Functional and proteomic analysis of submandibular saliva in rats exposed to chronic stress by immobilization or constant light.

OBJECTIVE: In this study, we have evaluated the effects of stress on functional and proteomic changes in submandibular saliva of rats. DESIGN: Male adult rats were divided in three groups: IMO (2 h/day of immobilization for 7 days), LL (constant light during 20 days), C (unstressed controls submitted to 14 h light-10h dark cycle). Body weight, food intake and the dry weight of submandibular gland were recorded. Saliva samples, collected under anaesthesia following i.p. administration of isoproterenol and pilocarpine (5 mg/kg), were assayed for total proteins (TP), amylase activity and SDS-PAGE electrophoresis. RESULTS: Body weight, food intake and the dry weight of submandibular gland of IMO rats were lower than those of C and LL groups. The salivary volumes secreted in IMO and LL rats, were significantly higher than in controls. The TP output (µg protein/µg saliva/mg of dry tissue) and amylase activity output (AU/µg of saliva/mg of dry tissue) in IMO were significantly higher than in C and LL animals. The electrophoretic pattern of saliva proteins of LL rats, revealed the absence of a protein band of approximately 25 kDa. This band was composed by the common salivary protein-1 and a prolactin-induced protein as identified by peptide mass fingerprinting. CONCLUSIONS: Differences in body weight and food intake between IMO and LL might be attributed to the sort and intensity of stressors stimuli. The changes in the volume of secreted saliva could be a compensatory mechanism in response to stressors. The increase of total protein in IMO rats and the absence of 25 kDa proteins in LL, would suggest that the submandibular glands respond to the sympathetic nervous system stimuli induced by the stress with an increase of activity of the sympathetic nerves in IMO and a reduction in LL rats.

Trypsin/acrosin inhibitor activity of rat and guinea pig caltrin proteins. Structural and functional studies.

Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 microM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca(2+) uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca(2+) influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.

Androgen-dependent synthesis/secretion of caltrin, calcium transport inhibitor protein of mammalian seminal vesicle.

Effects of androgen status on the synthesis and secretion of rat caltrin have been studied by three different procedures: a) immunocytochemistry in seminal vesicle tissues; b) polyacrylamide gel electrophoresis and Western immunostaining of seminal vesicle secretion; and c) evaluation of trypsin inhibitory activity of the seminal vesicle secretion. Rat caltrin has been immunolocalized in cells of the secretory epithelium, specifically in the electron-lucent halo of secretory granules which store and transport proteins to the lumen. No caltrin immunoreaction was detected 14 days postcastration, and the ultrastructure of the epithelial cells was markedly altered. SDS-PAGE and Western blotting of the seminal vesicle secretion revealed alterations in the protein pattern and loss of the caltrin-related immunoreactive bands. The 54-kDa caltrin-precursor protein and the 6.2-kDa active caltrin were absent. Trypsin inhibitory activity of the seminal secretion was reduced about 50% in castrated animals. Daily testosterone administration restored both the protein pattern and immunoreactivity of the seminal vesicle secretion, and, as expected, reversed the morphological alterations of the gland after 7 days of treatment. Trypsin--inhibitor effect of the secretion also returned to normal levels after fourteen days of testosterone administration. Data suggest that the synthesis and secretion of caltrin are testosterone-dependent processes.

Electron microscopic immunolocalization of caltrin proteins in guinea pig seminal vesicles.

Caltrins, the small, basic proteins of the seminal vesicle secretion that inhibit calcium transport into epididymal spermatozoa, and consequently the onset of the acrosome reaction and the hyperactivated motility, were localized in the epithelial cells and the lumen of the seminal vesicles of the guinea pig by an immunocytochemical procedure and electron microscopy. Rabbit antisera against each protein (caltrin I or II), and goat anti-rabbit IgG antiserum labeled with colloidal gold were used to detect the caltrin immunoreaction. The subcellular distribution of the gold labeling was occasionally localized in the rough endoplasmic reticulum but mainly within big secretory vacuoles containing low electron-dense material, which are components of the Golgi complex known as condensing vacuoles. These are involved in the intracellular transport, storage, and discharge of secretory proteins. Gold-labeled material released to the lumen was also detected. There was no clear evidence that the discharge was mediated by an exocytotic process. Immunoreaction was observed neither in the electron-dense core nor in the electron-lucent halo of the typical secretory granules of the epithelial cells of the seminal vesicles. Using light microscope immunocytochemistry, intense positive immunoreactivity was detected in the material secreted to the lumen but not on the epithelial cell layer. Only those cells undergoing a degenerative process and showing a picnotic nucleus and condensed cytoplasmic matrix exhibited detectable immunoreaction when gold label and silver intensification were applied. The same distribution of the immunoprobes was obtained by electron or light microscopy when antiserum to either I or II was used. It would appear that the two caltrin proteins of the guinea pig are synthesized in the rough endoplasmic reticulum of the epithelial cells and transported quickly to the Golgi complex where the secretory vacuoles (condensing vacuoles) are formed. The proteins are transported by the secretory vacuoles to the apical ends of the cells to be discharged into the lumen.

The lactate/pyruvate shuttle in spermatozoa: operation in vitro.

The operation of a shuttle for the transfer of reducing equivalents in the special mitochondria present in the middle piece of spermatozoa (sperm-type mitochondria, STM) was studied in reconstituted systems in vitro with mouse, rat, and rabbit STM. The redox couple lactate/pyruvate and the lactate dehydrogenase isozyme C4 are involved in the shuttle. It is active with rat and rabbit STM, while it does not work with mouse STM, probably because the influx of lactate into the mouse organelles is relatively poor. Ratios of consumption of pyruvate/lactate by STM were 21.6, 1.28, and 1.6 for mouse, rat, and rabbit organelles, respectively. The shuttle is inhibited by 0.6 mM mersalyl, a blocker of lactate transport. The operation of the shuttle would oxidize cytosolic NADH produced during aerobic glycolysis (or fructolysis) in spermatozoa of those species having an efficient lactate carrier in mitochondria.

Isolation and characterization of a 54-kilodalton precursor of caltrin, the calcium transport inhibitor protein from seminal vesicles of the rat.

A basic 54-kDa protein (pI approximately 8.8) that cross-reacts with anti-caltrin antisera has been detected and isolated by gel filtration and cation exchange chromatography from seminal vesicle content of the rat. The soluble protein spontaneously precipitated in NaHCO3-buffered solution at pH 7.8, but it was kept soluble in imidazole buffer containing EDTA and dithiothreitol at pH 7.0. In addition to the main band of 54 kDa, two faint immunoreactive fractions with molecular weights around 45,000 and 14,000 were also revealed by Western blotting. The presence of the rat caltrin sequence within the primary structure of the 54-kDa molecule has been investigated by sequencing the peptides generated by trypsin digestion. The sequence of the first 46 amino acid residues of rat caltrin has been found in one of the fragments produced by enzymatic cleavage. However, the exact location of the caltrin sequence in the whole 54-kDa protein has not been determined. The purified 54-kDa protein did not inhibit Ca2+ uptake by epididymal spermatozoa. Results indicated that this molecule represents an inactive precursor of caltrin and is enzymatically processed in the lumen of the seminal vesicle to the small and active calcium transport inhibitor protein. The immunoreactive proteins with intermediate molecular weights (45,000 and 14,000) could represent partially degraded products of the precursor. The lack of inhibitory activity of the precursor may be related to the molecule's having a size and conformation that would make it unable to interact with caltrin receptors on the sperm surface.

Purification, structure, and characterization of caltrin proteins from seminal vesicle of the rat and mouse.

Caltrins, small basic proteins that inhibit calcium uptake by epididymal spermatozoa, have been purified from seminal vesicle content of the mouse and rat. Mouse caltrin (M(r) 8,476) contains 75 amino acid residues, 14 basic, 5 acidic, and 7 cysteines while rat caltrin (M(r) 6,217) has 56 residues, 10 basic, 5 acidic, and 6 cysteines; their pI values are 10.2 and 9.3, respectively. The proteins did not react with Ellman's reagent unless the cystine residues were previously reduced. The primary structures were determined by sequencing fragments generated by trypsin, clostripain, and endoproteinase Lys-C digestion. The sequences were ordered to give the total structural formula. The two molecules have no sequence similarity and are different from those of the bull and guinea pig previously reported. Only rat caltrin has a sequence of 13 residues nearly identical to that in guinea pig caltrin I. Both rat and mouse caltrin react with antibodies against bovine and guinea pig caltrins. Reduction and alkylation of cysteine residues suppressed the immunologic response of mouse caltrin; however, modified rat caltrin retained partially its immunoreactivity with the antiserum against guinea pig caltrin I. The same treatment abolished the calcium transport inhibitory activity of mouse caltrin and greatly reduced that of rat caltrin. It is likely that rat and mouse caltrins have the same physiological function as proposed for bovine caltrin; namely, to regulate the development of the Ca(2+)-dependent processes that "capacitate" sperm for fertilization.

Functional properties of caltrin proteins from seminal vesicle of the guinea pig.

Caltrin proteins from seminal vesicle content of the guinea pig bind with great specificity to different regions of the spermatozoa. Indirect immunofluorescence studies with polyclonal antibodies showed that caltrin I binds to the head, on the acrosomal cup, while caltrin II binds on the principal tail and the neck. No fluorescence was detected either in the midpiece or in the post-acrosomal area of the head when sperm were exposed to either of the caltrins. Calcium-induced hyaluronidase release, which occurs during the acrosomal reaction, was dramatically inhibited by caltrin I (approximately 85% inhibition). Caltrin II was less effective in preventing the enzyme release (approximately 50% inhibition). Chemical modification of the structure modified the biological activity of the two caltrins. Reduction and carboxymethylation of the cysteine residues diminished the inhibitory activity on 45Ca2+ uptake and reduced the ability of the proteins to react with their antibodies. Removal of the carbohydrate portion by chemical deglycosylation transformed the inhibitor proteins into enhancers of calcium uptake into the spermatozoa. Caltrin proteins from the guinea pig appear to play the same physiological role as bovine caltrin, regulating specifically calcium transport across the spermatozoal membranes related with the acrosome reaction and hyperactivation process. The dual behavior of caltrins to inhibit or enhance Ca2+ uptake enables them to fulfill this function. Nevertheless, molecular mechanisms different from those described for bovine caltrin seem to be involved in the control of the functional activity of the guinea pig caltrins.

Purification and structure of caltrin-like proteins from seminal vesicle of the guinea pig.

Two different small proteins that cross-react with the antiserum against bovine caltrin (calcium transport inhibitor) have been purified from the seminal vesicle contents of the guinea pig. The primary structure and some molecular characteristics of the pure proteins are reported. The two proteins interact with concanavalin A indicating the presence of carbohydrates in their molecules. Chemical deglycosylation with trifluoromethanesulfonic acid, after reduction and carboxymethylation, results in complete loss of affinity for the lectin. Removal of sugar components from the structure destroys the ability of caltrin-like proteins to react with antibodies to bovine caltrin. The protein moving faster on polyacrylamide gel electrophoresis is designated guinea pig caltrin I, the other is II. They contain 45 and 55 amino acids, and the molecular weights of the peptide portions are 5082 and 6255, respectively. Although they have entirely different amino acid sequences, they share some common features: recognition by rabbit antibodies to bovine caltrin, the predominance of basic residues and the presence of 3 cysteine residues in fraction I and 8 in fraction II. The proteins have pI values of 9.5 and 10.2, respectively, which are consistent with the amino acid composition. The two pure fractions are approximately equally effective, on a weight basis, as inhibitors of 45Ca2+ uptake by guinea pig spermatozoa. The data presented reinforce the hypothesis that caltrin-like proteins are responsible for the previously reported (Coronel, C.E., San Agustin, J., and Lardy, H.A. (1988) Biol. Reprod. 38, 713-722), calcium-transport inhibitor activity detected in reproductive tract fluid from adult male guinea pigs.

Identification and partial characterization of caltrin-like proteins in the reproductive tract of the guinea pig.

The presence of caltrin-like proteins in reproductive tract fluid (RTF) and seminal vesicle content from male guinea pigs has been determined. Two fractions with electrophoretic mobility corresponding to Mr = 6200 (main band) and 5100 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Isoelectric focusing in thin-layer agarose gels revealed three bands with acidic pIs of 5.3, 6.0, and 6.2, respectively. RTF prevented the enhancement of calcium permeability induced by incubating guinea pig epididymal spermatozoa in medium for capacitation. Spermatozoa incubated for 2 h in minimal culture medium plus pyruvate and lactate containing RTF accumulated less than 30% of the 45Ca2+ accumulated by cells maintained in absence of this fluid. Calcium uptake by preincubated spermatozoa was also inhibited by RTF. Inhibition of calcium transport activity by RTF and seminal vesicle proteins was not decreased by heating the dialyzed preparations at 60 degrees C for 5 min. After this treatment, the inhibitory activity and the protein pattern were stable for 3 wk when stored at 4 degrees C. Unheated extracts lost calcium transport inhibitory activity after 2 or 3 days at 4 degrees C. In spite of the differences in pIs among the proteins from the guinea pig reproductive tract and bovine caltrin, several features indicate they may play a similar role in both species by controlling Ca2+ movement across the plasma membrane. By this mechanism, these proteins could regulate physiologic events essential for the fertilization process.

Characterization of Ca2+ uptake by guinea pig epididymal spermatozoa.

Comparative studies of Ca2+-uptake by guinea pig spermatozoa were performed with fresh epididymal sperm and with cells preincubated in a chemically defined, Ca2+-free medium for capacitation. Calcium uptake was negligible in fresh spermatozoa, but increased dramatically after 20 min of incubation at 37 degrees C in the presence of pyruvate and lactate. Spermatozoa incubated in the absence of these substrates accumulated only 34% as much 45Ca2+ as was taken up by cells in complete medium. The monosaccharides glucose, fructose, and mannose and the nonmetabolizable sugars 2-deoxyglucose and sucrose inhibited the enhancement of Ca2+-permeability. In the presence of 6 mM sucrose 45Ca2+ uptake was not influenced by external sodium chloride concentration between 0 mM and 145 mM. The respiratory activity of the capacitated spermatozoa not only was higher than that of uncapacitated cells, but it was stimulated by Ca2+. No effect of Ca2+ on respiration of fresh spermatozoa was detected. An increase in calcium uptake was associated with increasing pH of the medium. It is possible that a regulatory mechanism through the calcium permeability of the plasma membrane of guinea pig spermatozoa exists and controls the development of physiological events related with the fertilization process. The sugar composition, the availability of the energy substrates lactate and pyruvate, and the pH of the reproductive tract fluids could play an important role in the accessibility of Ca2+ into the cells in vivo, as has been demonstrated in vitro. The enhancement of calcium permeability during the preincubation could be a useful indicator to verify if capacitation has occurred.

Properties of the branched-chain 2-hydroxy acid/2-oxo acid shuttle in mouse spermatozoa.

Operation of the branched-chain 2-hydroxy acid/2-oxo acid shuttle for the transfer of reducing equivalents in mitochondria of mouse spermatozoa was studied in vitro in reconstituted systems. Results show that the branched-chain 2-oxo acids within the mitochondria are offered several metabolic pathways. (a) Decarboxylation: mouse sperm mitochondria possess high branched-chain 2-oxo acid decarboxylase activity. (b) Recycling to the cytosol by using a transport system which can be inhibited by alpha-cyano-3-hydroxycinnamate and pH 6.8. (c) Transamination to the corresponding amino acids: experiments presented indicate that leucine formed from 4-methyl-2-oxopentanoate may pass to the external phase, re-initiating the cycle. These two last possibilities would allow autocatalytic operation of the shuttle. The branched-chain 2-hydroxy acids apparently do not utilize the monocarboxylate carrier to penetrate the mitochondria.

Effect of temperature upon catalytic properties of alpha-hydroxyacid dehydrogenase from Trypanosoma cruzi.

A comparative study of the effect of temperature (10, 20, 30 and 37 degrees C) upon Km and V of alpha-hydroxyacid dehydrogenase (HADH), isozyme I and II, from Trypanosoma cruzi, a parasite whose life cycle comprises stages in an insect vector, and of another enzyme with analogous substrate specificity, the lactate dehydrogenase, isozyme X (LDH X) from mouse, a homeotherm, is presented. The Km for alpha-ketoisocaproate of HADH is markedly reduced as temperature decreases. This effect can compensate the reduction in thermal energy and produce stabilization of the reaction rate. This compensation does not occur with mouse LDH X. The activation energy for both HADH isozymes is about half the value determined for mouse LDH X. Results indicate that HADH from T. cruzi is able to adjust instantaneously to thermal changes of the environment, behaving as other enzymes of terrestrial poikilothermic animals.

Catalytic properties of the sperm-specific lactate dehydrogenase (LDH X or C4) from different species.

A comparative study of catalytic properties of the sperm-specific lactate dehydrogenase (EC 1.1.1.27) isozyme X or C4 from a variety of animals (boar, bull, goat, Guinea pig, man, mouse, pigeon, rabbit, and rat) is presented. Optimum concentration and Km values for pyruvate, inhibition by substrate, and activity against analog substrates (alpha-ketoacids with linear and branched chains from 4 to 6 carbon atoms) for isozyme X of different species showed significant differences. The observed properties are correlated with available evidence on the metabolic role of the enzyme.

Studies in vitro on shuttle systems of mouse spermatozoa.

Observations on systems reconstituted in vitro with different starting substrates (2-hydroxy-acids, 2-oxo-acids or leucine) indicate that a branched-chain 2-hydroxy-acid/2-oxo-acid shuttle for the transfer of reducing equivalents from cytosol to mitochondria may be operational in mouse sperm. Evidence is presented suggesting that the 2-oxo-acids produced by intramitochondrial oxidation of 2-hydroxy-acids ingressed from the cytosol can recycle back into the external phase. Observations in vitro demonstrate that, in addition to the branched-chain 2-hydroxy-acid/2-oxo-acid shuttle, the malate/aspartate system is also active in mouse sperm. On the contrary, the lactate/pyruvate redox couple does not appear to function as part of a shuttle system in mouse sperm mitochondria. The glycerol 3-phosphate shuttle probably is not functionally significant in mouse spermatozoa, since the activity of the 'soluble' glycerol 3-phosphate dehydrogenase is very low.

Substrate specificity of the lactate dehydrogenase isoenzyme C4 from human spermatozoa and a possible selective assay.

The activity of lactate dehydrogenase (EC 1.1.1.27) in normal human sperm lysates and in human heart and liver homogenates was determined by using a variety of 2-oxoacids as substrates. Sperm preparations were active with pyruvate, 2-oxobutanoate, 2-oxopentanoate and 2-oxohexanoate, while heart and liver extracts utilized only pyruvate and 2-oxobutanoate. Selective staining after gel electrophoresis indicated that the fraction corresponding to lactate dehydrogenase C4, the sperm-specific isoenzyme, was responsible for the utilization of substrates with a linear chain of 3 to 6 carbon atoms. The use of 5 mM 2-oxohexanoate allowed the selective determination of isoenzyme C4 in preparations containing different lactate dehydrogenase molecular forms.

Correlation of lactate dehydrogenase isoenzyme C4 activity with the count and motility of human spermatozoa.

The activity of lactate dehydrogenase isoenzyme C4 was determined on 90 human semen samples. The correlation between the isoenzyme activity and sperm count and motility was good (r = 0.74 for values of U/ml semen against sperm count).