Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

Particulate and soluble adenylate cyclase activities of mouse male germ cells

Germ cells from the mouse testis possess both a particulate and a soluble form of adenylate cyclase (EC 4.6.1.1). Germ cell adenylate cyclase activity is Mn⁺⁺ dependent and is not stimulable with either NaF or 5′guanylylimidodiphosphate. Both particulate and soluble adenylate cyclase specific activities increase as germ cells progress through their differentiative stages, but epididymal spermatozoa seem to lack a significant amount of soluble activity. Somatic cells of the seminiferous tubule possess only a membrane bound activity, which is Mg⁺⁺ and Mn⁺⁺ dependent, NaF and 5′guanylylimidodiphosphate stimulable. It is suggested that germ cell adenylate cyclases represent incomplete forms of the enzyme, devoid of regulative subunits.     

Changes in adenylate cyclase and phosphodiesterase activities during the growth cycle of adult rat hepatocytes in primary culture

Long-term primary adult rat hepatocyte cultures show growth-state-dependent changes in adenylate cyclase and cAMP phosphodiesterase activities. Cellular adenylate cyclase activity decreases to undetectable levels within 1 day postplating, reappears on Days 4-5, and becomes maximal on Day 9. Membrane adenylate cyclase and cellular cAMP formation are insensitive to glucagon during log phase (Days 4-8) but not during lag (Day 1) or stationary phase (Day 12). Cyclic AMP phosphodiesterase activities (soluble and particulate) fall approximately equal to 70% by Day 2 but recover as proliferation begins. By contrast, the particulate phosphodiesterase assayed at 100 microM cAMP, decreased during Days 0-2. These observations simulate changes seen during liver proliferative transitions in vivo and, therefore, further support the use of these cultures as a developmental model.     

Activation of spermatozoan adenylate cyclase by a low molecular weight factor in porcine seminal plasma

The ejaculated porcine spermatozoa were fractionated into the cytosol, membrane, midpiece plus tail (flagella) and head fractions, and their adenylate cyclase activities were measured. About 65% of the total activity was located in the flagella fraction. For all the fractions, Mn2+-dependent adenylate cyclase activity was about 20 times higher than Mg2+-dependent activity, and guanine nucleotides, fluoride, and other reagents tested did not activate adenylate cyclase. The results suggest that the GTP-dependent regulatory subunit is absent in porcine spermatozoa. The porcine seminal plasma was found to stimulate the adenylate cyclase activity in spermatozoa. The stimulating factor in porcine seminal plasma was partially purified by gel filtration and the molecular weight of the factor appeared to be between 200 and 300. The partially purified factor is heat stable and is not inactivated by treatment with Pronase, trypsin, phospholipase A2 or D but is inactivated by acid hydrolysis. It is easily soluble in water, partially soluble in methanol, and insoluble in ethanol, ethyl ether, chloroform, or acetone. The activation of sperm adenylate cyclase by the factor occurred without a time lag. The activating effect was dose-dependent, saturated at high dose, and ascribed to the increase of the maximal velocity (Vmax). The effect of the factor appears to be limited to adenylate cyclase in spermatozoa; the factor activated adenylate cyclase both in porcine and bovine spermatozoa but failed to activate those in other porcine tissues. The factor was shown to activate the enzyme not only in the ejaculated spermatozoa but also in the epididymal sperm. The factor was also found to elevate the cAMP level in the intact porcine spermatozoa. The factor enhanced the motility of corpus and cauda epididymal spermatozoa. These findings indicate the possibility that this factor initiates the spermatozoan motility upon ejaculation through directly activating adenylate cyclase.     

The subcellular localization of calmodulin, cyclic AMP phosphodiesterase, and adenylate cyclase in bovine adrenal medulla

The subcellular localization of calmodulin, cyclic nucleotide phosphodiesterase, and adenylate cyclase was studied in bovine adrenal medulla. Approximately 70% of the calmodulin and 90% of the cAMP phosphodiesterase activities were found colocalized in the cytoplasm. The subcellular distribution of adenylate cyclase closely paralleled the distribution of acetylcholinesterase, a marker for plasma membranes. The fraction of calmodulin which is particulate in nature has a distribution profile very similar to that of adenylate cyclase. The chromaffin granule fraction contained only 0.86% of the total cAMP phosphodiesterase, 0.41% of the total adenylate cyclase, and 1.4% of the total calmodulin.     

Isolation of cyclic AMP and cyclic GMP by thin-layer chromatography. Application to assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

A procedure is described for isolation of cAMP and cGMP by thin-layer chromatography on polyethylenimine cellulose. Chromatographs are developed (descending) twice in the same direction with two different solvents. This procedure separates cAMP and cGMP from other radioactive metatolites of [³H] or [¹⁴C] ATP or GTP. Application of this isolation method to assay of adenylate cyclase, (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2), and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) has proven convenient and provides results of unusual quality.     

Characterization of soluble uterine cyclic nucleotide phosphodiesterase.

Soluble cyclic nucleotide phosphodiesterase of rat uterus displays distinct structural and regulatory properties. Like phosphodiesterases from many mammalian sources the soluble uterine enzyme system exhibits nonlinear Lineweaver--Burk kinetics with cyclic adenosine 3':5'-monophosphate (cAMP) as substrate (apparent Kms congruent to 3 and 20 micron) and linear kinetics with cyclic guanosine 3':5'-monophosphate (cGMP) as substrate (apparent Km congruent to 3 micron). Unlike most other mammalian phosphodiesterases, however, numerous separation procedures reveal only a single form of uterine phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP. A single form of the enzyme is observed upon sucrose gradient centrifugation (7.9 S), agarose gel filtration, and DEAE-cellulose chromatography at either pH 8.0 OR 6.0. Heat denaturation (50 degrees C) of soluble uterine phosphodiesterase causes the loss of both cAMP and cGMP hydrolytic activities at the same rate. Isoelectric focusing reveals major (pI = 5.2) and minor forms (pI = 5.8) of phosphodiesterase which both catalyze the hydrolysis of the two cyclic nucleotide substrates. In vivo administration of estradiol produces identical decreases in the activities of cAMP and cGMP phosphodiesterase. These results raise the possibility that the uterus contains a single form of soluble phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP.     

Cell-cycle-specific activity of cyclic nucleotide phosphodiesterase in Physarum polycephalum

The cell-cycle-related activities of the cAMP- and cGMP-dependent phosphodiesterases of Physarum polycephalum were assayed. The activities of plasmodial homogenate and of selected subcellular fractions were measured. The results suggested the presence of both cAMP- and cGMP-dependent phosphodiesterase in the isolated nuclei of P. polycephalum. In addition, they reveal that the cAMP- and cGMP-dependent phosphodiesterase activities of the subcellular fractions fluctuate throughout the cell cycle. The whole-cell homogenates exhibit no cell-cycle-related changes in the presence of 5 X 10(-4) M cGMP. Kinetic data suggest the presence of multiple phosphodiesterase activities in the homogenate and its particulate fractions for the cGMP-dependent enzyme. Multiple cAMP activities are also suggested for the particulate fractions. The Km values indicate that the substrate affinities of the phosphodiesterases from P. polycephalum are similar to those found previously in mammalian systems.     

Aggregation states of cyclic nucleotide phosphodiesterase of murine thymocytes

In murine thymocytes cyclic nucleotide phosphodiesterase is represented by cAMP- and cGMP-specific forms. cAMP and cGMP phosphodiesterase activities showed anomalous kinetic behaviour indicative of 'low' and 'high' affinity enzyme forms. Sucrose density gradient centrifugation resolved only 'low' affinity forms of cAMP and cGMP phosphodiesterases. Gel filtration on Ultragel Aca 34 column showed that cAMP and cGMP phosphodiesterases are probably oligomeric enzymes. Storage of enzyme preparation at 4 degrees C for 24-48 h led to a decrease of higher molecular weight form and enhancement of cAMP and cGMP phosphodiesterase activities.     

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.     

Changes in the system of cyclic nucleotides in irradiated body tissues

It has been shown that the content of cAMP in the rat thymus, spleen, and liver 1 and 3 days after gamma-radiation with 7.5 Gy decreases, and that of cGMP increases. Analogous dynamics has been revealed when studying adenylate cyclase and guanylate cyclase activities. The activity of cAMP and cGMP phosphodiesterases increased during the first period of study but subsequently it showed no distinction from the initial data level. The revealed postradiation changes in the content of cyclic nucleotides seem to be basically caused by the cyclases activity alterations.     

Cyclic AMP affects the haemocyte responses of larval Galleria mellonella to selected antigens

Signal transduction of the innate immediate responses of insect haemocytes to foreign matter is rarely considered. Herein using a combination of adenylate cyclase inhibitors and activators and phosphodiesterase inhibitors we determined that cyclic adenosine monophosphate (cAMP) at high levels normally impairs non-self response. Haemocyte contact with glass and bacteria lowered cAMP in vitro. Inactive phosphodiesterases, including type 4, impaired haemocyte reactions in vitro. Using the drugs in vivo to modulate adenylate cyclase and phosphodiesterases altered the total and types of haemocytes. Adenylate cyclase inhibitors and etazolate (a type 4 phosphodiesterase inhibitor) alone produced changes in the haemograms similar to those caused by Bacillus subtilis. Sequential injections of an enzyme modulator followed by B. subtilis impaired bacterial removal due (1) in the case of enzyme inhibitors, to the removal of haemocytes prior to bacterial challenge and (2) in the case of forskolin and IBMX to the shut-down of the haemocytes. Activating adenylate cyclase or inhibiting phosphodiesterase impaired bacterial removal when co-injecting the compounds and bacteria.     

Sperm maturation in the domestic cat

The epididymis is essential for sperm development and maturation, and, subsequently, the ability of spermatozoa to penetrate and fertilize the female gamete. Functional differences in segments of the long tubule are reflected by histological differences among epididymal regions. The feline epididymis can be divided into six different regions according to their histological differences. A marked increase in sperm concentration occurs between regions 2 and 3, indicating resorption of fluid in region 2, a concept supported by the histological characteristics of the epithelium. At the transition between regions 4 and 5, located between the caput and corpus epididymides, histological characteristics change from being that of a maturation function to being typical of a storage function. Migration of the cytoplasmic droplet and induction of motility occur in this same region. Proteins are secreted from epithelial cells in the feline epididymis by merocrine and apocrine secretion, although the functions of different feline epididymal proteins have not been determined. Hypotaurine, taurine and, probably, alkaline phosphatase are produced by the feline epididymis. During epididymal transit the percentage of immature, unviable and morphologically abnormal spermatozoa decreases, indicating the existence of a mechanism that removes abnormal spermatozoa. In contrast, the percentage of spermatozoa with abnormal tails increases slightly during epididymal transit. Most of the distal droplets present on spermatozoa in the cauda epididymis are lost at or after ejaculation. Additional knowledge of the feline epididymis should be beneficial for developing sperm preservation protocols and advance the prospects for effective male contraceptive methods.     

cAMP levels and in situ measurement of cAMP related enzymes during yeast-to-hyphae transition in Candida albicans

Intracellular levels of cAMP and specific activities of adenylate cyclase, cAMP phosphodiesterase and cAMP-dependent protein kinase were measured during filamentation in the dimorphic fungus Candida albicans. Enzymatic assays were performed in permeabilized cells under conditions prevented endogenous proteolysis. The variations observed in cAMP levels were mainly accounted for by variations in the specific activities of adenylate cyclase and cAMP phosphodiesterase at different stages during germ tube formation. cAMP-dependent protein kinase, measured with kemptide as exogenous substrate, was developmental regulated. Some properties of the enzymatic activities from cell-free extracts are described.     

Reduction in soluble guanylyl cyclase-specific activity following prolonged treatment of porcine pulmonary artery with nitric oxide

In a newly characterized cultured porcine pulmonary artery (PA) preparation, 24-h treatment with the nitric oxide (NO) donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) decreased the response to acutely applied DETA-NO compared with 24-h control (-log EC(50) 6.55 +/- 0.12 and 5.02 +/- 0.21, respectively). Treatment of PA with the cell-permeable superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride, did not change NO responsiveness in either freshly prepared or 24-h DETA-NO-treated PA. cGMP and cAMP phosphodiesterase activities were approximately equal in PA. Twenty-four-hour DETA-NO treatment did not change either cGMP or cAMP phosphodiesterase activities. Twenty-four hours in culture had no significant effect on soluble guanylyl cyclase (sGC) subunit mRNA expression, but 24-h DETA-NO treatment significantly decreased the expression of both sGCalpha(1) and sGCbeta(1). sGCbeta(1) protein expression was 42 +/- 4 ng/mg soluble protein. Twenty-four hours in culture without and with DETA-NO reduced sGCbeta(1) protein expression (36 +/- 3 and 31 +/- 3 ng/mg soluble protein, respectively, P < 0.025). Basal tissue cGMP [(cGMP)(i)] was significantly increased, and NO-induced (cGMP)(i) was significantly decreased by 24-h DETA-NO treatment. (cGMP)(i) normalized to the amount of sGC protein expressed in PA was significantly lower in PA treated for 24 h with DETA-NO compared with both freshly isolated and 24-h cultured PA. We conclude that prolonged NO treatment induces decreased acute NO responsiveness in part by decreasing both sGC expression and sGC-specific activity.     

Rapid nitric oxide-induced desensitization of the cGMP response is caused by increased activity of phosphodiesterase type 5 paralleled by phosphorylation of the enzyme

Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.     

Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited by rat spermatozoa

As mammalian spermatozoa migrate through the epididymis, they acquire functionality characterized by the potential to express coordinated movement and the competence to undergo capacitation. The mechanisms by which spermatozoa gain the ability to capacitate during epididymal transit are poorly understood. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation, because this process is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility. Western blot and immunocytochemical analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues from the sperm head. As cells pass from the caput to the cauda epididymis, tyrosine phosphorylation becomes confined to a narrow band at the posterior margin of the acrosomal vesicle. Epididymal maturation of rat spermatozoa was also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. Immature caput spermatozoa were incapable of exhibiting this response, despite the apparent availability of cAMP and protein kinase A. These findings help to clarify the biochemical changes associated with the functional maturation of spermatozoa during epididymal transit.     

Substrate and effector specificity of a guanosine 3':5'-monophosphate phosphodiesterase from rat liver.

Guanosine 3':5'-monophosphate phosphodiesterases, which appear to be under allosteric control, have been partially purified from rat liver supernatant and particulate fractions. The preferred substrate for both phosphodiesterases was cGMP (Km values: cGMP less than cIMP less than cAMP). At subsaturating concentrations of substrate, the phosphodiesterases were stimulated by purine cyclic nucleotides. The order of effectiveness for activation of cyclic nucleotide hydrolysis was cGMP greater than cIMP greater than cAMP greater than cXMP. Using cAMP derivatives as activators of cIMP hydrolysis, modifications in the ribose, cyclic phosphate, and purine moieties were shown to alter the ability of the cyclic nucleotide to activate the supernatant enzyme. cGMP, at concentrations that stimulated cyclic nucleotide hydrolysis, enhanced chymotryptic inactivation of the supernatant phosphodiesterase. At similar concentrations, cAMP was not effective. It appears that on interaction with appropriate cyclic nucleotides, this phosphodiesterase undergoes conformational changes that are associated with increased catalytic activity and enhanced susceptibility to proteolytic attack. Divalent cation may not be required for the nucleotide-phosphodiesterase interaction and resultant change in conformation.     

Influence on adipocyte plasma membrane bound protein kinase by feedback regulator.

Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.     

Presence of cyclic nucleotide phosphodiesterases PDE1A,existing as a stable complex with calmodulin,and PDE3A in human spermatozoa

Mammalian sperm motility, capacitation, and the acrosome reaction are regulated by signal transduction systems involving cAMP as a second messenger. Levels of cAMP are controlled by two key enzymes, adenylyl cyclase and phosphodiesterases (PDEs), the latter being involved in cAMP degradation. Calmodulin-dependent PDE (PDE1) and cAMP-specific PDE (PDE4) activities were previously identified in spermatozoa via the use of specific inhibitors. Here we report that human sperm PDEs are associated with the plasma membrane (50%-60%) as well as with the particulate fraction (30%-50%) and have more affinity for cAMP than cGMP. Immunocytochemical data indicated that PDE1A, a variant of PDE1, is localized on the equatorial segment of the sperm head as well as on the mid and principal pieces of the flagellum, and that PDE3A is found on the postacrosomal segment of the sperm head. Immunoblotting confirmed the presence of PDE1A and PDE3A isoforms in spermatozoa. Milrinone, a PDE3 inhibitor, increased intracellular levels of cAMP by about 15% but did not affect sperm functions, possibly because PDE3 represents only a small proportion of the sperm total PDE activity (10% and 25% in Triton X-100 soluble and particulate fractions, respectively). PDE1A activity in whole sperm extract or after partial purification by anion-exchange chromatography was not stimulated by calcium + calmodulin. Results obtained with electrophoresis in native conditions indicated that calmodulin is tightly bound to PDE1A. Incubation with EGTA + EDTA, trifluoperazine, or urea did not dissociate the PDE1A-calmodulin complex. These results suggest that PDE1A is permanently activated in human spermatozoa.