Isolation of rat Leydig cells by density gradient centrifugation

A rapid method for preparing Leydig cells from rat testes is described. An interstitial cell suspension, prepared by collagenase treatment of decapsulated testes, was centrifugal for 10 min over a cushion of 60% (v/v) Percoll to remove red blood cells, and then centrifuged for 20 min in a 0-60% linear density gradient of Percoll. Seventy-four per cent of the cells present in that fraction of the gradient comprising 35-50% Percoll were Leydig cells; the yield from each testis was about 1.5 x 10(6) cells. The Leydig cells appeared viable, excluded Trypan blue, possessed high-affinity binding sites for human chorionic gonadotrophin (hCG) and synthesized increased quantities of testosterone in response to hCG. The cells could be stored overnight in 20% (v/v) glycerol at -20 degrees C, with only minimal effect on the specific activities of a number of enzymes used as markers of subcellular components. Testosterone production in vitro by the cells after storage for 20 h was greater than that of hCG-stimulated fresh cells and was not further increased by hCG.