Characterization of oviductal aromatase in the northern leopard frog (rana pipiens).

Abstract

Estrogens increase the growth of the frog oviduct. Testosterone also has a growth promoting effect in the northern leopard frog, Rana pipiens, while dihydrotestosterone does not. To investigate the possibility that the testosterone effect is due to its conversion to estradiol, the oviduct was examined for the presence of an aromatase, first by looking for metabolites with thin layer chromatography, and then by using an enzymatic technique. The characteristics and kinetic parameters of the amphibian enzyme were then examined. Human placental aromatase was also studied for comparative purposes. Oviducts were obtained from summer frogs in various stage of oogenesis. After the animals were sacrificed, the oviducts were quickly removed, frozen on dry ice, and later homogenized in Tris-EDTA-sucrose buffer, pH 7.4, at 4 dc. Microsomes were isolated by ultracentrifugation, and aromatase activity was assayed by a tritiated water release method using [1.e-3H(N)]androstenedione as a substrate. Incubation time totaled 7.5 hours. Under such conditions, oviductal aromatase produced estrogen at a constant rate. The optimum temperature for the enzyme appears to lie between 33 and 37°C. The pH has a broad optimum between 7.4 and 10.4. The Km and the Vmax for androstenedione were 188.1±30.2 nM and 1.42±0.11 fmol of estrogen produced/min/mg microsomal protein, respectively. Comparison of these parameters with those of human placental aromatase (Km =123.0 nM, Vmax =113.57 fmol of estrogen produced/min/mg microsomal protein) shows that both enzymes had similar affinity for androstenedione, but frog oviduct had much less aromatase activity than human placenta. To further characterize the frog enzyme, the effects of two mammalian aromatase inhibitors, 10-propargylestr-4ene- 3,17-dione (PED) and 4-hydroxyandrostenedione (4-0H-A) were tested. Since progesterone has been found to inhibit estrogen production in the frog ovary, it was also tested for inhibitory activity. Although PED did not inhibit the aromatase activity, 4-0H-A inhibited it in a dose-dependent fashion. Progesterone also showed inhibitory ability. Since testosterone has been reported to be the primary androgen in the circulation of female frogs, serum testosterone levels in frogs with mature ovaries were measured and compared with the Km of the oviductal aromatase. The testosterone levels showed a wide range from 17.4 to 176.6 nM with a mean value of 90.6± 17.6 nM. These results suggest that the oviductal aromatase could function in converting blood androgens to estrogens to stimulate oviduct growth.