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Yali Xu Department of Histology, Embryology, Genetics and Developmental Biology, Shanghai Key Laboratory for Reproductive Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Yong Fan Department of Histology, Embryology, Genetics and Developmental Biology, Shanghai Key Laboratory for Reproductive Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Weimin Fan Department of Histology, Embryology, Genetics and Developmental Biology, Shanghai Key Laboratory for Reproductive Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Reproductive Medicine Center, Shanghai Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Jia Jing Department of Histology, Embryology, Genetics and Developmental Biology, Shanghai Key Laboratory for Reproductive Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Ke Xue Department of Histology, Embryology, Genetics and Developmental Biology, Shanghai Key Laboratory for Reproductive Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Xing Zhang Department of Clinical Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Bin Ye Department of Clinical Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Yingjie Ji Department of Clinical Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Yue Liu Department of Histology, Embryology, Genetics and Developmental Biology, Shanghai Key Laboratory for Reproductive Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Zhide Ding Department of Histology, Embryology, Genetics and Developmental Biology, Shanghai Key Laboratory for Reproductive Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Asthenozoospermia is one of the leading causes of male infertility owing to a decline in sperm motility. Herein, we determined if there is a correlation between RNASET2 content on human spermatozoa and sperm motility in 205 semen samples from both asthenozoospermia patients and normozoospermia individuals. RNASET2 content was higher in sperm from asthenozoospermia patients than in normozoospermia individuals. On the other hand, its content was inversely correlated with sperm motility as well as progressive motility. Moreover, the inhibitory effect of RNASET2 on sperm motility was induced by incubating normozoospermic sperm with RNase T2 protein. Such treatment caused significant declines in intracellular spermatozoa PKA activity, PI3K activity and calcium level, which resulted in severely impaired sperm motility, and the sperm motility was largely rescued by cAMP supplementation. Finally, protein immunoprecipitation and mass spectrometry identified proteins whose interactions with RNASET2 were associated with declines in human spermatozoa motility. AKAP4, a protein regulating PKA activity, coimmunoprecipated with RNASET2 and they colocalized with one another in the sperm tail, which might contribute to reduced sperm motility. Thus, RNASET2 may be a novel biomarker of asthenozoospermia. Increases in RNASET2 can interact with AKAP4 in human sperm tail and subsequently reduce sperm motility by suppressing PKA/PI3K/calcium signaling pathways.

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Qian Chen Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiaotong University, School of Medicine, Shanghai Key Laboratory of Reproductive Medicine, Huangpu, Shanghai, China
Center of Reproductive Medicine, Ruijin Hospital, Shanghai Jiaotong University, School of Medicine, Huangpu, Shanghai, China

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Yong Fan Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University, School of Medicine, Huangpu, Shanghai, China

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Xiaowei Zhou Center of Reproductive Medicine, Ruijin Hospital, Shanghai Jiaotong University, School of Medicine, Huangpu, Shanghai, China

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Zheng Yan Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University, School of Medicine, Huangpu, Shanghai, China

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Yanping Kuang Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University, School of Medicine, Huangpu, Shanghai, China

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Aijun Zhang Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiaotong University, School of Medicine, Shanghai Key Laboratory of Reproductive Medicine, Huangpu, Shanghai, China
Center of Reproductive Medicine, Ruijin Hospital, Shanghai Jiaotong University, School of Medicine, Huangpu, Shanghai, China

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Chen Xu Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiaotong University, School of Medicine, Shanghai Key Laboratory of Reproductive Medicine, Huangpu, Shanghai, China

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Some studies have demonstrated that the implantation rate of fresh transfer cycles is lower in the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol than in the GnRH agonist (GnRH-a) protocol during in vitro fertilization (IVF). This effect may be related to endometrial receptivity. However, the mechanisms are unclear. Here, endometrial tissues obtained from the mid-secretory phase of patients treated with GnRH-a or GnRH-ant protocols and from patients on their natural cycle were assessed. Endometrial expression of B-type creatine kinase (CKB), which plays important roles in the implantation phase, was significantly reduced in the GnRH-ant group. At the same time, expression of the endometrial receptivity marker HOXA10 was considerably reduced in the GnRH-ant group. GnRH-ant exposure in endometrial epithelial cells (EECs) in vitro decreased CKB expression and ATP generation and blocked polymerization of actin. Furthermore, in vitro GnRH-ant-exposed Ishikawa cells showed enhanced F-actin depolymerization, and these effects were rescued by CKB overexpression. Similar effects were observed after CKB knockdown, and these effects were rescued by CKB overexpression. Moreover, cell migration was decreased in CKB-knockdown Ishikawa cells compared with that in control cells, and this effect was also rescued by CKB overexpression. Overall, these findings showed that GnRH-ant affected CKB expression in EECs, resulting in cytoskeletal damage and migration failure. These results provide insight into the roles and molecular mechanisms of GnRH-ant treatment in the endometrium.

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Yang Yu State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Chenhui Ding State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Eryao Wang State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Xinjie Chen State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Xuemei Li State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Chunli Zhao State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Yong Fan State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Liu Wang State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Nathalie Beaujean State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Qi Zhou State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Alice Jouneau State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Weizhi Ji State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China, Graduate University of Chinese Academy of Sciences, Beijing, China and INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas 78350, France

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Even though it generates healthy adults, nuclear transfer in mammals remains an inefficient process. Mainly attributed to abnormal reprograming of the donor chromatin, this inefficiency may also be caused at least partly by a specific effect of the cloning technique which has not yet been well investigated. There are two main procedures for transferring nuclei into enucleated oocytes: fusion and piezoelectric microinjection, the latter being used mostly in mice. We have, therefore, decided to compare the quality and the developmental ability, both in vivo and in vitro, of embryos reconstructed with electrofusion or piezoelectric injection. In addition, the effect of piezo setups of differing electric strengths was investigated. Along with the record of the rate of development, we compared the nuclear integrity in the blastomeres during the first cleavages as well as the morphological and cellular quality of the blastocysts. Our results show that the piezo-assisted micromanipulation can induce DNA damage in the reconstructed embryos, apoptosis, and reduced cell numbers in blastocysts as well as a lower rate of development to term. Even if piezo-driven injection facilitates a faster and more efficient rate of reconstruction, it should be used with precaution and with as low parameters as possible.

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Guo-Min Zhang College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China

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Ming-Tian Deng Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China

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Zhi-Hai Lei College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China

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Yong-Jie Wan Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China

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Hai-Tao Nie Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China

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Zi-Yu Wang Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China

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Yi-Xuan Fan Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China

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Feng Wang Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China

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Yan-Li Zhang Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, China

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During goat follicular development, abnormal expression of nuclear respiratory factor 1 (NRF1) in granulosa cells may drive follicular atresia with unknown regulatory mechanisms. In this study, we investigated the effects of NRF1 on steroidogenesis and cell apoptosis by overexpressing or silencing it in goat luteinized granulosa cells (LGCs). Results showed that knockdown of NRF1 expression significantly inhibited the expression of STAR and CYP19A1, which are involved in sex steroid hormones synthesis, and led to lower estrogen levels. Knockdown of NRF1 resulted in an increased percentage of apoptosis, probably due to the release of cytochrome c from mitochondria, accompanied by upregulating mRNA and protein levels of apoptosis-related markers BAX, caspase 3 and caspase 9. These data indicate that NRF1 might be related with steroidogenesis and cell apoptosis. Furthermore, NRF1 silence reduced mitochondrial transcription factor A (TFAM) transcription activity, mtDNA copy number and ATP level. Simultaneously, knockdown of NRF1 suppressed the transcription and translation levels of SOD, GPx and CAT, decreased glutathione level and increased 8-OHdG level. However, the overexpression of NRF1 in LGCs or gain of TFAM in NRF1 silenced LGCs increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the antioxidant stress system and steroids synthesis. Taken together, aberrant expression of NRF1 could induce mitochondrial dysfunction and disturb the cellular redox balance, which lead to disturbance of steroid hormone synthesis, and trigger LGC apoptosis through the mitochondria-dependent pathway. These findings will be helpful for understanding the role of NRF1 in goat ovarian follicular development and atresia.

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