FSH and LH/hCG Responsive Adenylyl Cyclases in Adult Rat Testes: Methodology and Assay Conditions

Abstract

The activities of the membrane bound testicular adenylyl cyclases (AC) and their responsiveness to LH/hCG and FSH were examined in membrane particles from adult rat testes. The assay requires the use of an ATP regenerating system consisting of creatine kinase, creatine phosphate, and myokinase for the maintenance of ATP levels. LH/hCG and FSH responsive AC in the rat testes showed the following properties:1. GTP and GMP‐P(NH)P showed a concentration‐dependent stimulation of AC activity with an apparent Km of 2 times 10‐6M.2. The relative response to hCG and FSH was not influenced by ATP concentration if the free magnesium (Mg2+) concentration was kept constant. The AC activitity increased with increasing concentration of ATP (with an apparent Km of approximately 1.0 mM) using Mg2+ of 0.4 mM in excess of ATP and EDTA.3. At low ATP (0.15 mM) Mg2+ caused a concentration‐dependent increase in both basal and hormone stimulated activities. At high ATP (1.65 mM) Mg2+ caused a concentration‐dependent increase in basal AC activity, but a gradual decrease in hCG and FSH stimulated AC activities at Mg2+ concentrations higher than 5 mM.4. Decreasing Mg2+ was associated with increasing relative responses to hCG and FSH. Optimal relative stimulation by FSH and hCG was obtained when Mg2+ was 0.4‐1.0 mM in excess of the added Mg2+ binding ingredients (ATP and EDTA). Maximal relative stimulation by fluoride was achieved at much higher Mg2+ (5mM).5. Increasing pH from 7.0 to 8.2 was associated with increasing basal activity. Optimal hormonal effects (relative response) were achieved using an incubation medium of pH of 7.00–7.40.6. Time curves with GTP and GMP‐P(NH)P with or without hormones showed linearity up to 20 min.7. Hormonal activation by gonadotropins (hCG and FSM) was seen using both membrane particles and homogenates, and in the presence of either GTP or GMP‐P(NH)P.8. Relative responses to hCG and FSH were slightly better in membrane particles than in homogenates, and GMP‐P(NH)P reinforced the FSH response better than GTY. Similar relative stimulation by hCG was obtained using both nucleotides.9. Complete maintenance of AC activity as well as hormonal responses were seen after freezing at ‐70°C for two weeks.10. LH/hCG responsive AC activity was associated with Leydig cells, whereas FSH responsive AC was entirely localized in the seminiferous tubules. The finding of an FSH responsive AC in seminiferous tubules of adult rats does not support the notion that “uncoupling” of the FSH receptors from the AC is a primary reason for the loss of FSH response occurring during sexual maturation.The finding of an FSH responsive AC in seminiferous tubules of adult rats does not support the notion that “uncoupling” of the FSH receptors from the AC is a primary reason for the loss of FSH response occurring during sexual maturation.