ISOLATION AND CULTURE OF HIGHLY ENRICHED POPULATIONS OF LEYDIG-CELLS FROM GUINEA-PIG (CAVIA-PORCELLUS) TESTES

Leydig cells were isolated from adult male guinea-pig testes using a m ulti-step procedure involving enzymatic dissociation and Percoll-gradi ent centrifugation, The following description is the first account of a successful isolation of adolescent guinea-pig Leydig cells. The enri ched Leydig-cell preparation routinely isolated from six intact testic les yielded approximately 5.0 x 10(6)+/-0.7 x 10(6) (+/-SEM) Leydig ce lls with a viability of 98.0+/-0.4% as determined using the trypan-blu e exclusion method. The purity of the isolated cell population as asse ssed by 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) staining aver aged 82.5 +/- 0.8%. Under light microscopy, guinea-pig Leydig cells we re polyhedral in shape with a large prominent nucleus and a distinct n ucleolus. The acidophilic cytoplasm contained numerous lipid-filled ve sicles. Ultrastructurally, guinea-pig Leydig cells displayed an eccent rically located ovoid nucleus with dark-staining peripheral heterochro matin. Large quantities of mitochondria, smooth endoplasmic reticulum and particulate-laden lipid droplets were also evident. The steroidoge nic potential of the isolated Leydig cells was verified using a maxima lly stimulating dose of ovine LH (100 ng ml(-1)) and human CG (200 mIU ml(-1)). Leydig cells incubated in a shaking (120 cycles min(-1)) wat er bath for 3 h at 37 degrees C in capped polypropylene microcentrifug e tubes produced 233 +/- 21 ng and 223 +/- 18 ng testosterone per 1x10 (6) eels when maximally stimulated with oLH or hCG, respectively. The inclusion of low (1-5 mu M) levels of sodium ascorbate during culture enhanced significantly Leydig-cell viability vs. control values.