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Bo Zheng, Jun Yu, Yueshuai Guo, Tingting Gao, Cong Shen, Xi Zhang, Hong Li and Xiaoyan Huang

The cellular nucleic acid-binding protein (CNBP), also known as zinc finger protein 9, is a highly conserved zinc finger protein that is strikingly conserved among vertebrates. Data collected from lower vertebrates showed that CNBP is expressed at high levels and distributed in the testes during spermatogenesis. However, the location and function of CNBP in mammalian testes are not well known. Here, by neonatal mouse testis culture and spermatogonial stem cells (SSC) culture methods, we studied the effect of CNBP knockdown on neonatal testicular development. Our results revealed that CNBP was mainly located in the early germ cells and Sertoli cells. Knockdown of CNBP using morpholino in neonatal testis culture caused disruption of seminiferous tubules, mislocation of Sertoli cells and loss of germ cells, which were associated with the aberrant Wnt/β-catenin pathway activation. However, knockdown of CNBP in SSC culture did not affect the survival of germ cells. In conclusion, our study suggests that CNBP could maintain testicular development by inhibiting the Wnt/β-catenin pathway, particularly by influencing Sertoli cells.

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Yuansong Yu, Jun Yong, Xiangyun Li, Tingting Qing, Han Qin, Xiaoran Xiong, Jiefang You, Mingxiao Ding and Hongkui Deng

In this study, we cloned mice from ES cells by a post-electrofusion MG132 treatment and improved development of cloned embryos with a sequential cultivation protocol. When 5 μM MG132, a proteasome inhibitor, were used to treat the reconstructed embryos, the capacity of in vitro development, implantation and full-term development were significantly improved. Blastocyst formation rates of the reconstructed embryos from X4 ES cells (F1 strain derived from C57BL/6 × 129sv) and J1 ES cells obtained with or without MG132 treatment were 66.9% and 26.6%, and 66.1% and 34.5% respectively (P < 0.05). A total of 146 two-cell embryos cloned from X4 ES cells with MG132 treatment were transferred to recipients, and five cloned pups (3.4%) were born, of which four survived. When the same numbers of two-cell embryos cloned from X4 ES cells without MG132 treatment were transferred, however, no live-born mice were obtained. When embryos cloned from J1 ES cells without MG132 treatment were cultured in KSOM medium for 54 h followed by culture in CZB medium containing 5.6 mM glucose for 42 h, the blastocyst rate was significantly higher than when they were cultured in KSOM continuously for 96 h (34.5% vs 17.1%). However, sequential cultivation did not improve the development of embryos cloned with MG132 treatment and that of parthenotes. In conclusion, MG132 treatment increased the developmental potential of reconstructed mouse embryos, and sequential cultivation improved development of the embryos cloned by electrofusion without MG132 treatment.

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Zhengkai Wei, Tingting Yu, Jingjing Wang, Chaoqun Wang, Xiao Liu, Zhen Han, Xu Zhang, Yong Zhang, Hongsheng Ouyang and Zhengtao Yang

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.