Development of a Mn-2+-sensitive, "soluble" adenylate cyclase in rat testis.

Abstract

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.

Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

“…Sperm must swim, find the egg and undergo the acrosome reaction (AR) to fertilize it [1]. cAMP levels influence sperm ionic fluxes, motility and the AR [2].Two types of adenylyl cyclases (ACs) are expressed in mammals, transmembrane ACs (tmACs) and soluble AC (sAC) molecularly identified in rat testis [3] and sperm [4]. Restricted to the plasma membrane, tmACs are regulated by heterotrimeric G-proteins and stimulated by forskolin [5].…”

Manganese inhibits spontaneous nuclear maturation in bovine cumulus-enclosed oocytes

“…The adenylate cyclase enzyme of mature mammalian spermatozoa is not activated by known hormones and is not sensitive to stimulation by cholera toxin and sodium fluoride (Cheng and Boettcher 1979;Garbers and Kopf 1980;Forte et al 1983). Moreover, Braun and Dods (1975) have identified an adenylate cyclase activity that has an obligatory requirement for Mn 2+ . These observations in oocytes and spermatozoa led to the initial hypothesis for the present study that male and female gametes might possess an adenylate cyclase enzyme with common characteristics and that manganese could also stimulate the activity of the oocyte adenylate cyclase and, therefore, affect oocyte maturation.…”

“…It is possible that male and female gametes share an adenylate cyclase with common characteristics because the adenylate cyclase enzyme of mature mammalian spermatozoa is not activated by known hormones, sodium fluoride, or cholera toxin (Cheng and Boettcher 1979;Garbers and Kopf 1980;Forte et al 1983). The activity of the adenylate cyclase in spermatozoa is increased by manganese and magnesium; Braun and Dods (1975) have also identified an adenylate cyclase activity that has an obligatory requirement for Mn 2+ . If Mn 2+ also activates oocyte adenylate cyclase, it could potentially have an effect on nuclear maturation.…”

Inhibitors of adenylate cyclase from ejaculated human spermatozoa

“…The incuba¬ tion tube was immediately transferred to a boiling water bath for 3 min. (Braun & Dods (1975) suggested the use of EDTA to prevent non-enzymic formation of cyclic AMP in the presence of Mn2+ during the boiling process.) The tube was then allowed to cool.…”