Pei-mediated Mouse Spermatogenic Cell Gene Transfection And Its Applications In The Nyd-sp12 Gene Function Studies

To study the biological mechanisms of spermatogenesis, functional characterization of the testis-specific genes especially the spermatogenic cell-specific genes is very important. As in vitro cell culture is very difficulty in reproducing the spermatogenesis and the present in vivo research method has these or those deficiency, we are exploring a new in vivo gene transfer method to develop a convenient and efficient gene functional research assay for the spermatogenic-cell specific genes. In this study, the linear 22 kDa form of polyethylenimine (PEI) was used to mediate the transfection of spermatogenic cell-specific gene NYD-SP12 (GFP tagged) into spermatogenic cells via intra-testicular injection. Direct fluorenscence analysis of GFP indicated that the transfection was occurred in the spermatogenic cells, no Sertoli cell had been observed to be transfected, which was quite different from that of previous in vivo gene transfer methods and need to be further investigated. As for long-term examination of the GFP-NYD-SP12 fusion gene expression, the fusion protein was expressed in spermatocytes in the early transfection day (3 day), whereas it seemed to be transported into daughter cells as the spermatocytes developed and differentiated. The subcellular location of the fusion protein turned out to be dynamic as compared with the GFP alone protein that was dispersed in all types of spermatogenic cells. It dispersed in the cytoplasm of spermatocytes and congregated as acrosome-like structure beside the condensed nuclear of roundspermatids. In elongated spermatids, the fusion protein seemed to began to draw itsself up along the nuclear, and in sperms it was obviousely located in the acrosomes. The presentation of the fusion protein's location in spermatogenic cells of different stage was quite similar to the Golgi apparatus. And as NYD-SP12 was previously reported to be located in Golgi, we now declear that NYD-SP12 is quite like to be involved in acrosome formation. This result also supports that PEI mediated in vivo gene transfection method was convenient and efficient for the functional research of spermatogenic-cell specific genes.