Bone morphogenetic proteins (BMPs) are known to play an indispensable role in preventing the precocious luteinization of granulosa cells within growing ovarian follicles. In this study, we found that the transcripts of BMP8 genes are enriched in the ovaries of humans and rodents. When analyzing transcriptomic datasets obtained from human mature granulosa cells, we further found that the BMP8 transcripts not only show the highest abundance among the searchable BMP-related ligands but also decrease significantly in women of advanced age or women with polycystic ovarian syndrome. The correlation between the BMP8 levels in granulosa cells and the decline in ovarian function in these subjects suggests that BMP8 protein may be involved in the regulation of granulosa cell function(s). Using a rat model, we demonstrated that human BMP8A protein activates the SMAD1/5/8 and the SMAD2/3 pathways simultaneously in both immature and mature granulosa cells. Furthermore, the expression of potential type I and type II receptors used by BMP8 in rat granulosa cells was characterized. We found that BMP8A treatment can significantly inhibit gonadotropin-induced progesterone production and steroidogenesis-related gene expression in granulosa cells. Pathway dissection using receptor inhibitors further revealed that such inhibitory effects occur specifically through the BMP8-activated SMAD1/5/8, but not SMAD2/3, pathway. Taken together, considering its abundance and possible functions in granulosa cells, we suggest that BMP8 may act as a novel luteinization inhibitor in growing follicles.
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Fang-Ju Wu, Ying-Wen Wang, and Ching-Wei Luo
Kezhen Yao, Yu Sun, Xiaoqun Ye, and Ying Wu
IFN-λs participate in the fetal–maternal immune interaction, involving in immune regulation, uterine receptivity, cell migration and adhesion, and endometrium apoptosis. Our study helps to elucidate the underlying causes of the IFN-λs deficiency to spontaneous pregnancy loss in women.
Immunotherapy has been commonly used to prevent recurrent pregnancy loss in women with inadequate uterus receptivity or immunological imbalance. Many immune regulators are now identified as having crucial roles at the embryo–maternal interface. However, the clinical efficacy of immunity-related markers during the peri-implantation period remains to be explored in depth. Here, we demonstrated that endometrial expression of interferon-λ (IFN-λ), regarded as a newer class of interferons, is aberrantly lower in women who suffered from recurrent implantation failure than that in fertile control. We further uncovered genetic and biochemical evidence that IFN-λ is induced directly by estrogen in the endometrial cells, and IFN-λ pathway may play multiple roles involving the inflammatory response, uterine receptivity, cell migration, and blastocyst adhesion. Furthermore, we indicated IFN-λ lessens the sensitivity of endometrium to FASL-mediated apoptosis. In addition to uncovering this IFN-λ as a novel nonredundant regulator that participates in the fetal–maternal immune interaction, our study helps to elucidate the underlying causes of spontaneous pregnancy loss in women.
Xiaokui Yang, Ying Zhou, Sha Peng, Liang Wu, Hai-Yan Lin, Shuyu Wang, and Hongmei Wang
Recent studies implicate the regulatory function of microRNAs (miRNAs) in oocyte maturation and ovarian follicular development. Differentially expressed miRNAs are found in the plasma of premature ovarian failure (POF) patients and normal cycling women. In this study, miRNA-regulated signaling pathways and related genes were described using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The effect of mir-23a on granulosa cell apoptosis was also studied by examining the protein expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3, followed by subsequent counting of apoptotic cells after Hoechst 33258 staining. Both GO analysis and pathway analysis suggested that many signaling pathways, including the AKT signaling pathway, steroid hormone receptor signaling pathways, and others, were regulated by this group of differentially expressed miRNAs. A decrease in XIAP expression (mRNA and protein level) and caspase-3 protein levels and an increase in cleaved caspase-3 protein were observed in human ovarian granulosa cells transfected with pre-mir-23a, along with an increased occurrence of apoptosis. In conclusion, differentially expressed miRNAs in the plasma of POF patients may have regulatory effects on proliferation and apoptosis of granulosa cells by affecting different signaling pathways. Mir-23a may play important roles in regulating apoptosis via decreasing XIAP expression in human ovarian granulosa cells.
Liu Shi-fan, Wang Zhong-xing, Yuan Yao-e, Bing Sheng-min, Zhang Bei-zhu, Wu Jin-zhi, Wu Yi-e, and Peng Xin-ying
Summary. The concentrations of LH, FSH, prolactin, oestradiol and progesterone in serum were measured daily during the menstrual cycle of 100 normal Chinese women. The cyclic changes in LH, FSH, oestradiol and progesterone were typical of ovulatory cycles in women of other ethnic groups as reported in the literature. The geometric mean of the LH midcycle peak value was 51·64 i.u./l, the FSH mid-cycle peak value was 11 ·52 i.u./l, the preovulatory oestradiol peak was 1229·12 pmol/1, and the progesterone luteal maximum was 53·27 nmol/1. The cyclic changes of prolactin concentrations were irregular: the value at mid-cycle was significantly higher than that at the follicular or luteal phases.
A correlation between the length of the cycle and mean concentrations of LH and oestradiol at different stages throughout the cycle was shown.
Wei-Bin Wu, Yue-Ying Xu, Wei-Wei Cheng, Bo Yuan, Jiu-Ru Zhao, Yan-Lin Wang, and Hui-Juan Zhang
Fetal growth restriction (FGR) threatens perinatal health and is correlated with increased incidence of fetal original adult diseases. Most cases of FGR were idiopathic, which were supposed to be associated with placental abnormality. Decreased circulating placental growth factor (PGF) was recognized as an indication of placental deficiency in FGR. In this study, the epigenetic regulation of PGF in FGR placentas and the involvement of PGF in modulation of trophoblast activity were investigated. The expression level of PGF in placental tissues was determined by RT-qPCR, immunohistochemistry and ELISA. DNA methylation profile of PGF gene was analyzed by bisulfite sequencing. Trophoblastic cell lines were treated with ZM-306416, an inhibitor of PGF receptor FLT1, to observe the effect of PGF/FLT1 signaling on cell proliferation and migration. We demonstrated that PGF was downregulated in placentas from FGR pregnancies compared with normal controls. The villous expression of PGF was positively correlated with placental and fetal weight. The CpG island inside PGF promoter was hypomethylated without obvious difference in both normal and FGR placentas. However, the higher DNA methylation at another CpG island downstream exon 7 of PGF was demonstrated in FGR placentas. Additionally, we found FLT1 was expressed in trophoblast cells. Inhibition of PGF/FLT1 signaling by a selective inhibitor impaired trophoblast proliferation and migration. In conclusion, our data suggested that the PGF expression was dysregulated, and disrupted PGF/FLT1 signaling in trophoblast might contribute to placenta dysfunction in FGR. Thus, our results support the significant role of PGF in the pathogenesis of FGR.
Cheng Zeng, Pei-Li Wu, Zhao-Tong Dong, Xin Li, Ying-Fang Zhou, and Qing Xue
Endometriosis is an estrogen-dependent disease, and estrogen receptor 2 (ESR2) plays a critical role in the pathogenesis of ovarian endometriosis by promoting cell invasion. Yes-associated protein 1 (YAP1) plays suppressive roles in several types of tumors. However, the relationship between YAP1 and ESR2 is not fully understood. The aim of this study was to investigate the regulatory mechanism of YAP1 in terms of ESR2 and YAP1 regulation of endometriotic stromal cell (ECSC) invasion in ovarian endometriosis. Our results demonstrated that YAP1 mRNA and protein levels in eutopic endometrium (EU) tissues were higher than those in paired ectopic endometrium (EC) tissues. ECSCs transfected with siYAP1 exhibited a significant increase in both ESR2 mRNA levels and protein expression. Simultaneously, YAP1 overexpression in ECSCs yielded the opposite results. Co-IP assays demonstrated YAP1-NuRD complex formation by YAP1, CHD4 and MTA1 in ECSCs. YAP1 bound to two sites, (-539, -533) and (-158, -152), upstream of the ESR2 transcription initiation site. YAP1 binding to the two sites of the ESR2 promoter in ECSCs was significantly lower than that in eutopic endometrial stromal cells (EUSCs) from EU tissues. ECSCs transfected with siYAP1 exhibited increased invasion activity, while ECSCs transfected with siESR2 showed inhibition of invasion. However, transfection with siYAP1 and siESR2 together decreased the number of invading cells compared with transfection with siYAP1 alone. Therefore, we conclude that decreased levels of YAP1 in ovarian endometriomas enhance ESR2 expression via formation of a YAP1-NuRD complex, which further binds to the ESR2 promoters. Furthermore, YAP1 inhibits ECSCs invasion.
Lanlan Fang, Zhen Wang, Ze Wu, Yang Yan, Yibo Gao, Yuxi Li, Jung-Chien Cheng, and Ying-Pu Sun
Matrix metalloproteinases (MMPs) play a pivotal role in the regulation of cell invasion. Placental trophoblast cell invasion is a precisely regulated event. Dysregulation of MMPs has been linked to various placental diseases. Growth differentiation factor-8 (GDF-8), also known as myostatin, is a member of the transforming growth factor-beta (TGF-β) superfamily. GDF-8 and its putative receptors are expressed in human extravillous cytotrophoblast cells (EVTs). Although the pro-invasive effect of GDF-8 in human EVT cells has been recently reported, the underlying molecular mechanism remains largely unknown. In this study, we investigate the effects of GDF-8 on the expression of the two most important MMPs, MMP2 and MMP9, in the HTR-8/SVneo human EVT cell line. Our results show that GDF-8 significantly upregulates the expression of MMP2. The expression of MMP9 is not affected by GDF-8. Using a siRNA-mediated knockdown approach, we reveal that the stimulatory effect of GDF-8 on MMP2 expression is mediated by the ALK5-SMAD2/3 signaling pathway. Additionally, the knockdown of MMP2 attenuates the GDF-8-induced cell invasiveness. These findings deepen our understanding of the biological roles of GDF-8 in the regulation of human trophoblast cell invasion.
Kun Lei, Quanwei Wei, Ying Cheng, Zhe Wang, Haoze Wu, Fang Zhao, Wei Ding, and Fangxiong Shi
The apoptosis of granulosa cells (GCs) is the main reason for porcine follicular atresia. This study provides a novel mechanism for peroxynitrite anion-mediated GC apoptosis and follicular atresia in porcine ovary.
Granulosa cells play a crucial role in the development of follicles, and their cell apoptosis in the porcine ovary is a major contributor to follicular atresia. Here, we provide a new mechanism for follicular atresia by describing a crucial mechanism by which peroxynitrite anion (OONO–) may cause GC death. We discovered that nitric oxide, oxidative stress level, and OONO– were positively correlated with porcine follicular atresia, which was accompanied by high expression of matrix metalloproteinase 2 (MMP2) and MMP9. We created a model of OONO–-induced apoptosis in GCs and discovered that OONO– could boost the expression of MMP2 and MMP9 and increase the expression of pro-apoptotic proteins and DNA damage. Furthermore, by inhibiting the activities of MMP2 and MMP9, we found that SB-3CT (a specific inhibitor for MMP2 and MMP9) alleviated the decrease in cell survival rates and DNA damage caused by OONO–, which may have been impacted by reducing the cleavage of PARP1 by MMP2 and MMP9. Therefore, our findings imply that OONO– can cause DNA damage to GCs, participating in mediating the expression of pro-apoptotic proteins and inhibiting DNA repair by preventing the activity of PARP1 through MMP2 and MMP9. These results help explain how OONO–/MMP2/MMP9 affects porcine follicular atresia and GC apoptosis.
Rui Chen, Jian Du, Lin Ma, Li-qing Wang, Sheng-song Xie, Chang-ming Yang, Xian-yong Lan, Chuan-ying Pan, and Wu-zi Dong
MicroRNAs (miRNAs) are 18–24 nucleotides non-coding RNAs that regulate gene expression by post-transcriptional suppression of mRNA. The Chinese giant salamander (CGS, Andrias davidianus), which is an endangered species, has become one of the important models of animal evolution; however, no miRNA studies on this species have been conducted. In this study, two small RNA libraries of CGS ovary and testis were constructed using deep sequencing technology. A bioinformatics pipeline was developed to distinguish miRNA sequences from other classes of small RNAs represented in the sequencing data. We found that many miRNAs and other small RNAs such as piRNA and tsRNA were abundant in CGS tissue. A total of 757 and 756 unique miRNAs were annotated as miRNA candidates in the ovary and testis respectively. We identified 145 miRNAs in CGS ovary and 155 miRNAs in CGS testis that were homologous to those in Xenopus laevis ovary and testis respectively. Forty-five miRNAs were more highly expressed in ovary than in testis and 21 miRNAs were more highly expressed in testis than in ovary. The expression profiles of the selected miRNAs (miR-451, miR-10c, miR-101, miR-202, miR-7a and miR-499) had their own different roles in other eight tissues and different development stages of testis and ovary, suggesting that these miRNAs play vital regulatory roles in sexual differentiation, gametogenesis and development in CGS. To our knowledge, this is the first study to reveal miRNA profiles that are related to male and female CGS gonads and provide insights into sex differences in miRNA expression in CGS.
Shijia Ying, Ziyu Wang, Changlong Wang, Haitao Nie, Dongyang He, Ruoxin Jia, Yongcong Wu, Yongjie Wan, Zhengrong Zhou, Yibo Yan, Yanli Zhang, and Feng Wang
This study investigated the effects of short-term food restriction or supplementation on folliculogenesis and plasma and intrafollicular metabolite and hormone concentrations. Ewes were randomly assigned to three groups: the control group received a maintenance diet (M) while the supplemented group and restricted group received 1.5×M and 0.5×M respectively on days 6–12 of their estrous cycle. Estrus was synchronized by intravaginal progestogen sponges for 12 days. On days 7–12, blood samples were taken. After slaughter, the ovarian follicles were classified and the follicular fluid was collected. Compared with restriction, supplementation shortened the estrous cycle length, decreased the number of follicles 2.5–3.5 mm and follicular fluid estradiol (E2) concentration, increased the number of follicles >3.5 mm and plasma glucose, insulin and glucagon concentrations, and augmented the volume of follicles >2.5 mm. Restricted ewes had higher intrafollicular insulin concentration, but it was similar to that of supplemented ewes. Compared with follicles ≤2.5 mm, the intrafollicular glucose and E2 concentrations were increased and the testosterone, insulin, and glucagon concentrations and lactate dehydrogenase (LDH) activity were decreased in follicles >2.5 mm. Only in restricted ewes were intrafollicular LDH and testosterone concentrations in follicles ≤2.5 mm not different from those in follicles ≤2.5 mm. In conclusion, the mechanism by which short-term dietary restriction inhibits folliculogenesis may involve responses to intrafollicular increased E2, testosterone, and LDH levels in late-stage follicles. This may not be due to the variation of intrafollicular insulin level but rather due to decreased circulating levels of glucose, insulin, and glucagon.