FSH and LH (6,7). Since mature spermatozoa have no protein synthetic ability, the adenylate cyclase in this cell type must be synthesized at a specific developmental stage in which this distinctive Mn2+-sensitive adenylate cyclase appears in the testis. The properties of the distinctive adenylate cyclase system have been studied during its development in testis and epididymal sperm. It has been found that the properties of the Mn2+-sensitive adenylate cyclase in testis and sperm homogenates are similar. However, upon centrifugation, the bulk of activity in testis is in the soluble fraction of cytoplasm whereas in sperm, the adenylate cyclase system is firmly associated with membranes.
MATERIALS AND METHODSRats (Charles River CD®) of various age groups were used. Immature rats younger than 21 days were kept in groups of ten with their mothers. Purina chow diet and water ad lib were provided to nursing mothers, suckling and weaned (more than 21 days old) rats. They were maintained at a temperature of 22-23°and a 14-hr light and a 10-hr dark period each day.[a-32P]ATP was purchased from International Chemical and Nuclear Corp., creatine phosphate and creatine phosphokinase (skeletal muscle, rabbit) from Calbiochem.The rats were sacrificed by decapitation and the testes were removed, decapsulated, freed of visible blood vessels and placed in ice-cold 5 mM Tris HCl buffer (pH 7.2) containing 3 mM MgCl2 and 1 mM EDTA (5 mM Tris buffer). In experiments where seminiferous tubules and interstitial cells were separated, the testes, after being excised, were placed in icecold Krebs-Ringer phosphate buffer (pH 7.2) containing 10 mg of bovine serum albumin per ml (KRP-BSA), with half of the usual calcium ion content replaced by the equivalent amount of sodium.Seminiferous tubules were isolated from testes by microdissection using procedures previously described (9).Interstitial cells were isolated by collagenase (Worthington; 1 mg/ml per 100 mg of tissue) treatment of testes for 5 min in a metabolic shaker at 60 cycles/min; cells were separated from the undigested tubules by filtration through a double layer of nylon mesh, and washed four times with KRP-BSA buffer. The cell fraction thus obtained is enriched with interstitial cells and is usually composed of 65% interstitial, 15% tubular, and 20% erythrocytes. Spermatozoa were collected in 5 mM Tris buffer from the caput, corpus, and cauda of epididymis. The epididymis was ligated in situ into these portions and separated accordingly after excision.
The soluble form of rat germ cell adenylate cyclase was inhibited by compounds with a catechol moiety. Among the naturally occurring catechols tested, catechol estrogens were the most potent inhibitors. Catechol estrogens at 2-6 microM inhibited enzyme activity by 50% and almost completely at 30-100 microM concentration. The inhibitory activity of catechol estrogens depends on the catechol moiety of the molecule. Catechol per se also inhibited the activity of this enzyme, 50% inhibition being achieved at about 11 microM. The two hydroxyls of the catechol moiety are essential for the inhibitory interaction with the enzyme. Thus, aromatic compounds containing only one hydroxyl group in the benzene ring, such as tyrosine, phenylephrine, estradiol, and 6 alpha-hydroxyestradiol were either completely inactive or had marginal inhibitory activity at concentrations up to 0.3-1 mM. Moreover, methylation of the hydroxyl groups of the catechol moiety of the catechol estrogens as in 2-methoxyestradiol 3-methyl ether rendered the catechol estrogens inactive. The inhibitory potency of these compounds varied greatly depending on the structure associated with the catechol ring. Thus, compounds in which catechol is associated with an aliphatic side chain, such as dopamine, L-dopa, norepinephrine, and isoproterenol, were about 11- to 34-fold less potent than catechol. On the other hand, compounds in which catechol is associated either with a hydroaromatic ring system, as in apomorphine, or with an alicyclic ring system, as in catechol estrogens, were about 2- to 5-fold more potent than catechol. The inhibitory effect of dopamine, apomorphine, and catechol estrogens was not affected by specific D-1 or D-2 antagonist, indicating that they do not act via receptors for dopamine.
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