5alpha-reductase, an enzyme regulating glucocorticoid action in the testis of the toad Rhinella arenarum

TESONE AMALIA J., REGUEIRA ELEONORA, CEBALLOS NORA R

Ann Arbor

Simposio; ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology; 2011

The sensitivity of Leydig cells to glucocorticoids (GC) is determined by several factors: plasma levels of GC and corticosteroid binding globulins, the amount of GC receptors (GR) and the activating/inactivating enzyme 11β-hydroxysteroid dehydrogenase, among others. In the toad Rhinella arenarum, breeding levels of corticosterone (B) inhibit testosterone synthesis by suppressing the cytochrome P450 17-hydroxylase, C17,20-lyase (Cyp450c17) via GR-mediated mechanism. Anurans express a high testicular activity of 5alpha-reductase (5alphaRed), enzyme that could contribute to modulate GC action by the irreversible reduction of the A-ring of GC. The objective of this work was to study the role of 5alphaRed on GC action in toad testes. Enzymatic activity was assayed with [3H]B and [3H]testosterone as substrates. The binding capacity of 5alpha-dihydrocorticosterone (5alphaDHB) to testicular GR was determined by the displacement of [3H]dexamethasone with radioinert B and 5alphaDHB. For analyzing the agonistic/antagonistic properties of 5alphaDHB, the effect of dexamethasone and 5alphaDHB on the activity of Cyp450c17 was measured after culturing testes with both steroids. Results indicate that 5alphaRed localizes in microsomes with the highest activity at pH between 6.0 and 8.0. Kinetic parameters, Km and Vmax, do not change along the year. The affinity of the enzyme for B was significantly higher than for testosterone, indicating that B is the preferred substrate. Competition studies show that 5alphaDHB displaces the binding of [3H]dexamethasone with a similar potency than B (Kd for B and 5alphaDHB, 31.33 and 35.24 nM, respectively), suggesting that 5alphaDHB could compete with B for GR. Moreover, long-term incubation of testes with 5alphaDHB (150 and 1500 nM) inhibits the activity of the key enzyme of androgen biosynthesis, Cyp450c17, similarly than dexamethasone. This result is consistent with an agonistic activity and suggests that 5alphaDHB could be part of the activating mechanism.