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C Gagnon

Publications and source records attributed to C Gagnon.

At least 145 records · Page 8Linked to original sources
PubMed1986

Effects of protease inhibitors and substrates on motility of mammalian spermatozoa.

A series of protease inhibitors were tested on the motility of human, rat, bull, and rabbit demembranated reactivated spermatozoa. Some inhibitors, including aprotinin, boc-gln-leu-lys-H, and D-phe-pro-arg-H, could inhibit motility as well as prevent initiation of motility. In general, with the exception of aprotinin, protease inhibitors were more potent in preventing the initiation of movement than in blocking motility of demembranated spermatozoa. Protease substrates could also block sperm motility. Of the substrates tested only those with arg or lys ester bonds were active. The inhibition of motility by protease substrates was reversible, as once spermatozoa hydrolyzed the added exogenous protease substrates, motility reappeared. The importance of ester bond in the inhibitory action of protease substrates was confirmed by experiments that showed the lack of effect of pre-hydrolyzed protease substrates. The results suggest that a serine protease with lys and arg ester bond specificity is involved in the control of sperm motility. The fact that protease substrates also block motility of intact spermatozoa further emphasizes the physiological relevance of this new regulatory system.

Animals↗
PubMed1985

The relationship between glycolysis, mitochondrial respiration, protein-carboxyl methylation and motility in hamster epididymal spermatozoa.

Endogenous protein-carboxyl methylase activity can be measured in intact motile spermatozoa by using [3H]methionine as a precursor of the methyl donor, S-adenosylmethionine (SAM). Since the conversion of methionine to SAM requires ATP, the relationship between the energy metabolism of spermatozoa and methylation was investigated using inhibitors of glycolysis and mitochondrial respiration. When hamster spermatozoa from cauda epididymides were incubated in 12.2 mM glucose, glycolysis was progressively inhibited as concentrations of 2-deoxyglucose (2-DOG) increased. On the other hand, endogenous protein-carboxyl methylation showed a biphasic response being stimulated at low concentrations of 2-DOG and inhibited at higher concentrations. Sperm movement was also altered by 2-Dog. Increasing concentrations of 2-DOG in the incubation medium resulted in an increase in beat amplitude and a corresponding decrease in beat frequency. When the glucose concentration of the medium was reduced to 5 mM, protein methylation was inhibited at all concentrations of 2-DOG. The biphasic effect of 2-DOG on methylation at 12.2 mM and its monophasic effect at 5 mM suggested that this reaction was related to the effective glucose concentration. To investigate this possibility, endogenous protein-carboxyl methylation was measured after incubation of sperm in glucose concentrations ranging from 0-48.8 mM. Low glucose concentrations stimulated protein methylation (up to 6.1 mM for maximal effect), but further increases in the glucose concentration (10-48.8 mM) suppressed the rate of protein methylation to that observed in the absence of glucose. 2-Deoxyglucose decreased conversion of pyruvate to CO2 consistent with diminished mitochondrial respiration. The inhibition of mitochondrial respiration by antimycin A also inhibited endogenous methylation and motility. The results of these studies suggest that maximal endogenous protein-carboxyl methylation in intact spermatozoa from hamster epididymides depends upon active glycolysis as well as mitochondrial respiration.

Animals↗
PubMed1984

Purification and characterization of protein methylesterase from rat kidney.

Protein methylesterase, an enzyme that hydrolyzes protein methyl esters, has been purified. The purification procedure includes ammonium sulfate and acid precipitations, and chromatographies on Sephadex G-100, Polybuffer exchanger 94, and matrex gel Green A. With this procedure, protein methylesterase was purified 1190-fold with a 28% recovery in activity. Its molecular weight has been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 31,000. Protein methylesterase has an optimum pH of 4.0 and an isoelectric point of 4.45. The enzyme is stable over a wide range of pH (2-10). The Km for ovalbumin methyl esters was estimated at 5.9 microM. Monovalent ions had little effect on activity while divalent ions at concentrations above 50 mM inhibited protein methylesterase activity in a concentration-dependent manner.

Animals↗
PubMed1984

Phosphorylation of zymogen granule membrane proteins in intact rat pancreatic acinar cells.

Phosphorylated substrates of molecular weights 130,000, 70,000, and 29,000, were identified by SDS-gel electrophoresis in zymogen granule membranes of rat pancreatic acinar cells incubated in vitro with protein kinase catalytic subunit. However, when intact cells were incubated with [32P]-orthophosphate, only the 29,000 molecular weight protein was phosphorylated.

Animals↗
PubMed1984

Sensitive assay detects protein methylesterase in spermatozoa: decrease in enzyme activity during epididymal maturation.

A more sensitive assay for protein methylesterase (PME) was developed. The new assay measures the methanol formed from hydrolysis of protein-methyl esters instead of methyl esters remaining on proteins. The formation of methanol is linear with time and enzyme concentration up to 20% of substrate hydrolysis. With this assay, we have detected very low PME activity in rat spermatozoa from cauda epididymidis. However, PME activity in spermatozoa from caput epididymidis was 10-fold higher. In a more detailed study on bull spermatozoa during epididymal maturation, we observed that PME activity was low in testicular spermatozoa, increased in spermatozoa from caput epididymidis to a maximum of 1.29 pmol/mg protein, and progressively decreased to a very low level (0.06 pmol/mg protein) in spermatozoa from cauda epididymidis. The drop in PME activity during epididymal transit parallels the progressive acquisition of motility and fertilizing capacity by spermatozoa.

Animals↗
PubMed1983

Aprotinin and a seminal plasma factor inhibit the motility of demembranated reactivated rabbit spermatozoa.

The demembranation and reactivation of ejaculated rabbit spermatozoa have been studied. ATP, Mg, glutamate, dithiothreitol (DTT), and Tris-HCl were found to be essential for a good reactivation. With this experimental model, we investigated the effects of protease inhibitors on the reinitiation of movement by ATP and on the movement of already motile spermatozoa. Soybean trypsin inhibitor (STI) prolonged the length of reactivation by 7- to 8-fold, whereas pepstatin, antithrombin III, phenylmethylsulfonyl fluoride (PMSF), and alpha-1-antitrypsin had no significant effect. Aprotinin (1.5 micrograms/ml) and leupeptin (50 micrograms/ml) completely prevented the reinitiation of movement by ATP; aprotinin at the same concentration even blocked the movement of motile spermatozoa. A tissue-specific seminal plasma factor could also prevent both the reinitiation of movement and the movement of motile spermatozoa. However, it took 2-3 times the amount of seminal plasma to stop the movement than to prevent the reinitiation of movement. The inhibitor in the seminal plasma is most probably not a protease or an aprotinin-like protease inhibitor since a partially purified preparation of the seminal plasma inhibitor does not hydrolyze a trypsin substrate, is not inhibited by protease inhibitors and has no significant capacity to inhibit trypsin. The data suggest that aprotinin and the seminal plasma inhibitor block movement through different mechanisms. Aprotinin and the seminal plasma inhibitor represent two new tools to study the regulation of sperm movement.

Adenosine Triphosphate↗
PubMed1983

Endogenous protein carboxyl methylation in hamster spermatozoa: changes associated with capacitation in vitro.

Protein carboxyl methylase (PCM) and its substrate(s), methyl acceptor protein(s) (MAP), are present in the spermatozoa of rat, rabbit and man. In the present study, PCM activity and MAP capacity were measured in homogenates of hamster testes and isolated spermatozoa, and found to be similar to those of other species. Using solubilized preparations of spermatozoa from the cauda epididymis of hamster, PCM activity was twice as high in sperm tails as in heads while the reverse was true for MAP capacity. Since in this and in previous studies this enzyme reaction has been measured in broken cells, we thought it pertinent to measure methylation under physiological conditions (i.e., in motile spermatozoa). To accomplish this, an assay was developed which depends upon the conversion of [3H-methyl]methionine to S-adenoxyl-L [methyl-3H]methionine which, in turn, serves as a methyl donor for PCM. In sperm that had been labelled with [3H]methionine the MAP for the endogenous methylation reaction was not solubilized with Triton X-100, and was found primarily in sperm tails. When hamster spermatozoa were incubated in medium containing taurine, epinephrine and bovine serum albumin to induce capacitation, endogenous methylation was stimulated 8- to 9-fold; when taurine was omitted from this medium, methylation was stimulated 14-fold. Taurine, however, was essential for in vitro fertilization as the number of eggs fertilized declined from 92% in complete medium to 0% in medium minus taurine. The decrease in fertilization rate was also associated with a decrease in sperm motility. In previous studies we have demonstrated an association between PCM and sperm motility, and have suggested that this enzyme system could be involved in other functions such as the acrosome reaction. From the results in the present study we conclude that the conditions that lead to capacitation in vitro are associated with a marked change in endogenous protein carboxyl methylation.

Animals↗
PubMed1983

Enzymatic carboxyl methylation of calcium-binding proteins.

The enzymes protein-carboxyl methylase and protein methylesterase reversibly modify the charge and structure of proteins by adding and removing methyl groups on free carboxyl groups of proteins. Since this posttranslational system has been implicated in biological processes that required calcium, the carboxyl methylation of calmodulin was investigated. Calmodulin was an excellent substrate for both protein-carboxyl methylase and protein methylesterase. Carboxyl methylation of calmodulin resulted in inactivation, since methylated calmodulin was less capable of activating cyclic nucleotide phosphodiesterase. To determine whether the carboxyl methylation of calmodulin was simply a test tube reaction or a biochemical reaction normally occurring in intact cells, two different cell lines were labeled with [methyl-3H]methionine. Calmodulin was isolated by affinity chromatography and was found to be carboxyl methylated. Finally, calcineurin was also an excellent substrate for the methylase, suggesting that other calcium-binding proteins may be affected by protein methylation-demethylation.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1983

Aprotinin and a seminal plasma factor provide two new tools to study the regulation of sperm motility.

A seminal plasma factor and aprotinin are capable of inhibiting the movement of demembranated reactivated rabbit spermatozoa. These inhibitors can be overcome by the addition of ATP. This reversibility could be explained by a common binding receptor for the inhibitors and MgATP. The respective affinities for the receptor are dependent on whether the microtubules are sliding or resting.

Adenosine Triphosphate↗
PubMed1982

Deficiency of protein-carboxyl methylase in immotile spermatozoa of infertile men.

We studied protein-carboxyl methylase, an enzyme involved in the regulation of cellular locomotion in both bacteria and leukocytes, in semen from 22 normal fertile men, 10 vasectomized volunteers, and nine infertile patients with nonmotile spermatozoa. In normally motile spermatozoa protein-carboxyl methylase activity was 68.8 +/- 5.5 pmol per milligram of protein (mean +/- S.E.). On the other hand, the enzyme activity in nonmotile spermatozoa from the infertile patients was low (17.4 +/- 3.4 pmol per milligram of protein) and similar to that in the cellular debris from the vasectomized volunteers (10.4 +/- 1.3 pmol per milligram of protein). The low enzyme activity in the infertile patients was not caused by the presence of dead spermatozoa or spermatozoa with leaky plasma membranes, since mitochondrial protein synthesis and lactate dehydrogenase activity were normal in these patients. The deficiency of protein-carboxyl methylase activity in nonmotile sperm is probably not due to a primary genetic defect, since the enzyme activity is normal in the red cells of these patients and spontaneous recovery of motility is associated with the return of enzyme activity.

Adult↗
PubMed1981

erythro-9-[3-(2-Hydroxynonyl)]adenine is an inhibitor of sperm motility that blocks dynein ATPase and protein carboxylmethylase activities.

Protein carboxylmethylase (S-adenosyl-L-methionine:protein O-methyltransferase, EC 2.1.1.24.) is believed to be involved in the regulation of sperm motility. To test this hypothesis, we investigated the effects of erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) which, in combination with adenosine and homocysteine thiolactone, inhibits protein carboxylmethylase activity in monocytes. This group of compounds inhibited sea urchin sperm motility. Unexpectedly, EHNA alone inhibited the motility., This observation was confirmed in intact spermatozoa from rats, rabbits, and humans. EHNA also inhibited the motility of demembranated, reactivated sea urchin and rat spermatozoa from which protein carboxylmethylase had been extracted. In these preparations, motility was restored by ATP. These observations suggested that EHNA arrests sperm motility by inhibiting the axonemal dynein ATPase on which motility depends. Kinetic analysis demonstrated that EHNA produced mixed inhibition of both the axonemal ATPase and the partially purified dynein 1 from sea urchin sperm tails, as well as the axonemal ATPase of rat sperm tails. These observations also provide evidence for the similarity of the active site of the dynein ATPase in sea urchin and rat spermatozoa.

Adenine↗