NYD-SP12 Role In Acrosome Formation

NYD-SP12 was a novel human testis-specific gene cloned by our lab. In-situ hybridization revealed that the mouse homologous NYD-SP12 mRNA was detected primarily in the spermatocyte. NYD-SP12-GFP (green fluorescent protein) fusion protein was localized to the Golgi apparatus in vitro transfection, and congregated as acrosome-like structure beside the nuclear of spermatids by in vivo gene transfer via intra-testicular injection. The Golgi apparatus is actively engaged in sorting and delivering proteins and membranes to the developing acrosome. Preliminary studies predicted that NYD-SP12 was related to the acrosome formation. In this study, we investigated the role and mechanism of NYD-SP12 in the process of acrosome formation.First of all, we examined the localization and expression pattern of NYD-SP12 during spermatogenesis. NYD-SP12 open reading fragment was cloned into the pET28a+ expression vector. The recombinant protein expression was induced with isopropyl-1-thio-b-D-galactopyranoside. The purified recombinant NYD-SP12-His protein was used as an immunogen and injected into New Zealand rabbits for polyclonal antibody development. Western Blot studies revealed that NYD-SP12 protein was existed in human sperm. Indirect immunofluorescence of human sperm revealed the presence of NYD-SP12 in acrosome. Together with initial studies, this result indicated that NYD-SP12 may play a role in acrosome formation during spermatogenesis.Subsequently, we investigated the function of NYD-SP12 in formation of sperm acrosome using spermatogenic cell line. Because NYD-SP12 was highly conserved between human and mouse, we characterized the alterations of the Golgi apparatus and vesicles in the mouse spermatocyte cell stably expressing the mouse NYD-SP12. Swelling of the Golgi apparatus and congregation of vesicles at the edge of nucleus were observed. The altered Golgi morphology was restored in mouse spermatocyte cell after knock-down of overexpressional NYD-SP12. Thus, NYD-SP12 regulated the formation and transport of Golgi-derived vesicles during acrosome formation.NYD-SP12 contained a TPR (tetratricopeptide repeat) -like motif which mediated protein-protein interactions and the assembly of multiprotein complexes. We identified the physical interaction between NYD-SP12 and Lamin A in mouse spermatocyte cell line by co-immunoprecipitation coupled with MALDI-TOF. The function of this interaction in acrosome formation remained to be resolved.Globozoospermia is a rare but severe disorder of male infertility characterized by sperm possessing a round head, lack of an acrosome. At last, we confirmed that NYD-SP12 was involved in the pathogenesis of human globozoospermia. Indirect immunofluorescence showed the lack of NYD-SP12 in globozoospermia. Western blot analysis indicated that expression of NYD-SP12 was greatly diminished in globozoospermia. Acrosome formation of the round-headed spermatozoon was abnormal in different developmental stages. Electron micrographs showed residual Golgi-like membrane and unfused acrosomal vesicles in globozoospermia.In conclusion, NYD-SP12 was a testis-specific gene involved in the acrosome formation. It was localized in the acrosome of human sperm. It facilitated the formation and trafficking of acrosomal vesicles by the Golgi pathway. It interacted with Lamin A in mouse spermatocyte cell line. It was also involved in the pathogenesis of human globozoospermia. Functional study of NYD-SP12 provided the invaluable information to clinical diagnosis and treatment of male infertility.