Differential regulation of leydig cell 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴-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 (1×10⁶ cells)^-1) or 3,5,3'-triiodothyronine (T₃, 50 ng (1×10⁶ cells)^-1) or T₃-induced protein (TIP, 2 μg (1×10⁶ cells)^-1). 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴-isomerase (3βHSD) activity was determined by the conversion of [³H]Δ⁵-dehydroepiandrosterone (DHEA) to [³H]Δ⁴-androstenedione or [³H]Δ⁵-pregnenolone to [³H]Δ⁴-progesterone (P₄) or by spectrophotometric estimation of NADH formation from NAD. T₃ significantly increased (P<0.01) both Δ⁵-DHEA to Δ⁴-androstenedione and Δ⁵-pregnenolone to Δ⁴-P₄ conversion in Leydig cells indicating stimulation of 3β-HSD activity. T₃ stimulation of 3β-HSD activity could be inhibited by cycloheximide (50 μg ml^-1) suggesting the involvement of T₃-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 T₃ and GTH stimulated perch testicular Leydig cell 3β-HSD activity, T₃ 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.