SEPT12基因表現會影響SUN蛋白質在細胞核膜上的定位,與哺乳類精蟲生成有關
葉忠信1,2,江漢聲2,3,林盈宏3
1新光醫院 泌尿科,外科部 2天主教輔仁大學 醫學系,3生物醫學暨藥學研究所
estis-specific SEPT12 expression affects SUN protein localization and is involved in mammalian spermiogenesis
Chung-Hsin Yeh1, 2, Han-Sun Chiang2,3 , Ying-Hung Lin3
1Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
2School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
3Graduate Institute of Biomedical and Pharmaceutical Science, Fu-Jen Catholic University, New Taipei City, Taiwan
Purpose: To investigate whether SEPT12 affects SPAG4(SUN4) during mammalian spermiogenesis.
Material and Methods: 1.Isolated murine testicular germ cells: Testes was excised from 2-momth-old C57BL/6 male mice (n=3)..Different developmental stages of male germ cells were isolated based on diverse centrifugal forces. 2.Immunofluorescence assay and Western blotting were performed as our previously established protocol. Images at different developmental stages were captured. 3.Cloning and transfection: The full-length coding sequences of SEPT1,6,7,11,12 and SPAG4 were amplified from a human RNA panel using rt-PCR and cloned into the pEGFP-N1 or pFLAG-CMV2 vector. Subsequently, the cell line was transfected with the vectors by using Lipofectamine (Invitrogen, USA), and the cells were analyzed using Western blotting or immunostaining. 4.Cell culture: NTERA-2 cl.D1 (NT2/D1), a pluripotent human testicular embryonal carcinoma cell line, was used for Western blotting and immunostaining experiments. The cells were sub-cultured by scraping every 2–3 days.
Results:1. we found that SPAG4 and SEPT12 exhibited similar localizations in the post-acrosomal region of elongating spermatids and at the neck of mature sperm through isolated murine male germ cells. 2.SEPT12 expression altered the nuclear membrane localization of SPAG4, as observed through confocal microscopy, in a human testicular cancer cell line. 3. SEPT12 expression also altered the localizations of nuclear membrane proteins LAMINA/C in the cells. This effect was specifically due to the expression of
SEPT12 and not that of SEPT1, SEPT6, SEPT7, or SEPT11.
Conclusion: Based on these results, we suggest that SEPT12 is one of the moderators of SPAG4/LAMIN complexes and involved in the morphological formation of sperm during mammalian spermiogenesis.