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Isolation and culture of highly enriched populations of Leydig cellsfrom guinea-pig (Cavia porcellus) testes

Isolation and culture of highly enriched populations of Leydig cellsfrom guinea-pig (Cavia porcellus) testes - Cell Biology and Cell Culture

Isolation and culture of highly enriched populations of Leydig cellsfrom guinea-pig (Cavia porcellus) testes - Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


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Old 11-29-2008, 11:15 PM
tocopherol
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Default 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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