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

BACKGROUND: Golgin 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. METHODS: Immunofluorescence co-localization in cells and protein dephosphorylation experiments were performed. Golga3( S461L/S461L)mice 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. RESULTS: Immunofluorescence co-localization showed reduced Golgi localization of GOLGA3(S465L) with some protein scattered in the cytoplasm of HeLa cells .In addition, protein dephosphorylation experiments indicated a reduced band shift of the dephosphorylated GOLGA3(S465L), confirming S461 as the phosphorylation site. Golga3 is an evolutionarily conserved gene and Golga3(S461L/S461L)mice 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. CONCLUSIONS: Golga3 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 GOLGA3(S465L)showed 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.