Isolation and enrichment of putative spermatogonial stem cells from ram (Ovis aries) testis

The present study aimed to isolate and enrich putative SSCs from ram testes, which are positive for promyelocytic leukaemia zinc-finger protein (PLZF). The putative SSCs were isolated using a combination of enzymes with different concentrations, collagenase (1 and 2 mg/ml), hyaluronidase (1 mg/ml) and trypsin (0.25 and 0.5 mg/ml). The isolated SSCs were purified using an extracellular matrix such as laminin (20 μg/ml), DSA-lectin (5 μg/ml) and gelatin (0.2%) in combination with BSA (0.5 mg/ml). The number of putative SSCs/ tubule was significantly (p < 0.05) higher in prepubertal (3.1 ± 0.51) and adult (3.45 ± 0.58) than the number of gonocytes/tubule in neonatal (0.59 ± 0.03) testis. Optimum enzyme combinations required for isolation of putative SSCs from prepubertal testis (collagenase; 2 mg/ml and trypsin; 0.5 mg/ml) were different from adult testis (collagenase; 1 mg/ml, trypsin; 0.25 mg/ml and hyaluronidase; 1 mg/ml). Though the number of putative SSCs/tubule was comparable in prepubertal and adult animals, a significantly (p < 0.05) higher percentage of putative SSCs (7.33 Vs 0.47%) were isolated from prepubertal testis than the adult. Differential plating using laminin along with BSA resulted in a significantly (p < 0.05) higher number of putative SSCs. The enzyme combinations suitable for isolation of putative SSCs from prepubertal testis are different from adult ram testis and the laminin has been found to be effective for purification of putative SSCs from testicular cells isolates.