Differential regulation of Leydig cell 3β-hydroxysteroid dehydrogenase/Δ 5–Δ 4-isomerase activity by gonadotropin and thyroid hormone in a freshwater perch, Anabas testudineus (Bloch)

Abstract

Leydig cells were isolated from the perch testes belonging to the pre-spawning stage by collagenase treatment and mechanical separation followed by percoll gradient. They were incubated in vitro either for 5 h or at different times in the absence (control) or presence of piscine gonadotropin (GTH, 2 μg (1×10 6 cells) −1) or 3,5,3′-triiodothyronine (T 3, 50 ng (1×10 6 cells) −1) or T 3-induced protein (TIP, 2 μg (1×10 6 cells) −1). 3β-hydroxysteroid dehydrogenase/Δ 5–Δ 4-isomerase (3β-HSD) activity was determined by the conversion of [ 3H]Δ 5-dehydroepiandrosterone (DHEA) to [ 3H]Δ 4-androstenedione or [ 3H]Δ 5-pregnenolone to [ 3H]Δ 4-progesterone (P 4) or by spectrophotometric estimation of NADH formation from NAD. T 3 significantly increased ( P<0.01) both Δ 5-DHEA to Δ 4-androstenedione and Δ 5-pregnenolone to Δ 4-P 4 conversion in Leydig cells indicating stimulation of 3β-HSD activity. T 3 stimulation of 3β-HSD activity could be inhibited by cycloheximide (50 μg ml −1) suggesting the involvement of T 3-induced protein (TIP) which was isolated and purified earlier in this laboratory from goat Leydig cells [15]. Addition of TIP or GTH significantly stimulated Leydig cell 3β-HSD activity ( P<0.01). However, there was a difference between TIP and GTH stimulation in time kinetic study where TIP enhanced 3β-HSD activity at 1 h ( P<0.05), reached its peak at 3 h ( P<0.01) and then plateaued till 8 h. GTH, on the other hand, did not show any stimulation of 3β-HSD activity for 2 h, stimulation was marked only at 3 h ( P<0.05), reached a peak at 6 h ( P<0.01) and then leveled off. Determination of K m and V max of the enzyme showed an increase in the velocity of reaction by GTH with unaltered K m, TIP increased both velocity and affinity of the enzyme. GTH significantly increased the synthesis of 3β-HSD protein at 3 h ( P<0.01) reaching maximal stimulation at 6 h which clearly coincided with the enzyme activity. In contrast, TIP had no effect on 3β-HSD protein synthesis, but its direct addition to 3β-HSD enzyme preparation in vitro caused significant augmentation of the enzyme activity ( P<0.01) suggesting thereby its modulatory effect on the enzyme. Results, therefore, show that although both T 3 and GTH stimulated perch testicular Leydig cell 3β-HSD activity, T 3 effect was not direct but mediated via TIP and there is a clear distinction between GTH and TIP stimulation. GTH increased the enzyme activity by stimulating 3β-HSD protein synthesis while TIP acts directly on the enzyme modulating it from less active to more active state.