Adenylyl Cyclase Activities in Ovarian Tissues. I. Homogenization and Conditions of Assay in Graafian Follicles and Corpora Lutea of Rabbits, Rats, and Pigs: Regulation by ATP, and Some Comparative Properties

Abstract

Responsiveness of ovarian adenylyl cyclases to luteinizing hormone (LH), found to be 5 to 10-fold in cell-free preparations under optimal conditions, required gentle homogenizations and storage in sucrose-containing media. Assay conditions required the use of an ATP-regenerating system consisting of creatine kinase, creatine phosphate, and myokinase for the preservation of ATP levels. LH-stimulated adenylyl cyclase (AC) in rabbit CL showed the following properties: 1) The pH optimum of basal activity was about 8.0; that of LH-stimulated activity was about 7.5. 2) The relative response to LH was low (1.5 to 2-fold) at 0.1 mM ATP and increased with increasing ATP, but not with increasing GTP. At low (0.1 mM) ATP, GTP increased catalytic efficacy of the system, both in the absence and in the presence of LH (no effect on relative stimulation). 3) The optimal relative stimulation by LH was obtained at about 1.0 mM MgCl2 in excess of added magnesium-binding ingredients. 4) The sensitivity to stimulation by LH (about 0.2 mug/ml NIH-LH-B8) was unaffected by either pH, nucleotides (ATP and GTP), or MgCl2 concentration. 5) Under the assay conditions used, activity was stimulated by prostaglandin E1 (PGE1) about 1.5 to 2-fold, and by epinephrine about 3 to 4-fold. In all aspects tested, LH-stimulated AC in rat CL resembled that in rabbit CL, except that about 5-fold higher concentrations of NIH-LH-B8 were needed for half-maximal stimulation. The AC activity in pig Graafian follicles, however, differed from that in rabbit CL in that 1) the ATP concentration needed for optimal stimulation by LH was lower (in the micromolar rather than the millimolar range); 2) catecholamines elicited only a 1.3 to 1.4-fold stimulation; and 3) NIH-LH-B8 elicited half-maximal stimulation at 0.008 to 0.020 mug/ml. We were unable to detect LH-responsive AC activity in either homogenates or washed particles of CL from either cycling or pregnant pigs. LH fractions of three origins (human, bovine, and ovine) and of varying specific activities (from 0.041 to 2.0 NIH-LH-S18 units/mg) were tested and the relative potencies by OAAD assay were found to correlate well with the relative potencies in the adenylyl cyclase assays (rat CL, rabbit CL, and pig follicles), consistent with the possibility that AC receptors are responsible for biologic actions of LH.

Soluble Mn2+-dependent Adenylate Cyclase Activity in the Testis of the Blue Fox (Alopex lagopus)

“…Enzyme activity, as in the rat and mouse, is approximately 10 times higher than that in the human testis [9].…”

“…The reduction in MnAC activity in the presence of NEM (> I mM) or PME (100 mM) suggests that free-SH groups and S-S bridges are essential enzyme components, as in the rat [7] and man [9]. The reduction in enzyme activity at high pH is most likely due to oxidation of Mn2+ to, for example, Mn4+.…”

“…The soluble MnZ+-dependent adenylate cyclase (MnAC) of the testis has been examined using broken cell systems in the rat [2,7] and man [9]. In both these species, soluble MnAC activity is associated exclusively with spermatids [2,13] and their physicochemical and kinetic properties are virtually identical [7,9,10].…”

“…In both these species, soluble MnAC activity is associated exclusively with spermatids [2,13] and their physicochemical and kinetic properties are virtually identical [7,9,10]. There is, however, no information available on soluble MnAC activity in the testis of seasonal breeders such as the blue fox, which show marked variations in reproductive function during the course of the year.…”

Changes in isoproterenol-stimulated adenylate cyclase activity in rat testicular tissue during cryptorchidism and after orchidopexy

“…At 42, 59 and 120 days of age, one group each of control, cryptorchid and surgically treated animals was killed. Surgical techniques and treatment of the animals were as described previously (Jahnsen et al, 1981 ;Karpe et al, 1981) (Salomon, Londos & Rodbell, 1974), with the modifications described by Birnbaumer, Yang, Hunzicker-Dunn, Bockaert & Duran (1976).…”