Search

A direct method for determination of delta 5 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity was employed in isolated Leydig cells (LC) derived from rats on fetal day 19 (Fig) and postnatal (N) days 1, 12, 24, 34 and 45 and adults. The activity of 3 beta-HSD in the adult LC was 1.15 +/- 0.02 (mumole/microgram DNA/hr, mean +/- SEM, n = 73). Activities in the other groups, expressed as a percentage of the respective adult control, were: Fig-38%; N1-39%; N12-8%; N24-89%; N34-166%; and N45-118%. A good correlation was found between histochemical staining for 3 beta-HSD and the quantitive method employed. Using (3H)-DHA as a substrate, LC isolated from F19, N1 and N12 produced testosterone in appreciable amounts (41%, 55% and 20% of the total products respectively) whereas at advanced stages of development (N24 to adulthood) the major product was androstenedione (93 +/- 1%). These findings may be explained by the observed decrease in 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity, due to an insufficient supply of NADPH, in the older vs. earlier stages of development. This study indicates the presence of steroidogenic enzymatic activity in LC throughout development in the rat. It also provides a relatively simple in vitro model for studies of testicular regulation during development.

A method for the determination of delta 5 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) activity in intact isolated Leydig cells was established. This method utilizes the conversion of [7-3H]dehydroepiandrosterone (1.04 mumole) to androstenedione and expresses the activity of the enzyme as mumoles of androstenedione produced/microgram DNA/h. The reaction is limited to 0.5 - 4 micrograms DNA of Leydig cells/ml (equivalent to 0.1-0.8 million of Leydig cells/ml) and to 1 h of incubation at 34 degree C. The 3 beta-HSD activity of 44 suspensions of Leydig cells isolated from adult rats was found to be 1.13 +/- 0.03 (SE) mumoles/microgram DNA/h. This new method for direct measurement of 3 beta-HSD activity in intact Leydig cells was found to be rapid, easy to perform and highly reproducible.

We studied the steroidogenic activity of isolated Leydig cells derived from rats on fetal day 19 (F19) and postnatal (N) days 1, 12, 24, 34, 45 and adults. Leydig cells, isolated at all ages by the collagenase method, increased in number throughout development with a doubling time of 8 days. Testicular content and serum concentrations of testosterone showed parallel changes during development. Moderate values were found at the early stages (F19 and N1), with a nadir on day 12, followed by a progressive increment to reach maximal values in adulthood. A reduction in steroidogenic activity of the testis during neonatal life was confirmed by in vitro studies with isolated Leydig cells. Maximal activity was found in group F19; testosterone production diminished after birth to reach a minimum in group N34 and rose thereafter to adulthood. Leydig cells were responsive to hCG stimulation at all ages in the following order: N1 greater than N34 greater than F19 greater than N24 greater than N45 greater than adult. The present study demonstrates the existence of an active and hCG-responsive population of Leydig cells in the rat testis from fetal life to adulthood.

Gossypol acetic acid is a polyphenolic compound present in the seed of cotton plants. Its antifertility activity by inhibition of spermatogenesis was proven in a large group of animals, including man. In the present study, the direct effect of gossypol acetic acid on collagenase isolated rat I-cells (interstitial cells) was investigated. It was shown that gossypol acetic acid depressed significantly the metabolic rate of the cells. Glucose utilization was abolished by a starting dose of 100 micrograms/ml. Oxygen consumption of I-cells was reduced even at a smaller dose of gossypol (50 micrograms/ml). At these doses, the vitality of the cells remained (proven by trypan blue exclusion test). 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) histochemical stain was slightly decreased. Increasing doses of gossypol caused a marked decrease in the vitality of I-cells and a dramatic drop in histochemical stain for 3 beta-HSD. The pH of the medium was not changed at any dose of treatment. In cultures of I-cells not stimulated by hCG, gossypol did not affect the tonic slow release of testosterone. Thus, gossypol acetic acid has a direct inhibitory effect on isolated rat I-cells, depressing cell metabolism. The failure of some of the other groups to show such an effect, especially in vivo, can be attributed to differences in the dose of treatment and strain of animals.