敲除基因Pabpc6对雄性小鼠精子发生的影响

doi:10.16352/j.issn.1001-6325.2023.04.0560

基础医学与临床 ›› 2023, Vol. 43 ›› Issue (4): 560-567.doi: 10.16352/j.issn.1001-6325.2023.04.0560

敲除基因Pabpc6对雄性小鼠精子发生的影响

汤洁琳, 柳俊, 邹定峰, 缪时英, 宋伟, 李凯^*

中国医学科学院基础医学研究所北京协和医学院基础学院生物化学与分子生物学系医学分子生物学国家重点实验室, 北京 100005

收稿日期:2023-01-06 修回日期:2023-02-15 出版日期:2023-04-05 发布日期:2023-04-03
通讯作者: ^*likai@ibms.pumc.edu.cn
基金资助:
国家自然科学基金(31970794,32000586)

Effect of Pabpc6 knockout on spermatogenesis of male mice

TANG Jielin, LIU Jun, ZOU Dingfeng, MIAO Shiying, SONG Wei, LI Kai^*

National Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China

Received:2023-01-06 Revised:2023-02-15 Online:2023-04-05 Published:2023-04-03
Contact: ^*likai@ibms.pumc.edu.cn

摘要/Abstract

摘要： 目的探究小鼠细胞质多聚腺苷酸结合蛋白-6(PABPC6)在雄性小鼠精子发生中的作用。方法利用CRISPR/Cas9靶向敲除C57BL/6J小鼠基因Pabpc6,通过PCR鉴定Pabpc6的敲除情况。通过交配繁殖得到Pabpc6^+/+、Pabpc6^+/-、Pabpc6^-/- 3种不同基因型的小鼠,进行睾丸称重、形态学观察和精子计数。RT-qPCR和Western blot检测不同组织中Pabpc6在mRNA和蛋白的表达水平;通过免疫荧光和苏木精-伊红染色观察该基因的定位以及睾丸曲细精管内部形态学变化。结果成功鉴定出敲除Pabpc6的小鼠;Pabpc6在睾丸组织中高表达(P＜0.001)。免疫荧光显示该基因主要表达于精母细胞和早期精子阶段,基因敲除小鼠与野生型小鼠相比在睾丸外形、精子形态、精子数量和曲细精管内部形态上没有明显差异。结论 Pabpc6在小鼠睾丸组织中高表达,敲除该基因后对雄性小鼠的精子发生无明显影响,说明该基因所编码的蛋白对于小鼠精子发生过程可能是非必需的。

关键词: 精子发生, PABPC6, 雄性生殖

Abstract: Objective To investigate the role of cytoplasmic poly (A) binding protein-6 (PABPC6) in spermatogenesis of male mice. Methods The Pabpc6 in C57BL/6J mice was knocked out by CRISPR/Cas9 targeting, and the Pabpc6 was identified by PCR. Three types of mice with different genotypes of Pabpc6^+/+, Pabpc6^+/- and Pabpc6^-/- were obtained by mating and breeding. Testis was weighed;Morphological observation and sperm counting were carried out. The mRNA and protein expression levels of Pabpc6 in different tissues were detected by real-time quantitative PCR and Western blot. Immunofluorescence and hematoxylin-eosin staining were used to observe the location of the gene and the morphological changes in the convoluted spermatic tubule of testis. Results The Pabpc6 knockout mice were successfully identified. Pabpc6 was highly expressed in testicular tissue(P＜0.001). Immunofluorescence showed that Pabpc6 was mainly expressed in spermatocyte at early sperm stage. Compared with wild-type mice, knockout mice showed no significant differences in testicular shape, sperm morphology, sperm count and the morphology of convoluted spermatic ducts. Conclusions Pabpc6 is found to be highly expressed in the testis tissue of mice. Pabpc6 has no obvious effect on spermatogenesis of male mice after knockout of Pabpc6. The results indicate that the protein encoded by Pabpc6 is not necessary for spermatogenesis of mice.

Key words: spermatogenesis, PABPC6, male reproduction

中图分类号:

汤洁琳, 柳俊, 邹定峰, 缪时英, 宋伟, 李凯. 敲除基因Pabpc6对雄性小鼠精子发生的影响[J]. 基础医学与临床, 2023, 43(4): 560-567.

TANG Jielin, LIU Jun, ZOU Dingfeng, MIAO Shiying, SONG Wei, LI Kai. Effect of Pabpc6 knockout on spermatogenesis of male mice[J]. Basic & Clinical Medicine, 2023, 43(4): 560-567.

参考文献 10

[1]	Tharakan T, Luo R, Jayasena CN, et al. Non-obstructive azoospermia: current an future perspectives[J]. Fac Rev, 2021,10:7. doi: 10.12703/r/10-7. eCollection 2021.
[2]	Ozturk S, Uysal F. Potential roles of the poly(A)-binding proteins in translational regulation during spermatogenesis[J]. J Reprod Dev, 2018,64:289-296.
[3]	Ozturk S, Sozen B, Uysal F, et al. The poly(A)-binding protein genes, EPAB, PABPC1, and PABPC3 are differentially expressed in infertile men with non-obstructive azoospermia[J]. J Assist Reprod Genet, 2016, 33:335-348.
[4]	Ozturk S, Uysal F. Potential roles of the poly(A)-binding proteins in translational regulation during spermatogenesis[J]. J Reprod Dev, 2018, 64:289-296.
[5]	Fouchécourt S, Picolo F, Elis S, et al. An evolutionary approach to recover genes predominantly expressed in the testes of the zebrafish, chicken and mouse[J]. BMC Evol Biol, 2019,19:137. doi: 10.1186/s12862-019-1462-8.
[6]	Schultz N, Hamra FK, Garbers DL. A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets[J]. Proc Natl Acad Sci U S A, 2003,100:12201-12206.
[7]	Hu X, Shen B, Liao S, et al. Gene knockout of Zmym3 in mice arrests spermatogenesis at meiotic metaphase with defects in spindle assembly checkpoint[J]. Cell Death & Disease. 2017, 8: e2910. doi: 10.1038/cddis.2017.228.
[8]	Ozturk S, Guzeloglu-Kayisli O, Demir N, et al. Epab and Pabpc1 are differentially expressed during male germ cell development[J]. Reprod Sci, 2012,19:911-922.
[9]	Wells SE, Hillner PE, Vale RD, et al. Circularization of mRNA by eukaryotic translation initiation factors[J]. Mol Cell, 1998, 2:135-140.
[10]	M Khan, N Jabeen, T Khan, et al. The evolutionarily conserved genes: Tex37, Ccdc73, Prss55 and Nxt2 are dispensable for fertility in mice[J]. Sci Rep, 2018, 8:4975. doi: 10.1038/s41598-018-23176-x.

敲除基因Pabpc6对雄性小鼠精子发生的影响

Effect of Pabpc6 knockout on spermatogenesis of male mice

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