Changes in testis cyclic nucleotide metabolism during trout spermatogenesis
Creators: name: Davis, Jillian Frances
Publisher: name: University of British Columbia
Year: 2010

description: The concentrations of cyclic GMP and cyclic AMP, in rainbow trout (Salmo gairdnerii) testis, were determined during hormonally-induced spermatogenesis. In immature trout testis, cyclic GMP and cyclic AMP concentrations were approximately equal (≃ 2 μmol/kg wet weight). An abrupt 10 fold decrease in cyclic GMP occurred during mitotic activity and testis growth prior to meiosis. Cyclic AMP decreased 2 fold during this time. A further gradual 5 fold decrease in cyclic GMP occurred during the remainder of spermatogenesis. No significant change in cyclic AMP occurred during development after the initial 2 fold decrease. Cyclic AMP and cyclic GMP phosphodiesterase activities, measured at both high (millimolar) and low (micromolar) substrate concentrations were determined in trout testis during hormonally-induced spermatogenesis. When measured at millimolar substrate, cyclic GMP phosphodiesterase activities did not change significantly throughout spermatogenesis. Cyclic AMP phosphodiesterase activities, measured at millimolar substrate, decreased about 50% prior to meiosis and then increased, during spermatid differentiation, to attain a final activity slightly greater than that observed in immature testis. When measured at micromolar substrate, in the presence of EGTA, cyclic GMP phosphodiesterase activities decreased about 50%, while cyclic AMP phosphodiesterase activities, measured under the same conditions, increased 20 fold during the course of spermatogenesis. A detailed study of cyclic GMP phosphodiesterase activities, measured at micromolar substrate, in the presence and absence of Ca²⁺, showed there was no change in Ca²⁺-dependent cyclic GMP phosphodiesterase activity at the time of development at which the large decrease in cyclic GMP is observed. DEAE-cellulose profiles of cyclic nucleotide phosphodiesterase activities, from trout testis at different stages of development, showed two peaks of cyclic AMP activity and one peak of cyclic GMP activity. The latter cochromato-graphed with the first cyclic AMP activity peak. A large increase in the first cyclic AMP phosphodiesterase peak occurred when spermatids were maturing, without a concurrent increase in cyclic GMP phosphodiesterase activity. This indicates the induction of a specific, high-affinity cyclic AMP phosphodiesterase during the meiotic stage of testicular development. Phosphodiesterase assays using micromolar substrate concentrations, on subcellular fractions, demonstrated that about 85% of cyclic AMP hydrolysis and 80% of cyclic GMP hydrolysis was soluble. Both peaks of cyclic AMP activity were observed in the DEAE-cellulose profile of a 100,000xg supernatant (soluble) fraction from trout testis. The small amount of particulate cyclic AMP phosphodiesterase activity, in the 100,000xg pellet fraction, was associated mainly with the second peak on the DEAE-cellulose profile. Kinetic analyses of homogenate phosphodiesterases from mature testis showed only high affinity cyclic AMP activity (apparent Kms for cyclic AMP of 1.1 μM and 0.3 μM) and both low and high affinity cyclic GMP activity (apparent Kms for cyclic GMP of 220 μM and 8 μM). Kinetic analyses of cyclic AMP hydrolysis by the two peaks of phosphodiesterase activities purified on DEAE-cellulose, confirmed the presence of high affinity activities. Guanylate cyclase activities were assayed in the soluble and particulate fractions from .immature testis and from testis at different stages of hormonally-induced development. There was an approximate 1:2 ratio of soluble to particulate guanylate cyclase activities in .immature and in mature trout testis. A 3 fold decrease in both soluble and particulate guanylate cyclase activities coincided with the 10 fold decrease in cyclic GMP concentration observed in maturing trout testis. Thus, in trout testis during spermatogenesis, cyclic GMP concentrations reflect the developmental modulation of guanylate cyclase activity, rather than that of cyclic GMP phosphodiesterase.