Carlos E. Coronel

β-microseminoprotein in human spermatozoa and its potential role in male fertility

beta-Microseminoprotein (MSMB) is one of the most abundant proteins in human seminal plasma. The objectives of this study were: (1) to purify MSMB from seminal plasma (SP) and generate antibodies against the pure protein; (2) to investigate the interaction of MSMB with ejaculated spermatozoa and its possible effect on the spontaneous acrosome reaction (AR); and (3) to quantify MSMB content in SP and examine its relationship with the clinical sperm parameters. MSMB was purified from SP and its presence on the sperm surface was examined by indirect immunofluorescence using a specific polyclonal antibody. The effect of MSMB on the AR was evaluated using guinea pig epididymal spermatozoa as a model. MSMB quantification assay was performed with a two-site binding ELISA using two polyclonal antibodies against MSMB. MSMB was assessed in semen samples from fertile donors (controls) and subfertile patients according to World Health Organization criteria. MSMB was detected on the sperm surface and mainly localized to the acrosomal region of the head and neck. A significant spontaneous AR inhibition was observed when guinea pig epididymal spermatozoa were preincubated with MSMB. Finally, MSMB was significantly increased in subfertile patients when compared with fertile controls (P<0.02). The association of MSMB to the sperm surface, the inhibitor effect on the spontaneous AR and the increased MSMB levels found in SP in subfertile men suggests a relationship between this protein and semen quality and a possible role in the process of fertilization.

Properties of α-hydroxyacid dehydrogenase isozymes from Trypanosoma cruzi

Whole cell extracts of culture epimastigotes of Trypanosoma cruzi (Tulahuen strain) have α-hydroxyacid dehydrogenase activity which catalyzes the NAD-linked reaction α-ketoacid ⇋ α-hydroxyacid, with a variety of substrates. Two molecular forms of the enzyme have been separated by means of gel electrophoresis. These isozymes were partially purified by DEAE-cellulose chromatography and ammonium sulfate precipitation. Molecular weights were estimated and some catalytic properties were determined with purified isozymes. The faster migrating fraction (isozyme I) has a molecular weight of 85 500 and showed significant activity against linear 3–5 carbon chain substrates. The lowest Km value was obtained for pyruvate. Isozyme II (MW 60 500) utilizes linear and branched chain substrates with 4–6 carbon atoms. Its highest activity and lowest Km value were recorded with α-keto-isocaproate as substrate.

Trypsin/Acrosin Inhibitor Activity of Rat and Guinea Pig Caltrin Proteins. Structural and Functional Studies

Abstract 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 μM 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 Ca2+ 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 Ca2+ influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.

Rat Caltrin Protein Modulates the Acrosomal Exocytosis During Sperm Capacitation

Abstract Caltrin is a small and basic protein of the seminal vesicle secretion that inhibits sperm calcium uptake. The influence of rat caltrin on sperm physiological processes related to fertilizing competence was studied by examining its effect on 1) spontaneous acrosomal exocytosis, 2) protein tyrosine phosphorylation, and 3) sperm-egg interaction. Results show that the presence of caltrin during in vitro capacitation both reduced the rate of spontaneous acrosomal exocytosis without altering the pattern of protein tyrosine phosphorylation, and enhanced the sperm ability to bind to the zona pellucida (ZP). The significantly higher proportion of sperm with intact acrosome observed in the presence of caltrin was accompanied by a strong inhibition in the acrosomal hyaluronidase release. Enhancement of sperm-ZP binding was evident by the increase in the percentage of eggs with bound spermatozoa as well as in the number of bound sperm per egg. Similar results were obtained when the assays were performed using spermatozoa preincubated with caltrin and then washed to remove the unbound protein, indicating that the sperm-bound caltrin was the one involved in both acrosomal exocytosis inhibition and sperm-ZP binding enhancement. Caltrin bound to the sperm head was partially released during the acrosomal exocytosis induced by Ca-ionophore A23187. Indirect immunofluorescence and immunoelectron microscopy studies revealed that caltrin molecules distributed on the dorsal sperm surface disappeared after ionophore exposure, whereas those on the ventral region remained in this localization after the treatment. The present data suggest that rat caltrin molecules bound to the sperm head during ejaculation prevent the occurrence of the spontaneous acrosomal exocytosis along the female reproductive tract. Consequently, more competent spermatozoa with intact and functional acrosome would be available in the oviduct to participate in 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.

Effect of temperature upon catalytic properties of α-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.

CEREBROSPINAL FLUID PURINES, PYRIMIDINES, ORGANIC ACIDS AND AMINO ACIDS IN NEONATAL CITRULLINAEMIA

Citrullinaemia is one of the inborn errors of urea synthesis caused by a deficiency of argininosuccinate synthase (EC 6.3.4.5). The classical neonatal presentation of the disorder is as a life threatening illness during the first week of life. Clinical symptoms develop between ages of 24 to 72 hours, starting as a poor suck, hypotonia, vomiting, lethargy, and hyperventilation with rapid progression to hyperammonemic coma and seizures. The mechanism of the ammonium-induced neuropathology remains unclear, but it is known that ammonia and glutamine accumulations have a number of toxic effects on the brain including disturbance of the blood brain barrier permeability, energy depletion and alteration of amino acid and neurotransmitter levels. In this paper, we report cerebrospinal fluid (CSF) purine, pyrimidine, amino acid and organic acid findings in two cases of Neonatal Type of Citrullinaemia in order to get a better insight about the physiopathology of this disorder.