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 multi-step procedure involving enzymatic dissociation and Percoll-gradient centrifugation. The following description is the first account of a successful isolation of adolescent guinea-pig Leydig cells. The enriched Leydig-cell preparation routinely isolated from six intact testicles yielded approximately 5.0 x 10(6) +/- 0.7 x 10(6) (+/- SEM) Leydig cells with a viability of 98.0 +/- 0.4% as determined using the trypan-blue exclusion method. The purity of the isolated cell population as assessed by 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) staining averaged 82.5 +/- 0.8%. Under light microscopy, guinea-pig Leydig cells were polyhedral in shape with a large prominent nucleus and a distinct nucleolus. The acidophilic cytoplasm contained numerous lipid-filled vesicles. Ultrastructurally, guinea-pig Leydig cells displayed an eccentrically located ovoid nucleus with dark-staining peripheral heterochromatin. Large quantities of mitochondria, smooth endoplasmic reticulum and particulate-laden lipid droplets were also evident. The steroidogenic potential of the isolated Leydig cells was verified using a maximally 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) water bath for 3 h at 37 degrees C in capped polypropylene microcentrifuge tubes produced 233 +/- 21 ng and 223 +/- 18 ng testosterone per 1 x 10(6) cells when maximally stimulated with oLH or hCG, respectively. The inclusion of low (1-5 microM) levels of sodium ascorbate during culture enhanced significantly Leydig-cell viability vs. control values.
Kukucka
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MA
Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg 24061-0442.
Misra
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HP
D015096:3-Hydroxysteroid Dehydrogenases / Q000032:analysis; D000818:Animals; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D002501:Centrifugation, Zonal; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D046508:Culture Techniques / Q000379:methods; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D006168:Guinea Pigs; D006570:Heterochromatin / Q000648:ultrastructure; D006801:Humans; D007700:Kinetics; D007985:Leydig Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007986:Luteinizing Hormone / Q000494:pharmacology; D008297:Male; D008928:Mitochondria / Q000648:ultrastructure; D011205:Povidone; D012756:Sheep; D012822:Silicon Dioxide; D013737:Testis / Q000166:cytology*; D013739:Testosterone / Q000096:biosynthesis
D006063:Chorionic Gonadotropin; D006570:Heterochromatin; D013739:Testosterone; C016039:Percoll; D012822:Silicon Dioxide; D007986:Luteinizing Hormone; D015096:3-Hydroxysteroid Dehydrogenases; D011205:Povidone