Variation in the End Products of Androgen Biosynthesis and Metabolism during Postnatal Differentiation of Rat Leydig Cells *

The amount of testosterone (T) secreted by Leydig cells is determined by a balance between T biosynthetic and metabolizing enzyme activities. It has been established that 5a-androstan-3a,17b-diol (3aDIOL) is the predominant androgen secreted by the testes of immature rats during days 20–40 postpartum, whereas T is the major androgen by day 56. However, the underlying changes in T biosynthetic and metabolizing enzymes during Leydig cell development and their magnitudes have remained unclear. The aim of the present study was to define the developmental trends for T biosynthetic and metabolizing enzymes in Leydig cells at three distinct stages of pubertal differentiation: mesenchymal-like progenitors on day 21, immature Leydig cells on day 35, and adult Leydig cells on day 90. Production rates for precursor androgen (androstenedione), T, and 5a-reduced androgens [androsterone (AO) and 3a-DIOL] were measured in progenitor, immature, and adult Leydig cells in spent medium after 3 h in vitro. Steady state messenger RNA (mRNA) levels and enzyme activities of biosynthetic and metabolizing enzymes were measured in fractions of freshly isolated cells at each of the three stages. Unexpectedly, progenitor cells produced significant amounts of androgen, with basal levels of total androgens (androstenedione, AO, T, and 3a-DIOL) 14 times higher than those of T alone. However, compared with immature and adult Leydig cells, the capacity for steroidogenesis was lower in progenitor cells, with a LH-stimulated production rate for total androgens of 84.33 6 8.74 ng/10 cellsz3 h (mean 6 SE) vs. 330.13 6 44.19 in immature Leydig cells and 523.23 6 67.29 in adult Leydig cells. The predominant androgen produced by progenitor, immature, and adult Leydig cells differed, with AO being released by progenitor cells (72.08 6 9.02% of total androgens), 3aDIOL by immature Leydig cells (73.33 6 14.52%), and T by adult Leydig cells (74.38 6 14.73%). Further examination indicated that changes in the predominant androgen resulted from differential gene expression of T biosynthetic and metabolizing enzymes. Low levels of type III 17b-hydroxysteroid dehydrogenase (17bHSD) mRNA and enzyme activity were present in progenitor cells compared with immature and adult Leydig cells. In contrast, levels of type I 5a-reductase (5aR) and 3a-hydroxysteroid dehydrogenase (3aHSD) mRNA and enzyme activities were dramatically lower in adult Leydig cells compared with those in progenitor and immature Leydig cells. Several T biosynthetic enzymes attained equivalent levels in immature and adult Leydig cells, but T was rapidly metabolized in the former to 3a-DIOL by high 5aR and 3aHSD activities, which were greatly reduced in the latter. Therefore, declines in 5aR and 3aHSD activities are hypothesized to be a major cause of the ascendancy of T as the predominant androgen end product produced by adult Leydig cells. These results indicate that steroidogenic enzyme gene expression is not induced simultaneously, but through sequential changes in T biosynthetic and metabolizing enzyme activities, resulting in different androgen end products being secreted by Leydig cells during pubertal development. (Endocrinology 139: 3787–3795, 1998) A stimulation is responsible for the maintenance of spermatogenesis and secondary sexual characteristics in the male. According to the literature, testosterone (T) produced by Leydig cells of the testis is the major androgen in the circulation of men and adult males of most mammalian species, including the rat. However, in rats, which have been studied more extensively than most other species, 5a-androstan-3a,17b-diol (3a-DIOL) and androsterone (AO) are abundant in the circulation between days 20–40 postpartum when T is still low (1). Testicular tissue of rats aged 15–40 days metabolizes radiolabeled progesterone and T to 3a-DIOL and, to a lesser extent, AO (2–5). Testicular enzyme activities of 3b-hydroxysteroid dehydrogenase (3bHSD), 17a-hydroxylase/C17–20 lyase (P450c17), and 17bhydroxysteroid dehydrogenase (17bHSD) increase gradually between the ages of 20 and 60 days and plateau thereafter (6, 7). The amount of testicular 5a-reductase (5aR) activity, on the other hand, sharply increases between days 20 and 40, and then falls between days 40 and 60 (7). Further analysis demonstrated that type I 5aR and 3a-hydroxysteroid dehydrogenase (3aHSD) messenger RNA (mRNA) and protein are abundantly present in progenitor and immature Leydig cells during days 15–35 (8, 9). These findings suggest that the Leydig cell itself is a metabolizing site for androgens during puberty, at least via the 5a-reduction pathway. However, technical barriers prevented the testing of this hypothesis, because steroidogenic enzymes such as 17bHSD (10–12), 5aR (13, 14), and 3aHSD (14, 15) are present in other cell types in the testis. Therefore, purified Leydig cells were examined in the present study to delineate T biosynthesis and metabolism occurring in this cell type during pubertal