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
from
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


ABSTRACT

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.


CITATION
Kukucka, Mark A. and Hara P. Misra. 1994. Isolation and culture of
highly
enriched populations of Leydig cells from guinea-pig (Cavia
porcellus)
testes. Andrologia 26(4):217-24.


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