As the distal part of the crustacean male reproductive tract, terminal ampullae play important roles in sperm development and storage of mature spermatophores. In the present study, the novel gene terminal ampullae peptide (TAP) was cloned from terminal ampullae of the prawn, Macrobrachium rosenbergii. The cDNA sequence consists of 768 nucleotides, with an open-reading frame of 264 nucleotides which encodes a putative 88-amino acid precursor protein with a 17-amino acid residue signal peptide. Western blotting and immunohistochemical analysis revealed that TAP was distributed on terminal ampullae and sperm, and its expression was related to gonad development. To elucidate the functional role of TAP in vivo, we disrupted the TAP gene by RNA interference (RNAi) and evaluated the effect on fertility and several sperm parameters. Although there was no difference in fertility between RNAi-induced prawns and controls, RNAi treatment decreased the sperm gelatinolytic activity and blocked proteolytic activity on the vitelline coat. These data provide evidence that TAP participates in regulating sperm proteolytic activity, and performs a crucial role in sperm maturation and degradation of the vitelline coat during fertilization.
Wen-Ming Ma, Ye-Qing Qian, Mo-Ran Wang, Fan Yang, and Wei-Jun Yang
Shou-Bin Tang, Lei-Lei Yang, Ting-Ting Zhang, Qian Wang, Shen Yin, Shi-Ming Luo, Wei Shen, Zhao-Jia Ge, and Qing-Yuan Sun
It is demonstrated that repeated superovulation has deleterious effects on mouse ovaries and cumulus cells. However, little is known about the effects of repeated superovulation on early embryos. Epigenetic reprogramming is an important event in early embryonic development and could be easily disrupted by the environment. Thus, we speculated that multiple superovulations may have adverse effects on histone modifications in the early embryos. Female CD1 mice were randomly divided into four groups: (a) spontaneous estrus cycle (R0); (b) with once superovulation (R1); (c) with three times superovulation at a 7-day interval (R3) and (d) with five times superovulation at a 7-day interval (R5). We found that repeated superovulation remarkably decreased the fertilization rate. With the increase of superovulation times, the rate of early embryo development was decreased. The expression of Oct4, Sox2 and Nanog was also affected by superovulation in blastocysts. The immunofluorescence results showed that the acetylation level of histone 4 at lysine 12 (H4K12ac) was significantly reduced by repeated superovulation in mouse early embryos (P < 0.01). Acetylation level of histone 4 at lysine 16 (H4K16ac) was also significantly reduced in pronuclei and blastocyst along with the increase of superovulation times (P < 0.01). H3K9me2 and H3K27me3 were significantly increased in four-cell embryos and blastocysts. We further found that repeated superovulation treatment increased the mRNA level of histone deacetylases Hdac1, Hdac2 and histone methyltransferase G9a, but decreased the expression level of histone demethylase-encoding genes Kdm6a and Kdm6b in early embryos. In a word, multiple superovulations alter histone modifications in early embryos.
Xiao-Qian Meng, Ke-Gang Zheng, Yong Yang, Man-Xi Jiang, Yan-Ling Zhang, Qing-Yuan Sun, and Yun-Long Li
Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.
Yufen Zhao, Boyang Yu, Xinyu Liu, Jitu Hu, Yanyan Yang, Erge Namei, Bingxue Yang, Yue Bai, Yinghong Qian, and Haijun Li
Although urokinase-type plasminogen activator (PLAU) and urokinase-type plasminogen activator receptor (PLAUR) have been reported to play key roles in ovarian function, their precise contribution to mammalian follicular development remains unclear. In this study, we first observed that PLAU and PLAUR were present in bovine granulosa cells (GCs). Following culture of granulosa cells with PLAU (0.5 ng/mL) and PLAUR antibody (10 µg/mL) separately and together for 24 or 48 h, a proliferation assay showed that interaction between PLAU and PLAUR contributes to bovine GC proliferation. To study the potential pathways involved in PLAU/PLAUR-induced cell proliferation, ELISA and Western blotting were performed. We found that PLAU significantly increased the ratio of phosphorylated to non-phosphorylated ERK1/2 through PLAUR signaling. Further treatment with U0126, a specific ERK1/2 phosphorylation inhibitor, markedly suppressed PLAU/PLAUR-induced ERK1/2 phosphorylation and cell proliferation. In addition, we found that PLAU and PLAUR significantly increased the intracellular cAMP level and the use of Rp-cAMP, a specific PKA inhibitor, prevented PLAU/PLAUR from promoting activation of the ERK1/2 pathway and GC proliferation. Therefore, the interaction between PLAU and PLAUR may be involved in accumulating cAMP signals and enabling MAPK/ERK1/2 activation, affecting GC proliferation. Here, we provide new mechanistic insights into the roles of PLAU and PLAUR on promoting bovine GC proliferation. The finding that potential cross-points between PLAU/PLAUR-induced intracellular signals affect GC proliferation will help in understanding the mechanisms regulating early follicular development.
Wen-Wen Gu, Long Yang, Xing-Xing Zhen, Yan Gu, Hua Xu, Miao Liu, Qian Yang, Xuan Zhang, and Jian Wang
The invasion of maternal decidua by extravillous trophoblast (EVT) is essential for the establishment and maintenance of pregnancy, and abnormal trophoblast invasion could lead to placenta-associated pathologies including early pregnancy loss and preeclampsia. SEC5, a component of the exocyst complex, plays important roles in cell survival and migration, but its role in early pregnancy has not been reported. Thus, the present study was performed to explore the functions of SEC5 in trophoblast cells. The results showed that SEC5 expression in human placental villi at first trimester was significantly higher than it was at the third trimester, and it was abundantly localized in the cytotrophoblast (CTB) and the trophoblastic column. SEC5 knockdown was accompanied by reduced migration and invasion in HTR-8/SVneo cells. In addition, the expression and plasma membrane distribution of integrin β1 was also decreased. Furthermore, shRNA-mediated knockdown of SEC5 inhibited the outgrowth of first trimester placental explants. SEC5 and InsP3R were colocalized in the cytoplasm of HTR-8/SVneo cells, and the cell-permeant calcium chelator BAPTA-AM could significantly inhibit HTR-8/SVneo cell invasion. The Ca2+ imaging results showed that the 10% fetal bovine serum-stimulated cytosolic calcium concentration ([Ca2+]c) was not only reduced by downregulated SEC5 but also was blocked by the InsP3R inhibitor. Furthermore, either the [Ca2+]c was buffered by BAPTA-AM or the knockdown of SEC5 disrupted HTR-8/SVneo cell F-actin stress fibers and caused cytoskeleton derangement. Taken together, our results suggest that SEC5 might be involved in regulating trophoblast cell migration and invasion through the integrin/Ca2+ signal pathway to induce cytoskeletal rearrangement.
Nan Meng, Xinyue Wang, Yan Shi, Yanyan Mao, Qian Yang, Baohui Ju, Qianxi Zhu, Tingting Zhang, Yan Gu, and Xuan Zhang
Decidualization is essential for the successful establishment of pregnancy, and the dysregulated decidualization may lead to early pregnancy loss. It was previously reported by us that miR-3074-5p could promote apoptosis but inhibit invasion of human extravillous trophoblast (EVT) cells in vitro, and the expression level of miR-3074-5p in villus tissues of recurrent miscarriage (RM) patients was significantly increased. The aim of this study was to preliminarily explore the role of miR-3074-5p played in the decidualization of human endometrial stromal cells (ESCs). It was found that the decidual expression level of miR-3074-5p in RM patients was remarkably higher than that in the control group. The overexpression of miR-3074-5p in the immortalized human ESC line, T-HESCs, showed suppressive effects not only on the cell proliferation, as well as the intracellular expression levels of cyclin B1 (CCNB1), CCND1 and CCNE1 but also on the in vitro-induced decidualization. CLN8 mRNA, encoding an endoplasmic reticulum (ER)-associated membrane protein, was validated to be directly targeted by miR-3074-5p. And, the expression level of CLN8 was continuously increased along with the decidualization process, whereas down-regulated CLN8 expression could inhibit the decidualization of T-HESCs in vitro. Furthermore, contrary to the increased expression level of miR-3074-5p, a significantly decreased CLN8 expression was observed in decidual tissues of RM patients. Collectively, these data suggested that an increased miR-3074-5p expression in ESCs might cause early pregnancy failure by disturbing decidualization of ESCs via the miR-3074-5p/CLN8 pathway, providing a potential diagnostic and therapeutic target for RM.