438. PLZF is a spermatogonia stem cell-specific marker in the sheep testis: application to enrichment of ovine spermatogonial stem cell

Abstract

Identification and isolation of spermatogonial stem cells (SSCs) are prerequisite for long-term culture, genetic manipulation, and transplantation research. The promyelocytic leukemia zinc-finger (PLZF) has been identified as a spermatogonia stem cell marker in rodent and other species, however its expression in sheep testis has not been reported yet. In this study, we validated an antibody that specifically binds to spermatogonia stem cell in sheep testis, thus demonstrated that PLZF is a spermatogonia stem cell marker and can be used for its identification. Testes from 12 Merino rams were selected to represent four stages of testis development at testis weights of 3–5 g (neonatal), 30 g (peripubertal), 50 g (prepubertal) and 100 g (mature). Three testes sections from 4 different developmental stage were stained with PLZF antibody and 25 individual tubules in each section were counted. In the sections, the percentage of PLZF positive cells/per tubule was increased nearly 2-fold from neonatal (6. 4 ± 0. 4%) to peripubertal (1 2.2 ± 2.8%), and then the percentage begin to decline in prepubertal (4.6 ± 0. 7%) and mature testes (3.1 ± 0.6%). A single cell suspension of testicular cells was generated by a two step enzymic digestion (n = 4) and spermatogonia stem cells were enriched by overnight differential plating with 0.2% gelatine coated flask. The percentages of spermatogonia stem cells in the single cell suspensions were assessed by PLZF antibody staining of smears. Compared with the initial isolation (3.1 ± 0.6%), spermatogonia were enriched 11-fold in overnight differential plating (34.0 ± 5.7,%) (P < 0.05). These data provide the basis for future studies aimed at refining conditions of spermatogonial stem cell culture and manipulation before male germ stem cell transplantation in sheep.