Isolation and culture of highly enriched populations of Leydig cellsfrom guinea-pig (Cavia porcellus) testes
Isolation and culture of highly enriched populations of Leydig cells
guinea-pig (Cavia porcellus) testes
by Mark A. Kukucka and Hara P. Misra
Department of Biomedical Sciences
Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute & State University, Blacksburg 24061
Leydig cells were isolated from adult male guinea-pig testes using a
multi-step procedure involving enzymatic dissociation and Percoll-
centrifugation. The following description is the first account of a
successful isolation of adolescent guinea-pig Leydig cells. The
Leydig-cell preparation routinely isolated from six intact testicles
approximately 5.0 x 10(6) +/- 0.7 x 10(6) (+/- SEM) Leydig cells with
viability of 98.0 +/- 0.4% as determined using the trypan-blue
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
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.
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
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
21 ng and 223 +/- 18 ng testosterone per 1 x 10(6) cells when
stimulated with oLH or hCG, respectively. The inclusion of low (1-5
levels of sodium ascorbate during culture enhanced significantly
viability vs. control values.
Kukucka, Mark A. and Hara P. Misra. 1994. Isolation and culture of
enriched populations of Leydig cells from guinea-pig (Cavia
testes. Andrologia 26(4):217-24.
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