192 IDENTIFICATION AND CHARACTERIZATION OF Oct4-EGFP EXPRESSING CELLS IN TRANSGENIC PIG TESTIS

Abstract

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.