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Hui Wang, Yansong Xue, Baolin Wang, Junxing Zhao, Xu Yan, Yan Huang, Min Du, and Mei-Jun Zhu

Accompanying the dramatic increase in maternal obesity, the incidence of type 1 diabetes (T1D) in children is also rapidly increasing. The objective of this study was to explore the effects of maternal obesity on the incidence of T1D in offspring using non-obese diabetic (NOD) mice, a common model for TID. Four-week-old female NOD mice were fed either a control diet (10% energy from fat, CON) or a high-fat diet (60% energy from fat) for 8 weeks before mating. Mice were maintained in their respective diets during pregnancy and lactation. All offspring mice were fed the CON to 16 weeks. Female offspring (16-week-old) born to obese dams showed more severe islet lymphocyte infiltration (major manifestation of insulitis) (P<0.01), concomitant with elevated nuclear factor kappa-light-chain-enhancer of activated B cells p65 signaling (P<0.01) and tumor necrosis factor alpha protein level (P<0.05) in the pancreas. In addition, maternal obesity resulted in impaired (P<0.05) glucose tolerance and lower (P<0.05) serum insulin levels in offspring. In conclusion, maternal obesity resulted in exacerbated insulitis and inflammation in the pancreas of NOD offspring mice, providing a possible explanation for the increased incidence of T1D in children.

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Dong Han, Xin-Yan Cao, Hui-Li Wang, Jing-Jing Li, Yan-Bo Wang, and Jing-He Tan

Although studies suggest that the low competence of oocytes from prepubertal animals is due to their insufficient cytoplasmic maturation and that FSH improves oocyte maturation possibly by retarding meiotic progression and allowing more time for cytoplasmic maturation, the mechanisms by which puberty and gonadotropins regulate meiotic progression require additional detailed studies. For the first time, we observed that while meiotic progression was significantly slower, the maturation-promoting factor (MPF) activity of oocytes was significantly higher in prepubertal than in adult mice. To resolve this contradiction, we specified the molecules regulating the MPF activity and their localization during oocyte maturation in prepubertal and adult mice primed with or without gonadotropins. Our tests using corresponding enzyme regulators suggested that while activities of protein kinase A were unaffected, the activity of adenylate cyclase (ADCY) and phosphodiesterase increased while cell division cycle 2 homolog A (CDC2A) decreased significantly after puberty. While most of the adult oocytes had CDC2A protein concentrated in the germinal vesicle (GV) region, the majority of prepubertal oocytes showed no nuclear concentration of CDC2A. Maximally priming mice with equine chorionic gonadotropin brought the above parameters of prepubertal oocytes close to those in adult oocytes. Together, the results suggest that puberty and gonadotropin control oocyte meiotic progression mainly by regulating the ADCY activity and the concentration of the activated MPF toward the GV region.

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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.

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Qian Zhang, Song Yu, Xing Huang, Yi Tan, Cheng Zhu, Yan-Ling Wang, Haibin Wang, Hai-Yan Lin, Jiejun Fu, and Hongmei Wang

Cullin 3 (CUL3), a scaffold protein, assembles a large number of ubiquitin ligase complexes, similar to Skp1-Cullin 1-F-box protein complex. Several genetic models have shown that CUL3 is crucial for early embryonic development. Nevertheless, the role of CUL3 in human trophoblast function remains unclear. In this study, immunostaining revealed that CUL3 was strongly expressed in the villous cytotrophoblasts, the trophoblast column, and the invasive extravillous trophoblasts. Silencing CUL3 significantly inhibited the outgrowth of villous explant ex vivo and decreased invasion and migration of trophoblast HTR8/SVneo cells. Furthermore, CUL3 siRNA decreased pro-MMP9 activity and increased the levels of TIMP1 and 2. We also found that the level of CUL3 in the placental villi from pre-eclamptic patients was significantly lower as compared to that from their gestational age-matched controls. Moreover, in the lentiviral-mediated placenta-specific CUL3 knockdown mice, lack of CUL3 resulted in less invasive trophoblast cells in the maternal decidua. Taken together, these results suggest an essential role for CUL3 in the invasion and migration of trophoblast cells, and dysregulation of its expression may be associated with the onset of pre-eclampsia.

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Xiaohui Cui, Yan Sun, Xiuge Wang, Chunhong Yang, Zhihua Ju, Qiang Jiang, Yan Zhang, Jinming Huang, Jifeng Zhong, Miao Yin, and Changfa Wang

The micromolar calcium-activated neutral protease gene (CAPN1) is a physiological candidate gene for sperm motility. However, the molecular mechanisms involved in regulating the expression of the CAPN1 gene in bulls remain unknown. In this study, we investigated the expression pattern of CAPN1 in testis, epididymis, and sperm at the RNA and protein levels by qRT-PCR, western blot, immunohistochemistry, and immunofluorescence assay. Results revealed that the expression of CAPN1 levels was higher in the sperm head compared with that in other tissues. Moreover, we identified a novel single-nucleotide polymorphism (g.-1256 A>C, ss 1917715340) in the noncanonical core promoter of the CAPN1 gene between base g.-1306 and g.-1012. Additionally, we observed greater sperm motility in bulls with the genotype CC than in those with the genotype AA (P<0.01), indicating that different genotypes were associated with the bovine semen trait. Furthermore, a higher fluorescence intensity of the C allele than that of the A allele at g. -1256 A>C was revealed by transient transfection in MLTC-1 cells and luciferase report assay. Finally, CAPN1 was highly expressed in the spermatozoa with the CC genotype compared with that with the AA genotype by qRT-PCR. This study is the first report on genetic variant g.-1256 A>C in the promoter region of CAPN1 gene association with the semen quality of Chinese Holstein bulls by influencing its expression. g.-1256 A>C can be a functional molecular marker in cattle breeding.

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Yue Li, Ru Zheng, Rui Wang, Xiaoyin Lu, Cheng Zhu, Hai-Yan Lin, Hongmei Wang, Xiaoguang Yu, and Jiejun Fu

The placenta has numerous functions, such as transporting oxygen and nutrients and building the immune tolerance of the fetus. Cell fusion is an essential process for placental development and maturation. In human placental development, mononucleated cytotrophoblast (CTB) cells can fuse to form a multinucleated syncytiotrophoblast (STB), which is the outermost layer of the placenta. Nephrin is a transmembrane protein that belongs to the Ig superfamily. Previous studies have shown that nephrin contributes to the fusion of myoblasts into myotubes in zebrafish and mice, presenting a functional conservation with its Drosophila ortholog sticks and stones. However, whether nephrin is involved in trophoblast syncytialization remains unclear. In this study, we report that nephrin was localized predominantly in the CTB cells and STB of human placenta villi from first trimester to term pregnancy. Using a spontaneous fusion model of primary CTB cells, the expression of nephrin was found to be increased during trophoblast cell fusion. Moreover, the spontaneous syncytialization and the expression of syncytin 2, connexin 43, and human chorionic gonadotropin beta were significantly inhibited by nephrin-specific siRNAs. The above results demonstrate that nephrin plays an important role in trophoblast syncytialization.

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Zhen Teng, Chao Wang, Yijing Wang, Kun Huang, Xi Xiang, Wanbao Niu, Lizhao Feng, Lihua Zhao, Hao Yan, and Hua Zhang

The reserve of primordial follicles determines the reproductive ability of the female mammal over its reproductive life. The primordial follicle is composed of two types of cells: oocytes and surrounding pre-granulosa cells. However, the underlying mechanism regulating primordial follicle assembly is largely undefined. In this study, we found that gap junction communication (GJC) established between the ovarian cells in the perinatal mouse ovary may be involved in the process. First, gap junction structures between the oocyte and surrounding pre-granulosa cells appear at about 19.0 dpc (days post coitum). As many as 12 gap junction-related genes are upregulated at birth, implying that a complex communication may exist between ovarian cells, because specifically silencing the genes of individual gap junction proteins, such as Gja 1, Gja4 or both, has no influence on primordial follicle assembly. On the other hand, non-specific blockers of GJC, such as carbenoxolone (CBX) and 18α-glycyrrhetinic acid (AGA), significantly inhibit mouse primordial follicle assembly. We proved that the temporal window for establishment of GJC in the fetal ovary is from 19.5 dpc to 1 dpp (days postpartum). In addition, the expression of ovarian somatic cell (OSC)-specific genes, such as Notch2, Foxl2 and Irx3, was negatively affected by GJC blockers, whereas oocyte-related genes, such as Ybx2, Nobox and Sohlh1, were hardly affected, implying that the establishment of GJC during this period may be more important to OSCs than to oocytes. In summary, our results indicated that GJC involves in the mouse primordial follicle assembly process at a specific temporal window that needs Notch signaling cross-talking.

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Yuan Yuan, Ling Zhao, Xiaoying Wang, Feng Lian, and Yan Cai

Preeclampsia (PE), a serious complication of pregnancy, is associated with abnormal trophoblast cell differentiation and autophagy. Herein, we investigated the molecular mechanism underlying the function of ligustrazine (2,3,5,6-tetramethylpyrazine, TMP), a constituent of the traditional Chinese plant medicine Ligusticum wallichii, in PE. Lipopolysaccharide (LPS) was applied to induce a PE rat model, followed by tail vein injection of TMP or lentiviral vector overexpressing microRNA-16-5p (miR-16-5p). Human trophoblast cell line JEG3 was cultured in vitro to construct a PE cell model, followed by t he treatment with different concentrations of TMP, miR-16-5p mimic/inhibitor, or shRNA (shRNA) against insulin growth factor-2 (IGF-2) (sh-IGF-2). Formation of autophagosomes and autophagy-related proteins were then examined. Cell counting kit-8 (CCK-8) and Transwell assays were applied to measure trophoblast cell viability and migration. The binding affinity between miR-16-5p and IGF-2 was verified by dual luciferase report assay. After TMP treatment, autophagosome formation was reduced in trophoblast cells of placental tissue of PE rats, along with downregulation of autophagy-related proteins Light Chain 3 (LC3)-II/LC3-I, Beclin1 (BECN1), and SQSTM1. Moreover, TMP repressed JEG3 cell autophagy, promoted viability and migration concentration-responsively. MiR-16-5p was upregulated in PE, and TMP inhibited miR-16-5p expression. Besides, miR-16-5p downregulated IGF-2 expression to promote cell autophagy and inhibit the viability and migration of JEG3 cells. Further, in vivo experiments validated that TMP impeded PE progression in rats by regulating the miR-16-5p/IGF-2 axis. In summary, TMP inhibits trophoblast cell autophagy and promotes its viability and migration in PE rat model through regulating the miR-16-5p/IGF-2 axis.

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Chulin Yu, Meiling Li, Yue Wang, Ying Liu, Chengzhi Yan, Jirong Pan, Jiali Liu, and Sheng Cui

The corticotropin-releasing hormone (CRH) signaling system is involved in numbers of stress-related physiological and pathological responses, including its inhibiting effects on estradiol (E2) synthesis and follicular development in the ovary. In addition, there are reports that microRNAs (miRNAs) can control the function of animal reproductive system. The aim of present study was to investigate the functions of miR-375 and the relationship between miR-375 and CRH signaling molecules in the porcine ovary. First, our common PCR results show that miR-375 and the CRH receptor 1 (CRHR1) are expressed in porcine ovary, whereas CRH receptor 2 (CRHR2) is not detected. We further have located the cell types of miR-375 and CRHR1 by in situ hybridization (ISH), and the results show that miR-375 is located only in the granulosa cells, whereas CRHR1 is positive in all of granulosa cells and oocytes, inferring that miR-375 and CRHR1 are co-localized in granulosa cells. Second, we show that overexpression of miR-375 in cultured granulosa cells suppresses the E2 production, whereas miR-375 knockdown demonstrates the opposite result. Besides, our in vitro results demonstrate that miR-375 mediates the signaling pathway of CRH inhibiting E2 synthesis. Finally, our data show that the action of miR-375 is accomplished by directly binding to the 3′UTR of specificity protein1 (SP1) mRNA to decrease the SP1 protein level. Thus, we conclude that miR-375 is a key factor in regulating E2 synthesis by mediating the CRH signaling pathway.

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Keqin Yan, Dingqing Feng, Jing Liang, Qing Wang, Lin Deng, Xiao Zhang, Bin Ling, and Daishu Han

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.