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Keqin Yan Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Dingqing Feng Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Jing Liang Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Qing Wang Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing, China

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Lin Deng Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Xiao Zhang Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Bin Ling Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Daishu Han Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing, China

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Viral infections of the ovary may perturb ovarian functions. However, the mechanisms underlying innate immune responses in the ovary are poorly understood. The present study demonstrates that cytosolic viral DNA sensor signaling initiates the innate immune response in mouse ovarian granulosa cells and affects endocrine function. The cytosolic DNA sensors p204 and cGAS and their common signaling adaptor stimulator of interferon (IFN) genes (STING) were constitutively expressed in granulosa cells. Transfection with VACV70, a synthetic vaccinia virus (VACV) DNA analog, induced the expression of type I interferons (IFNA/B) and major inflammatory cytokines (TNFA and IL6) through IRF3 and NF-κB activation respectively. Moreover, several IFN-inducible antiviral proteins, including 2′,5′-oligoadenylate synthetase, IFN-stimulating gene 15 and Mx GTPase 1, were also induced by VACV70 transfection. The innate immune responses in granulosa cells were significantly reduced by the transfection of specific small-interfering RNAs targeting p204, cGas or Sting. Notably, the VACV70-triggered innate immune responses affected steroidogenesis in vivo and in vitro. The data presented in this study describe the mechanism underlying ovarian immune responses to viral infection.

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Fang Yang
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Ye-Chun Ruan Shanghai First Maternity and Infant Hospital, Epithelial Cell Biology Research Centre, Shenzhen Research Institute, School of Medicine, Tongji University, Shanghai, China
Shanghai First Maternity and Infant Hospital, Epithelial Cell Biology Research Centre, Shenzhen Research Institute, School of Medicine, Tongji University, Shanghai, China

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Yun-jie Yang
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Kai Wang
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Shan-shan Liang
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Yi-bin Han
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Xiao-Ming Teng
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Jian-Zhi Yang
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Women with polycystic ovary syndrome (PCOS) undergoing IVF–embryo transfer based-assisted reproductive technology (ART) treatment show variable ovarian responses to exogenous FSH administration. For better understanding and control of PCOS ovarian responses in ART, the present study was carried out to compare the follicular hormones and the expression of granulosa cell genes between PCOS and non-PCOS women during ART treatment as well as their IVF outcomes. Overall, 138 PCOS and 78 non-PCOS women were recruited for the present study. Follicular fluid collected from PCOS women showed high levels of testosterone. The expression of aromatase was found significantly reduced in luteinized granulosa cells from PCOS women. In cultured luteinized granulosa cells isolated from non-PCOS women, their exposure to testosterone at a level that was observed in PCOS follicles could decrease both mRNA and protein levels of aromatase in vitro. The inhibitory effect of testosterone was abolished by androgen receptor antagonist, flutamide. These results suggest that the hyperandrogenic follicular environment may be a key hazardous factor leading to the down-regulation of aromatase in PCOS.

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Zhengkai Wei College of Life Sciences and Engineering, Foshan University, Foshan, Guangdong Province, People’s Republic of China

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Tingting Yu Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Animal Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China

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Jingjing Wang Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, People’s Republic of China

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Chaoqun Wang Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, People’s Republic of China

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Xiao Liu Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, People’s Republic of China

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Zhen Han Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, People’s Republic of China

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Xu Zhang Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, People’s Republic of China

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Yong Zhang Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, People’s Republic of China

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Hongsheng Ouyang Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Animal Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China

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Zhengtao Yang College of Life Sciences and Engineering, Foshan University, Foshan, Guangdong Province, People’s Republic of China

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Sperm motility, fertilization and embryo implantation are several important factors in reproduction. Except healthy state of sperm and embryo themselves, successful pregnancy is closely related to the status of female reproductive tract immune system. Increased immune cells in reproductive tract often leads to low sperm motility and low chance of embryo implantation, but the mechanisms remain not well clarified. The aim of this study is to investigate the direct effects of swine polymorphonuclear neutrophils (PMNs) on sperm or embryo in vitro and then try to clarify the molecular mechanisms undergoing the phenomenon. Swine sperm-triggered neutrophil extracellular traps (NETs) were observed by scanning electron microscopy (SEM). PMNs phagocytosis of sperms was examined by transmission electron microscopy (TEM). Sperm-triggered NETs were quantitated by Pico Green®. Vital staining of the interaction between PMNs and embryo were observed by using confocal microscope. It was showed that PMNs were directly activated by sperm in the form of phagocytosis or casting NETs and that sperm-triggered-NETs formation was made up with DNA co-located with citrullinated histone 3 (citH3) and myeloperoxidase (MPO). In addition, the potential mechanism of NETs release was relevant to NADPH oxidase, ERK1/2 or p38 MAPK signaling pathways. Of great interest was that swine embryo was first found entangled in NETs in vitro, but the function and mechanism of this action in vivo fertilization still needed further investigation. In conclusion, this is the first report about swine sperm-induced NETs that entangle sperm and embryo, which might provide an entirely understanding of swine reproductive physiology and immunology.

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Hui Peng College of Animal Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou, People’s Republic of China

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Haijun Liu Tianjin Institute of Animal Science and Veterinary Medicine, Tianjin, People’s Republic of China

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Fang Liu College of Animal Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou, People’s Republic of China

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Yuyun Gao College of Animal Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou, People’s Republic of China

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Jing Chen College of Animal Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou, People’s Republic of China

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Jianchao Huo College of Animal Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou, People’s Republic of China

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Jinglin Han College of Animal Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou, People’s Republic of China

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Tianfang Xiao College of Animal Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou, People’s Republic of China

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Wenchang Zhang College of Animal Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou, People’s Republic of China

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Nlrp2 is a maternal effect gene specifically expressed by mouse ovaries; deletion of this gene from zygotes is known to result in early embryonic arrest. In the present study, we identified FAF1 protein as a specific binding partner of the NLRP2 protein in both mouse oocytes and preimplantation embryos. In addition to early embryos, both Faf1 mRNA and protein were detected in multiple tissues. NLRP2 and FAF1 proteins were co-localized to both the cytoplasm and nucleus during the development of oocytes and preimplantation embryos. Co-immunoprecipitation assays were used to confirm the specific interaction between NLRP2 and FAF1 proteins. Knockdown of the Nlrp2 or Faf1 gene in zygotes interfered with the formation of a NLRP2–FAF1 complex and led to developmental arrest during early embryogenesis. We therefore conclude that NLRP2 interacts with FAF1 under normal physiological conditions and that this interaction is probably essential for the successful development of cleavage-stage mouse embryos. Our data therefore indicated a potential role for NLRP2 in regulating early embryo development in the mouse.

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Xiao Han State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Cong Zhang State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Xiangping Ma State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Xiaowei Yan State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Bohui Xiong State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Wei Shen College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Shen Yin College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Hongfu Zhang State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China

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Qingyuan Sun College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China
Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, People’s Republic of China

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Yong Zhao State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Muscarinic acetylcholine receptor (mAChR) antagonists have been reported to decrease male fertility; however, the roles of mAChRs in spermatogenesis and the underlying mechanisms are not understood yet. During spermatogenesis, extensive remodeling between Sertoli cells and/or germ cells interfaces takes place to accommodate the transport of developing germ cells across the blood-testis barrier (BTB) and adluminal compartment. The cell–cell junctions play a vital role in the spermatogenesis process. This study used ICR male mice and spermatogonial cells (C18-4) and Sertoli cells (TM-4). shRNA of control or M5 gene was injected into 5-week-old ICR mice testes. Ten days post-viral grafting, mice were deeply anesthetized with pentobarbital and the testes were collected. One testicle was fresh frozen for RNA-seq analysis or Western blotting (WB). The second testicle was fixed for immunofluorescence staining (IHF). C18-4 or TM-4 cells were treated with shRNA of control or M5 gene. Then, the cells were collected for RNA-seq analysis, WB, or IHF. Knockdown of mAChR M5 disrupted mouse spermatogenesis and damaged the actin-based cytoskeleton and many types of junction proteins in both Sertoli cells and germ cells. M5 knockdown decreased Phldb2 expression in both germ cells and Sertoli cells which suggested that Phldb2 may be involved in cytoskeleton and cell–cell junction formation to regulate spermatogenesis. Our investigation has elucidated a novel role for mAChR M5 in the regulation of spermatogenesis through the interactions of Phldb2 and cell–cell junctions. M5 may be an attractive future therapeutic target in the treatment of male reproductive disorders.

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