Identification of GFP expressing cells in Oct4-EGFP transgenic pig testis

Expression of the Oct4 gene, a marker for pluripotency was found in early embryos, germ cells throughout fetal development and in undifferentiated type A spermatogonial stem cells in testis. Here, we used Oct4-EGFP transgenic pigs as model to characterize GFP expressing cells in the adult testes. This animal model is unique because it allows in vivo and in vitro visualization of Oct4-EGFP positive cells. We isolated GFP fluorescent cells from adult testis using fluorescence-activated cell sorting (FACS)-based techniques. Two cell populations, i.e. GFP+ (14%) and GFP- (45%) could be collected. Analysis of isolated GFP positive cells with qRT-PCR demonstrated the presence of marker genes, specific for undifferentiated germ cells (OCT4, UTF1, FGFR3, PGP 9.5, THY-1, SALL4 and GFRα1). Moreover, expression of the markers BOLL and PRM2 was detected also in GFP+ cell population indicating that this cell population also contains differentiating spermatids. Markers specific for Sertoli cells (vimentin) and Leydig cells (LHCGR) were not observed. To verify the localization of GFP positive cells in seminiferous tubules, we performed immunohistochemical detection of GFP in adult pig testis. Unlike to the Oct4-EGFP reporter mouse model, GFP protein was not found in spermatogonia attached to the basement membrane of seminiferous tubules, but instead were found in differentiated germ cells, including spermatocytes and spermatids. These results show that the Oct4-EGFP expression in testis differs between mouse and porcine Oct4-EGFP transgenic models. To verify that the GFP expression driven by the mouse Oct4 promoter in porcine testis reflects the endogenous OCT4 expression profile, Western blot and histochemical analyses are currently underway.