Mutation of S461, in the GOLGA3 phosphorylation site, does not affect mouse spermatogenesis

PeerJ

article

Changtong Xu¹  Mingcong Zhu¹  Shuqin Zhao¹  Xin Zhang¹  Ying Wang²  Mingxi Liu³ 

[1] State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, School of Basic Medical Sciences, Nanjing Medical University;State Key Laboratory of Reproductive Medicine, Department of Reproduction, Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital;State Key Laboratory of Reproductive Medicine, The Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University

关键词: Golgin subfamily A member 3 (Golga3);    Protein phosphorylation;    Spermatogenesis;    Male fertility;   

DOI  :  10.7717/peerj.15133

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

BackgroundGolgin subfamily A member 3 (Golga3), a member of the golgin subfamily A, is highly expressed in mouse testis. The GOLGA3 protein, which contains eight phosphorylation sites, is involved in protein transport, cell apoptosis, Golgi localization, and spermatogenesis. Although it has been previously reported that nonsense mutations in Golga3 cause multiple defects in spermatogenesis, the role of Golga3 in the testis is yet to be clarified.MethodsImmunofluorescence co-localization in cells and protein dephosphorylation experiments were performed. Golga3 S461L/S461Lmice were generated using cytosine base editors. Fertility tests as well as computer-assisted sperm analysis (CASA) were then performed to investigate sperm motility within caudal epididymis. Histological and immunofluorescence staining were used to analyze testis and epididymis phenotypes and TUNEL assays were used to measure germ cell apoptosis in spermatogenic tubules.ResultsImmunofluorescence co-localization showed reduced Golgi localization of GOLGA3S465L with some protein scattered in the cytoplasm of HeLa cells .In addition, protein dephosphorylation experiments indicated a reduced band shift of the dephosphorylated GOLGA3S465L, confirming S461 as the phosphorylation site. Golga3 is an evolutionarily conserved gene and Golga3S461L/S461Lmice were successfully generated using cytosine base editors. These mice had normal fertility and spermatozoa, and did not differ significantly from wild-type mice in terms of spermatogenesis and apoptotic cells in tubules.ConclusionsGolga3 was found to be highly conserved in the testis, and GOLGA3 was shown to be involved in spermatogenesis, especially in apoptosis and Golgi complex-mediated effects. Infertility was also observed in Golga3 KO male mice. Although GOLGA3S465Lshowed reduced localization in the Golgi with some expression in the cytoplasm, this abnormal localization did not adversely affect fertility or spermatogenesis in male C57BL/6 mice. Therefore, mutation of the S461 GOLGA3 phosphorylation site did not affect mouse spermatogenesis.

【 授权许可】

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