MiR-21-5p改善白消安诱导的睾丸功能障碍并维持精子发生

Journal of Integrative Agriculture

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MiR-21-5p改善白消安诱导的睾丸功能障碍并维持精子发生

修回日期:2024-03-07

MiR-21-5p ameliorates Busulfan-induced testicular dysfunction and maintains spermatogenesis

ZHANG Meng-fei^1*, WAN Shi-cheng^1*, CHEN Wen-bo¹, YANG Dong-hui¹, WANG Cong-liang¹, LI Ba-lun¹, Aili Aierken^{1, 4}, DU Xiao-min¹, LI Yun-xiang¹, WU Wen-ping¹, WANG Yu-qi¹, XIE Fang-de¹, LUO Xuan¹, LI Na^{1, 3}, LI Xue-ling², YANG Lei², Ahmed Hamed Arisha^{5, 6}, HUA Jin-lian^{1, 3}

¹College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling 712100, China
²Key Laboratory for Mammalian Reproductive Biology and Biotechnology, Ministry of Education, Inner Mongolia University, Hohhot 010021, China
³Key Laboratory of Livestock Biology, Northwest A&F University, Yangling 712100, China
⁴State Key Laboratory on Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Medical University, Urumqi 830011, China
⁵Department of Animal Physiology and Biochemistry, Faculty of Veterinary Medicine, Badr University in Cairo, Badry 11829, Egypt
⁶Department of Physiology and Laboratory of Biotechnology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt

Revised:2024-03-07
About author:ZHANG Meng-fei, E-mail: zmf2049@163.com; #Correspondence HUA Jin-lian, Tel: +86-029-87091117, Fax: +86-029-87091117, E-mail: jinlianhua@nwsuaf.edu.cn *These authors contributed equally to this study.

摘要/Abstract

摘要：

近年来，精子质量下降、男性不育和繁殖障碍已成为国际关注的问题，发病率高达15%。精原干细胞（SSCs）位于雄性动物睾丸曲精管的基膜中，是维持精子发生、产生精子并将遗传信息从父母传递给后代的祖细胞。SSCs在动物遗传学、家畜育种和繁殖、治疗生殖损伤和不孕不育方面至关重要。因此，研究精原干细胞的调控机制对寻找治疗雄性不育的靶点和家畜育种均具有重要意义。然而，目前缺乏关于某些miRNA是否在SSC的自我更新、分化和凋亡过程中调节其表达的相关信息，也尚不清楚山羊的SSCs中保守表达的重要miRNA的功能。为了探索参与SSCs自我更新调控的miRNA、研究miRNA的靶基因和miRNAs在疾病治疗中的作用，我们进行了本研究，并得到以下发现：

（1）两步酶消化法分离了三月龄关中奶山羊的睾丸原代细胞，随后通过MACS纯化了CD49f阳性和CD49f阴性的睾丸细胞，并使用Illumina高通量测序技术对miRNA的表达进行了深入分析。发现在已鉴定的新的和已知的miRNA中，933个miRNA在CD49f阳性细胞中上调，916个miRNAs在CD49f阴性细胞中上调。富集的miRNA和被认为属于精原细胞的基因在奶山羊的CD49f阳性睾丸细胞中高度表达，包括miR-221、miR-23a、miR-29b、miR-24、miR-29a、miR-199b、miR-199a、miR-27a、miR-21等。（2）白消安诱导的小鼠生殖损伤模型显示出miR-21-5p对于生殖损伤具有很好的治疗效果。多物种的序列比对，证实miR-21-5p在山羊和小鼠的SSCs中都高度富集。EdU染色、CCK8检测和流式细胞术显示miR-21-5p可以增强SSCs的增殖和抗凋亡能力。因此miR-21-5p可以作为SSCs多物种保守的一个标志性miRNA。（3）RNA-seq和双荧光素酶分析表明，miR-21-5p抑制SPRY1活性，参与MAPK/ERK、PI3K-AKT等信号通路，增加Cyclin E表达，抑制P21表达，促进细胞周期，加速SSCs的细胞增殖。另一方面，miR-21-5p靶向并抑制FASLG的表达，以降低RHOB和P53的表达水平，从而增加BCL2的表达，降低活化型Caspase3/9的表达，抑制细胞凋亡，并降低SSC对白消安的敏感性。

总之，miR-21-5p在SSC的自我更新、增殖和治疗生殖损伤有着重要作用，这一发现扩展了miRNA调节精原干细胞的机制，为治疗生殖损伤、不孕不育、少精症、其他繁殖障碍疾病和生物育种提供了新的方法。

Abstract: Spermatogonial stem cells (SSCs) are the key to maintaining production of the sperms and healthy offsprings, and also treating breeding livestock’s reproductive damage and infertility. MicroRNAs act a decisive role in regulating gene expression in many cells and tissues, including in processes such as proliferation, self-renewal, differentiation, and apoptosis of stem cells. However, the miRNA mechanism in regulation of SSCs is still unclear. Here, high-throughput sequencing was used to identify specific miRNAs. We confirmed that miR-21-5p was concentrated in both goat and mouse SSCs, and enhanced the proliferation and antiapoptotic ability of SSCs. In vivo experiments have shown that miR-21-5p resisted the damage of the chemotherapy drug Busulfan to germ cells, ameliorated Busulfan-induced testicular dysfunction, and maintained spermatogenesis. Further RNA-seq and target gene prediction revealed that SPRY1 and FASLG are targets of miR-21-5p, thereby activating downstream signaling pathways such as MAPK/ERK, PI3K-AKT, and apoptosis. In summary, miR-21-5p is crucial for the self-renewal and maintenance of SSCs. This study provides new avenues for treating breeding livestock’s reproductive damages, infertility, oligospermia, and other conditions.

Key words: miR-21-5p , spermatogonial stem cells , infertility , Busulfan , apoptosis

. MiR-21-5p改善白消安诱导的睾丸功能障碍并维持精子发生[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.02.004.

ZHANG Meng-fei, WAN Shi-cheng, CHEN Wen-bo, YANG Dong-hui, WANG Cong-liang, LI Ba-lun, Aili Aierken, DU Xiao-min, LI Yun-xiang, WU Wen-ping, WANG Yu-qi, XIE Fang-de, LUO Xuan, LI Na, LI Xue-ling, YANG Lei, Ahmed Hamed Arisha, HUA Jin-lian. MiR-21-5p ameliorates Busulfan-induced testicular dysfunction and maintains spermatogenesis[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.02.004.

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