Reproduction Abstracts

Introduction: It is necessary to establish and culture of spermatogonia stem cells (SSCs) in vitro to perform the meiotic differentiation study and to generate transgenic spermatozoa in vitro or ex vivo. This study was focused on in vitro derivation and establishment of SSCs from porcine and canine.

Materials and methods: Five day-old porcine and 2-month-old canine testes were used to isolated SSCs and testicular somatic cells. For the establishment of porcine and canine SSCs, SSCs and testicular cells were cultured in stempro-34 and DMEM with 5% FBS medium, respectively, supplemented with GDNF, EGF, FGF, and LIF in 31 and 37 °C respectively. To identify the SSCs in culture, OCT4, NANOG, THY1, PLZF, and PGP9.5 were analyzed. Characteristics of SSCs were analyzed with xeno-transplantation of in vitro cultured porcine SSCs into immune deficient mice testes.

Result and discussion: SSCs established from both porcine and canine showed the strong alkaline phosphatase staining, pluripotency markers, and undifferentiated germ cell markers gene expressions. Both SSCs were stably maintained undifferentiated germ cell and stem cell characteristics for over 100 days on GATA4 expressed feeder cells, and xenotransplantation of pSGCs to immune deficient mice demonstrated the successful colonization in the recipient testes. In addition, GFRα-1 and UTF1 were identified as a putative specific markers for pSSCs, and total transcriptome analysis of pSSCs revealed the expressions of CD14, CD209, KLF9, IGFBP3, and IGFBP5 genes. This finding may facilitate the study on spermatogenesis and further application of spermatogonial stem cells in large domestic animals.