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Lihua Yao Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Mingyang Li Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Jingwen Hu Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Wangsheng Wang Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Minzhi Gao Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Polycystic ovary syndrome (PCOS) is a major cause of infertility in women of reproductive age. However, its exact etiology remains unknown. In this study, we sequenced miRNAs in human follicular fluid and identified 16 downregulated and 3 upregulated miRNAs in PCOS group compared with non-PCOS group. Among the differential expressed miRNAs, miR-335-5p was verified lower abundance in PCOS than non-PCOS group using quantitative real-time PCR. Besides, miR-335-5p negatively correlated with antral follicle count, anti-Müllerian hormone and total testosterone. Bioinformatics analysis identified serum/glucocorticoid-regulated kinase family member 3 (SGK3) as a potential target gene of miR-335-5p. SGK3 is involved in protein kinase B-mammalian target of rapamycin kinase (AKT-mTOR) signaling pathway and cell proliferation. Western blotting and cell counting kit-8 assays demonstrated that miR-335-5p mimic reduced, while miR-335-5p inhibitor increased, SGK3 abundance, AKT-mTOR pathway and cell proliferation in human granulosa-like tumor KGN cells. Dual-luciferase reporter assays showed that miR-335-5p binds to the 3′ untranslated region of SGK3 mRNA. Furthermore, miR-335-5p was decreased and SGK3 was elevated in human granulosa cells obtained from PCOS patients as compared with non-PCOS controls. These findings suggested that miR-335-5p is involved in granulosa cells proliferation by reducing SGK3 expression, which might provide a molecular target to improve dysfunctional granulosa cells in patients with PCOS.

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Yeping Wang Department of Obstetrics and Gynecology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China
Department of Obstetrics and Gynecology, Wenzhou People’s Hospital, Wenzhou Maternal and Child Health Care Hospital, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang, China

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Hongping Zhang Department of Obstetrics and Gynecology, Wenzhou People’s Hospital, Wenzhou Maternal and Child Health Care Hospital, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang, China

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Yuehui Zhang Department of Obstetrics and Gynecology, Wenzhou People’s Hospital, Wenzhou Maternal and Child Health Care Hospital, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang, China

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Xiaoqing Li Laboratory of Obstetrics and Gynecology, Wenzhou People’s Hospital, Wenzhou Maternal and Child Health Care Hospital, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang, China

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Xianqing Hu Department of Obstetrics and Gynecology, Wenzhou People’s Hospital, Wenzhou Maternal and Child Health Care Hospital, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang, China

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Xietong Wang Department of Obstetrics and Gynecology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China
Maternal and Child Health Care Hospital of Shandong Province, Jinan, Shandong, China
Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Jinan, Shandong, China

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Decorin (DCN) regulates a vast array of cellular processes including proliferation, migration, apoptosis, and autophagy, and its aberrant expression has been associated with poor extravillous trophoblasts (EVT) invasion of the uterus, which underlies the occurrence of preeclampsia (PE) and intrauterine growth restriction (IUGR). In this study, we aim to elucidate the molecular mechanism of how the DCN regulates the cell functions through the use of trophoblast cell line, HTR-8. Using a series of cell function assays, including CCK8, RTCA, transwell, scratch-wound assay, and Annexin V staining, we found that DCN suppressed proliferation and invasion, while promoted autophagy and apoptosis of HTR-8 in a dose-dependent manner. Transient stimulation of DCN have increased the activity of c-Met and its downstream effectors – Akt, FAK and m-TOR. However, a prolonged exposure to DCN have significantly downregulated the expression of c-Met, leading to suppression of its downstream effectors. Lentivirus that overexpressed c-Met targeting shRNA was used to knockdown c-Met expression and crizotinib was used to selectively inhibit the kinase activity of c-Met in HTR-8 cells. A combination of DCN and c-Met knockdown/inhibition have reduced the proliferation and invasion in HTR-8 cells; however, DCN-induced autophagy and apoptosis were not synergistically enhanced by c-Met inhibition. In conclusion, DCN promotes autophagy and apoptosis predominantly through downregulating c-Met/Akt/mTOR activity in human trophoblasts.

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Yan Shi Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, China

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Bingjie Hu Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, China

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Zizengchen Wang Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, China

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Xiaotong Wu Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, China

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Lei Luo Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, China

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Shuang Li Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, China

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Shaohua Wang Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, China

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Kun Zhang Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, China

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Huanan Wang Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, China

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In brief

The lineage specification during early embryonic development in cattle remains largely elusive. The present study determines the effects of trophectoderm-associated factors GATA3 and CDX2 on lineage specification during bovine early embryonic development.

Abstract

Current understandings of the initiation of the trophectoderm (TE) program during mammalian embryonic development lack evidence of how TE-associated factors such as GATA3 and CDX2 participate in bovine lineage specification. In this study, we describe the effects of TE-associated factors on the expression of lineage specification marker genes such as SOX2, OCT4, NANOG, GATA6, and SOX17, by using cytosine base editor system. We successfully knockout GATA3 or CDX2 in bovine embryos with a robust efficiency. However, GATA3 or CDX2 deletion does not affect the developmental potential of embryos to reach the blastocyst stage. Interestingly, GATA3 deletion downregulates the NANOG expression in bovine blastocysts. Further analysis of the mosaic embryos shows that GATA3 is required for NANOG in the TE of bovine blastocysts. Single blastocyst RNA-seq analysis reveals that GATA3 deletion disrupts the transcriptome in bovine blastocysts. Altogether, we propose that GATA3 plays an important role in maintaining TE lineage program in bovine embryos and the functional role of GATA3 is species-specific.

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Kun Li Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China

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Yue Liu Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China

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Xiaoyu Xia Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China

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Li Wang Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China

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Meige Lu Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China

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Yanqin Hu Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China

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Chen Xu Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China

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Bactericidal/permeability-increasing protein (BPI) is a 455-residue (∼55 kDa) protein found mainly in the primary (azurophilic) granules of human neutrophils. BPI is an endogenous antibiotic protein that belongs to the family of mammalian lipopolysaccharide (LPS)-binding and lipid transport proteins. Its major function is to kill Gram-negative bacteria, thereby protecting the host from infection. In addition, BPI can inhibit angiogenesis, suppress LPS-mediated platelet activation, increase DNA synthesis, and activate ERK/Akt signaling. In this study, we found that Bpi was expressed in the testis and epididymis but not in the seminal vesicles, prostate, and solidification glands. BPI expression in the epididymis increased upon upregulation of testosterone, caused by injection of GNRH. In orchidectomized mice, BPI expression was significantly reduced, but its expression was restored to 30% of control levels in orchidectomized mice that received supplementary testosterone. The number of sperm fused per egg significantly decreased after incubation with anti-BPI antiserum. These results suggest that BPI may take part in the process of sperm–oocyte fusion and play a unique and significant role in reproduction.

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Niuyi Zheng Department of Anatomy, Basic Medical College, Xuzhou Medical University, Xuzhou, Jiangsu, China.
Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogen Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu, China.

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Chaolong Wang Department of Anatomy, Basic Medical College, Xuzhou Medical University, Xuzhou, Jiangsu, China.
Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogen Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu, China.

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Yiqiu Li Department of Anatomy, Basic Medical College, Xuzhou Medical University, Xuzhou, Jiangsu, China.
Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogen Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu, China.

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Haiying Fu Department of Anatomy, Basic Medical College, Xuzhou Medical University, Xuzhou, Jiangsu, China.
Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogen Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu, China.

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Tao Hu Department of Anatomy, Basic Medical College, Xuzhou Medical University, Xuzhou, Jiangsu, China.
Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogen Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu, China.

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In brief

Brown adipose tissue impaired in polycystic ovary syndrome (PCOS) plays a crucial role in the treatment of PCOS. This study shows that myricetin potently improves PCOS by activating brown adipose tissue (BAT).

Abstract

PCOS is a complex endocrine disease characterized by hyperandrogenism, anovulation and polycystic ovary, and is often accompanied by metabolic disorder such as insulin resistance. BAT has been considered as a promising target for the treatment of obesity and other metabolic disease. In this study, we showed that 3 weeks of myricetin (a compound from natural product) treatment improved metabolic capacity and insulin sensitivity by activating BAT in dehydroepiandrosterone (DHEA)-induced PCOS mice. Furthermore, increased number of corpus luteum and decreased cystic formation were observed in PCOS mice. With the hormone levels such as luteinizing hormone (LH) were reversed, estrous cycle was also normalized after myricetin treatment. Eventually, myricetin markedly improved reproductive defects in PCOS mice. In short, our results suggest that myricetin treatment dramatically ameliorates ovarian dysfunction and metabolic disturbances in PCOS and provides a novel perspective for the treatment of PCOS.

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Xin Wen Department of Gynecology and Obstetrics, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
Department of Pediatric Urology, Key Laboratory of Children Genitourinary Diseases of Wenzhou City, Key Laboratory of Structural Malformations in Children of Zhejiang Province, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Jiexia Wang Department of Gynecology and Obstetrics, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
Department of Pediatric Urology, Key Laboratory of Children Genitourinary Diseases of Wenzhou City, Key Laboratory of Structural Malformations in Children of Zhejiang Province, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Mengjie Qin Department of Pharmacology, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Hu Wang Zhejiang Provincial Key Laboratory of Anesthesiology, Department of Anesthesiology, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Jingfeng Xu Zhejiang Provincial Key Laboratory of Anesthesiology, Department of Anesthesiology, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Xiaoju Guan Department of Pediatric Urology, Key Laboratory of Children Genitourinary Diseases of Wenzhou City, Key Laboratory of Structural Malformations in Children of Zhejiang Province, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Dan Shan Department of Gynecology and Obstetrics, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Panpan Chen Department of Gynecology and Obstetrics, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Jiajia Xie Department of Pharmacology, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Jingjing Shao Department of Pharmacology, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Ping Duan Department of Gynecology and Obstetrics, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Congde Chen Department of Pediatric Urology, Key Laboratory of Children Genitourinary Diseases of Wenzhou City, Key Laboratory of Structural Malformations in Children of Zhejiang Province, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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Haolin Chen Department of Gynecology and Obstetrics, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
Department of Pediatric Urology, Key Laboratory of Children Genitourinary Diseases of Wenzhou City, Key Laboratory of Structural Malformations in Children of Zhejiang Province, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
Department of Pharmacology, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China
Zhejiang Provincial Key Laboratory of Anesthesiology, Department of Anesthesiology, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

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In brief

Progenitor cells with ovulation-related tissue repair activity were identified with defined markers (LGR5, EPCR, LY6A, and PDGFRA), but their potentials to form steroidogenic cells were not known. This study shows that the cells can generate progenies with different steroidogenic activities.

Abstract

Adult mammalian ovaries contain stem/progenitor cells necessary for folliculogenesis and ovulation-related tissue rupture repair. Theca cells are recruited and developed from progenitors during the folliculogenesis. Theca cell progenitors were not well defined. The aim of current study is to compare the potentials of four ovarian progenitors with defined markers (LY6A, EPCR, LGR5, and PDGFRA) to form steroidogenic theca cells in vitro. The location of the progenitors with defined makers was determined by immunohistochemistry and immunofluorescence staining of ovarian sections of adult mice. Different progenitor populations were purified by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) techniques from ovarian cell preparation and were tested for their abilities to generate steroidogenic theca cells in vitro. The cells were differentiated with a medium containing LH, ITS, and DHH agonist for 12 days. The results showed that EPCR+ and LGR5+ cells primarily distributed along the ovarian surface epithelium (OSE), while LY6A+ cells distributed in both the OSE and parenchyma. However, PDGFRA+ cells were exclusively located in interstitial compartment. When the progenitors were purified by these markers and differentiated in vitro, LY6A+ and PDGFRA+ cells formed steroidogenic cells expressing both CYP11A1 and CYP17A1 and primarily producing androgens, showing characteristics of theca-like cells, while LGR5+ cells generated steroidogenic cells devoid of CYP17A1 expression and androgen production, showing a characteristic of progesterone-producing cells (granulosa- or lutea-like cells). In conclusion, progenitors from both OSE and parenchyma of adult mice are capable of generating steroidogenic cells with different steroidogenic capacities, showing a possible lineage preference.

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Xiaotong Wu Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Yan Shi Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Bingjie Hu Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Panpan Zhao Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Shuang Li Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Lieying Xiao Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Shaohua Wang Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Kun Zhang Key Laboratory of Dairy Cow Genetic Improvement and Milk Quality Research of Zhejiang Province, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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In brief

Lineage specification plays a vital role in preimplantation development. TEAD4 is an essential transcription factor for trophectoderm lineage specification in mice but not in cattle.

Abstract

Tead4, a critical transcription factor expressed during preimplantation development, is essential for the expression of trophectoderm-specific genes in mice. However, the functional mechanism of TEAD4 in mouse preimplantation development and its conservation across mammals remain unclear. Here, we report that Tead4 is a crucial transcription factor necessary for blastocyst formation in mice. Disruption of Tead4 through base editing results in developmental arrest at the morula stage. Additionally, RNA-seq analysis reveals dysregulation of 670 genes in Tead4 knockout embryos. As anticipated, Tead4 knockout led to a decrease in trophectoderm genes Cdx2 and Gata3. Intriguingly, we observed a reduction in Krt8, suggesting that Tead4 influences the integrity of the trophectoderm epithelium in mice. More importantly, we noted a dramatic decrease in nuclear Yap in outside cells for Tead4-deficient morula, indicating that Tead4 directly regulates Hippo signaling. In contrast, bovine embryos with TEAD4 depletion could still develop to blastocysts with normal expression of CDX2, GATA3, and SOX2, albeit with a decrease in total cell number and ICM cell number. In conclusion, we propose that Tead4 regulates mouse blastocyst formation via Krt8 and Yap, both of which are critical regulators of mouse preimplantation development.

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Xing Su Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China
Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China

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Yi Hu Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China
Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China

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Ying Li Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China
Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China

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Jing-Li Cao Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China
Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China

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Xue-Qin Wang Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China
Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China

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Xu Ma Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China
Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China

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Hong-Fei Xia Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China
Reproductive and Genetic Center of National Research Institute for Family Planning, Graduate School, Beijing 100081, China

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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.

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Xiaoli Chen The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China
The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Huabin Zhu The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Chuanhuo Hu The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Haisheng Hao The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Junfang Zhang The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Kunpeng Li The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China
The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Xueming Zhao The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Tong Qin The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Kan Zhao The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Huishan Zhu The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Dong Wang The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Animal Science and Technology College, Jilin Agriculture University, Beijing Protein Innovation Co., Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China

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Cryodamage is a major problem in semen cryopreservation, causing changes in the levels of proteins that influence the function and motility of spermatozoa. In this study, protein samples prepared from fresh and frozen–thawed boar spermatozoa were compared using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled to 2D LC–MS/MS analysis. A total of 41 differentially expressed proteins were identified and quantified, including 35 proteins that were present at higher levels and six proteins that were present at lower levels in frozen–thawed spermatozoa by at least a mean of 1.79-fold (P<0.05). On classifying into ten distinct categories using bioinformatic analysis, most of the 41 differentially expressed proteins were found to be closely relevant to sperm premature capacitation, adhesions, energy supply, and sperm–oocyte binding and fusion. The expression of four of these proteins, SOD1, TPI1, ODF2, and AKAP3, was verified by western blot analysis. We propose that alterations in these identified proteins affect the quality of cryopreserved semen and ultimately lower its fertilizing capacity. This is the first study to compare protein levels in fresh and frozen–thawed spermatozoa using the iTRAQ technology. Our preliminary results provide an overview of the molecular mechanisms of cryodamage in frozen–thawed spermatozoa and theoretical guidance to improve the cryopreservation of boar semen.

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Li-Juan Xiao State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China and Department of Physiology, the Cardiovascular Research Group, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5

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Jin-Xiang Yuan State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China and Department of Physiology, the Cardiovascular Research Group, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5

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Yin-Chuan Li State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China and Department of Physiology, the Cardiovascular Research Group, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5

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Rui Wang State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China and Department of Physiology, the Cardiovascular Research Group, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5

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Zhao-Yuan Hu State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China and Department of Physiology, the Cardiovascular Research Group, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5

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Yi-Xun Liu State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China and Department of Physiology, the Cardiovascular Research Group, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5

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The extracellular Ca2+-sensing receptor (CaR) is a member of the superfamily of G protein-coupled receptors (GPCRs). It is an important mediator of a wide range of Ca2+-dependent physiological responses in various tissues. In reproductive tissues it has been reported to play a significant role in promoting or maintaining placentation. Meanwhile, another Ca2+ regulated gene stanniocalcin-1 (STC-1) has been documented to be involved in decidualization and uterine remodelling. The phenomenon that CaR mediates STC-1’s transcription responding to extracellular calcium in fish urges us to suppose that CaR, like STC-1, may also play a role in implantation and decidualization. To resolve this conjecture, we have examined the expression and hormonal regulation of the CaR gene in rat uterus during peri-implantation period.

CaR mRNA was expressed at a moderate level in the luminal epithelium of the early stage of pregnancy (from day 1 to day 3). From day 2–3 it began to be expressed more strongly in the stromal cells immediately underneath the luminal epithelium, but decreased to a basal level on day 4. From day 6 to day 9 continuously, both CaR mRNA and protein were highly expressed in the primary decidua. Expression of CaR mRNA and protein in these cells was also observed when a delayed implantation was terminated by estrogen treatment to allow the embryo implantation. In contrast, only basal level expression of the molecules was detected in the cells of animals subjected to a normal-delayed implantation or the pseudopregnant condition.

Embryo transplantation experiment confirmed that CaR expression at the implantation site was induced by the implanting blastocyst. Consistent with the normal pregnant process, CaR mRNA and protein in the cells were also induced by an artificial decidualization procedure. Further experiments demonstrated that treatment of the ovariectomized rat with estrogen or/and progesterone stimulated a high level expression of CaR mRNA in the uterine epithelial and glandular epithelium. In conclusion, CaR was specifically induced during the processes of implantation and subsequent decidualization and may play a role in these processes.

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