Derivation, enrichment and characterization of goat (Capra hircus) spermatogonial stem cells from pre-pubertal testes

Abstract

Isolation of goat spermatogonial stem cells (gSSC) from pre-pubertal testes (3 to 6 months of age) by double enzymatic digestion is reported. The isolated cells were further enriched for spermatogonial stem cell population by filtration through 80- and 60-μm nylon mesh filters, followed by differential plating on DSA-Lectin coated dishes. After overnight incubation, the unattached cells (putative gSSCs) were cultured on sertoli cell feeder layers in SSC medium, composed of DMEM supplemented with 10% FBS, GDNF (Glial cell line derived neurotrophic factor, 40 ng mL-1), bFGF (Basic fibroblast growth factor, 10 ng mL-1) and EGF (Epidermal growth factor, 10 ng mL-1). After 10-15 days, the putative SSCs started to develop and the cultures were maintained till the colonies stopped growing. The colonies were characterized for their affinity to Dolichos biflorus Agglutinin (DBA), alkaline phosphatase (AP) activity and various spermatogonial stem cell markers (PLZF, THY1, UCHL1, BCL6B and ID4). The developed colonies were positive for DBA, AP and all other markers, which is an indication of their being spermatogonial stem cells. In the present study, goat SSCs have been successfully isolated and characterized and the culture conditions will be improved further for developing more efficient and long term in vitro spermatogonial stem cell culture system in goat.