation and polycystic ovaries, affects 5–7% of reproductive-age women. RAB5B, which is identified by a genome-wide association study as a risk locus for this syndrome, encodes a small GTPase involved in control of receptor internalization and early endosome fusion. We found that RAB5A mRNA levels in luteinized granulosa cells of obese patients with polycystic ovary syndrome were lower than in those of obese women without the syndrome. RAB5A regulated follicle-stimulating hormone (FSH)-mediated translocation of the FSH receptor (FSHR) from the membrane to the cytoplasm and the subsequent FSH–FSHR signaling pathway. We showed that RAB5A negatively regulated aromatase expression and estradiol synthesis in human granulosa cells in association with changes in FSHR levels by way of the cAMP/PKA/CREB pathway. The regulation of FSHR by RAB5A may have been associated with two transcription factors, USF1 and USF2. In conclusion, RAB5A gene was abnormally expressed in luteinized granulosa cells of obese patients with polycystic ovary syndrome, which may help explain high FSHR levels found in this syndrome.
Xu-Jing Geng, Dong-Mei Zhao, Gen-Hong Mao and Li Tan
Leydig cells are essential for male reproductive development throughout life. Production of androgens as well as intermediate steroids is tightly regulated. Although microRNAs (miRNAs) are suggested to play important roles in spermatogenesis, little is currently known regarding the regulation of steroidogenesis by miRNAs in Leydig cells. Here, we found that miR-150 was predominantly expressed in Leydig cells within mouse testis. Therefore, we determined steroidogenesis of the Leydig cells in which miR-150 was knocked down or overexpressed using miR-150 antagomir and agomir, respectively. Compared with negative control group, a significant increase of STAR expression was observed in miR-150 antagomir-treated Leydig cells. Conversely, STAR expression was significantly reduced in miR-150 agomir-transfected Leydig cells. Production of sex-steroid precursors and testosterone of Leydig cells was also negatively controlled by miR-150. We further identified Star as a target of miR-150 using luciferase reporter assay. Finally, we confirmed that miR-150 was necessary for steroidogenesis and spermatogenesis in vivo via intratesticular injection of miR-150 antagomir or agomir. Taken together, our studies suggest that miR-150 negatively regulates the expression of STAR and steroidogenesis of Leydig cells in mice.
Bo Zheng, Jun Yu, Yueshuai Guo, Tingting Gao, Cong Shen, Xi Zhang, Hong Li and Xiaoyan Huang
The cellular nucleic acid-binding protein (CNBP), also known as zinc finger protein 9, is a highly conserved zinc finger protein that is strikingly conserved among vertebrates. Data collected from lower vertebrates showed that CNBP is expressed at high levels and distributed in the testes during spermatogenesis. However, the location and function of CNBP in mammalian testes are not well known. Here, by neonatal mouse testis culture and spermatogonial stem cells (SSC) culture methods, we studied the effect of CNBP knockdown on neonatal testicular development. Our results revealed that CNBP was mainly located in the early germ cells and Sertoli cells. Knockdown of CNBP using morpholino in neonatal testis culture caused disruption of seminiferous tubules, mislocation of Sertoli cells and loss of germ cells, which were associated with the aberrant Wnt/β-catenin pathway activation. However, knockdown of CNBP in SSC culture did not affect the survival of germ cells. In conclusion, our study suggests that CNBP could maintain testicular development by inhibiting the Wnt/β-catenin pathway, particularly by influencing Sertoli cells.
Hong-Fei Xia, Jing-Li Cao, Xiao-Hua Jin and Xu Ma
MiR199a was found to be differentially expressed in rat uteri between the prereceptive and receptive phase via microRNA (miRNA) microarray analysis in our previous study. However, the role of miR199a in rat embryo implantation remained unknown. In the study, northern blot results showed that the expression levels of miR199a were higher on gestation days 5 and 6 (g.d.5–6) in rat uteri than on g.d.3–4 and g.d.7–8. In situ localization of miR199a in rat uteri showed that miR199a was mainly localized in the stroma or decidua. The expression of miR199a was not significantly different in the uteri of pseudopregnant rats and evidently increased in the uteri of rats subjected to activation of delayed implantation and experimentally induced decidualization. Treatment with 17β-estradiol or both 17β-estradiol and progesterone significantly diminished miR199a levels. Gain of function of miR199a in endometrial stromal cells isolated from rat uteri inhibited cell proliferation and promoted cell apoptosis. Loss of function of miR199a displayed opposite roles on cell proliferation and apoptosis. Further investigation uncovered a significant inverse association between the expression of miR199a and growth factor receptor-bound protein 10 (Grb10), an imprinted gene, and miR199a could bind to the 3′UTR of Grb10 to inhibit Grb10 translation. In addition, in vivo analysis found that the immunostaining of GRB10 was attenuated in the stroma or decidua from g.d.4 to 6, contrary to the enhancement of miR199a. Collectively, upregulation of miR199a in rat uterus during the receptive phase is regulated by blastocyst activation and uterine decidualization. Enforced miR199a expression suppresses cell proliferation partially through targeting Grb10.
Songcun Wang, Fengrun Sun, Mutian Han, Yinghua Liu, Qinyan Zou, Fuxin Wang, Yu Tao, Dajin Li, Meirong Du, Hong Li and Rui Zhu
There is delicate crosstalk between fetus-derived trophoblasts (Tros) and maternal cells during normal pregnancy. Dysfunctions in interaction are highly linked to some pregnancy complications, such as recurrent spontaneous abortion (RSA), pre-eclampsia and fetal growth restriction. Hyaluronan (HA), the most abundant component of extracellular matrix, has been reported to act as both a pro- and an anti-inflammatory molecule. Previously, we reported that HA promotes the invasion and proliferation of Tros by activating PI3K/Akt and MAPK/ERK1/2 signaling pathways. While lower HA secretion by Tros was observed during miscarriages than that during normal pregnancies, in the present study, we further confirmed that higher secretion of HA by Tros could induce M2 polarization of macrophages at the maternal–fetal interface by interacting with CD44 and activating the downstream PI3K/Akt-STAT-3/STAT-6 signaling pathways. Furthermore, HA could restore the production of IL-10 and other normal pregnancy markers by decidual macrophages (dMφs) from RSA. These findings underline the important roles of HA in regulating the function of dMφs and maintaining a normal pregnancy.
Ji-Wen Yang, Zi-Li Lei, Yi-Liang Miao, Jun-Cheng Huang, Li-Hong Shi, Ying-Chun OuYang, Qing-Yuan Sun and Da-Yuan Chen
This study was carried out to investigate the contributions of chromosomes to spindle assembly in mouse oocytes. We generated two groups of cytoplasts (holo- and hemi-cytoplasts) by enucleation of germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) oocytes using micromanipulation technology. After in vitro culture for 18 h, spindles with different shapes (bi-, mono-, or multipolar) formed in most of these cytoplasts except in hemi-GV cytoplasts. Two or more spindles were observed in most of holo-GV, holo-MI, and holo-MII cytoplasts (76.1, 77.0, and 83.7% respectively). However, the proportions of hemi-MI and hemi-MII cytoplasts with multiple sets of spindles decreased to 17.6 and 20.7% respectively. A single bipolar spindle was observed in each sham-operated oocyte generated by removing different volumes of cytoplasm from the oocytes and keeping nuclei intact. Localization of γ-tubulin showed that microtubule organizing centers (MTOCs) were dispersed at each pole of the multiple sets of spindles formed in holo-cytoplasts. However, most of the MTOCs aggregated at the two poles of the bipolar spindle in sham-operated oocytes. Our results demonstrate that chromosomes are not essential for initiating spindle assembly but for directing distinct MTOCs to aggregate to form a bipolar spindle. Some factors of undetermined nature may pre-exist in an inactive form in GV-stage ooplasm, serving as initiators of spindle assembly upon their activation. Moreover, GV materials released into the cytoplasm may facilitate spindle assembly in normal meiotic maturation.