The aim of this study was to characterize the testicular isoform of angio-tensin-converting enzyme (tACE) before and after semen cryopreservation, and in the acrosome reaction of sperm from Nelore bulls in vitro. Ejaculates of 10 sexually mature Nelore bulls were used. After semen was collected, 1.0 mL of the ejaculate was used for the analysis and the rest was subjected to cryopreservation. Fresh semen before freezing, and frozen/thawed semen were centrifuged twice and the pellet was resuspended intyrode’s albumin lactate pyruvate (TALP). Thereafter, 100 μL aliquots containing 100 × 10 6 spermatozoa were prepared. Aliquots of samples were used for western blot analysis, subjected to capacitation, and thereafter, acrosome reaction assays were performed </>in vitro</i>. With the help of an anti-ACE monoclonal antibody, a 100 kDa protein band was identified in the spermatozoa of Nelore bulls. Cryopreservation reduced the intensity of the protein bands obtained by western blot assay to less than half of that observed prior to freezing ( P < 0.05). Inhibition of ACE by captopril (10 μM), decreased the percentage of capacitated spermatozoa with a positive acrosome reaction ( P < 0.05), indicating the involvement of ACE in these processes. It is concluded that tACE can be found in the spermatozoa of Nelore bulls, and cryopreservation process decreases the intensity of bands of this enzyme; and that the inactivation of tACE reduces the capacity of spermatozoa to undergo the acrosome reaction.
Angiotensin-converting enzyme (ACE) is a zinc-dependent metalloprotease, which is anchored to the plasma membrane and exists in two isoforms. A somatic form (sACE150 - 180 kDa) is found in various organs and is involved in the control of blood pressure via the renin-angiotensin-aldosterone system. sACE converts inactive angiotensin I into angiotensin II, a potent vasoconstrictor that also stimulates the release of the hormone aldosterone, which induces sodium retention and increases blood pressure. In addition, it enhances this effect by inhibiting the action of the vasodilator bradykinin [
The second isoform is of lower molecular weight (110 kDa), although transcribed by the same gene, and is found exclusively in male germ cells; therefore, it was designated as a testicular isoform (tACE). This isoform is not expressed in animals before puberty, which suggests that hormonal stimulation is required for its synthesis [
tACE has an important function in the fertilization of the egg by sperm. In experiments with ACE-knockout mice, no changes in the number or morphology of sperm were observed, although they displayed defects when binding to the zonaepellucidae [
Part of ACE is released from spermatozoa during capacitation [
Several studies have revealed ACE activity in human [
Semen samples from 10 sexually mature Nelore bulls (Bos taurusindicus) were used. Semen was collected by the artificial vagina technique in an artificial insemination center. Only ejaculates with motility greater than 70% and vigour greater than three were used. Sperm motility (0% - 100%) and sperm vigour (score from 0 to 5) were subjectively assessed by the same technician in at least four fields in each sample, and the results were expressed in average of the fields.
All international guidelines for the care and use of animals for scientific purposes were followed in this experiment.
An ejaculate sample from each bull was diluted in tris-yolk buffer (tris 2.42%, citric acid 1.36%, fructose 1.0%, glycerol 7.0%, egg yolk 20.0%, and gentamic in 0.7%) at a concentration of 50 × 106 spermatozoids/mL, and packed in 0.5mLvials. Samples were frozen using a TK 4000 programmable portable semen cryopreservation system (TK Equipamentospara Reprodução, Uberaba, Brazil). In this system, the cooling rate was 0.25˚C/min up to 5˚C, where it remained constant for 5 hours. Then, the freezing rate was −15˚C /min from 5˚C to −80˚C, and then from −10˚C/min to −140˚C. Thereafter, the vials were racked and stored in cryogenic cylinders at −196˚C.
In order to prepare the fresh semen pellet, a 1.0 mL sample was withdrawn immediately after the ejaculate was collected and centrifuged twice at 700 g for 30 min (at room temperature) in Tyrode’s albumin lactate pyruvate medium (TALP [
To prepare a frozen/thawed semen pellet, 48 hours after of storage in cryogenic tanks, 10 vials were thawed in a water bath at 37˚C for 30 s. Then, the semen was placed in the same tube and centrifuged twice at 700 g for 30 min with TALP to remove the diluent. The supernatant was discarded and the pellet formed was used for subsequent analyses.
Western blot
To detect ACE in Nelore bull spermatozoa, the pellet concentration was determined after centrifugation with TALP, and protein was extracted from 100 µL aliquots containing 100 × 106 spermatozoa with 25 μL Laemmli buffer (4% SDS, 20% glycerol, 10% DTT, 0.004% bromophenol blue, and 0.125 M Tris HCl, pH 6.8) and heating at 100˚C for 5 min. The samples were then centrifuged at 11,200 g for 5 min.
The supernatant (protein extract) was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE 10%) to separate the proteins according to their molecular weights. After 1 h at a constant electric current of 100 V, the proteins traversed the entire length of the gel. The gel was then carefully removed from the cassette and submerged in 20% ethanol for 10 min to increase transfer efficiency. Proteins were transferred from the gel to a nitrocellulose membrane by electrophoresis in a sandwich system with transfer buffer (25 mM Tris, 0.2 M glycine, and 20% methyl alcohol, pH 8.5), at a constant voltage of 350 mA. The resulting nitrocellulose membranes were stained with 0.2% ponceau in acetic acid to determine the effectiveness of the electrotransfer. Subsequently, nonspecific binding sites were blocked with 3% skimmed milk powder, diluted in tris-buffered saline + tween 20 medium (TTBS), for 60 min at room temperature. The membranes were then incubated (overnight at 4˚C) with the primary antibody (anti-ACE, clone 2E2, Millipore®, 1:5000 in TTBS). Following overnight incubation the membranes were washed three times in TTBS, and incubated with secondary antibody (goat anti-mouse IgG, HRP conjugate, Millipore®, 1:10,000 in TTBS) for 1 h at room temperature. After washing three times with TTBS, the protein bands were identified by chemiluminescence [
To confirm that all samples contained the same amount of protein, another gel was prepared using the same samples, and a further western blot was performed using a monoclonal anti-β-tubulin antibody [
ACE was detected in seminal plasma by adding 25 μL of Laemmli buffer to 100-μL aliquots of plasma diluted in TALP (1:50) and heated at 100˚C for 5 min to extract protein. Then, the samples were centrifuged at 11,200 g for 5 min and the supernatant was used for western blotting, with the same methodology described for spermatozoa.
Aliquots of 50 µL containing 200 × 106 spermatozoa were used for each of the following treatments for each bull: control group (50 µL of semen + 50 µL of SP-TALP), heparin group (50 µL of semen + 49 µL of SP-TALP + 1.0 µL of heparin 100 µg/µL), captopril + heparin group (50 µL of semen + 49 µL of SP-TALP + 1.0 µL of heparin 100 µg/µL+ 10 µM captopril). The samples were incubated in a CO2 incubator at 39˚C for 4 h. Sperm motility and vigour were evaluated every hour in each experimental group to evaluate whether capacitation occurred at a similar time for each animal evaluated.
After 4-h incubation, an aliquot of each treatment was incubated for a further 30 min with L-α-lysophosphatidylcholine (LPC) to induce the acrosome reaction in the capacitated spermatozoids [
The size of the protein bands was subjected to analysis of variance and differences between groups were compared with a t-test, setting a significance level of P < 0.05. The percentage of cells exhibiting a positive acrosome reaction between treatments was compared by chi-square test. The level of significance set was P < 0.05.
The use of a monoclonal anti-ACE antibody revealed at leastone 100-kDa protein band in the spermatozoid suspension from Nelore bulls.
The pattern of the pre- (
Western blot analysis with an anti-β-tubulin antibody (
The freezing process increased the number of pixels in the ACE bands of samples from the 10 Nelore bulls after cryopreservation (P < 0.05), indicating reduced protein expression of this enzyme as a result of the protocol used (
Following induction of the acrosome reaction, the smears were analyzed under an epifluorescence microscope. The estimated percentage of spermatozoa with a positive acrosome reaction was 83% in the Heparin group (
Western blot analysis with a monoclonal anti-ACE antibody revealed a single protein band with a molecular mass of approximately 100 kDa in extracts prepared from the semen of sexually mature Nelore bulls. The molecular mass of the protein band was consistent with that reported for tACE, between 90 and 110 kDa [
Equal amounts of protein were loaded from each animal, as confirmed by the immunodetection of β-tubulin (
The location of tACE in sperm varies between species, and disagreement exists among researchers investigating the same species. Nikolaeva et al. [
study with human sperm, found tACE in the post-acrosomal region and in the midpiece, while Köhn et al. [
In pigs, tACE was detected in spermatids and cytoplasmic droplets of epidydimal sperm [
Changes in the structure of the plasma membrane (PM) occur during the freeze/thaw of semen, which can lead to cold thermal shock, loss of proteins on the spermatozoa surface, and damage to the sperm cell, such as rupture of the PM and, consequently, death of the spermatozoa [
As with tACE, BSP A1 and A2 are related to sperm capacitation, and sperm protein P25b is involved in the fusion of gametes [
In an experiment carried out comparing ACE activity in rooster, buffalo, and bull semen by spectrophotometry (using hippuryl-L-histidyl-L-leucine, as a substrate), Mohan et al. [
Mammalian sperm undergoes physiological changes during its trajectory in the reproductive system of the female, termed sperm capacitation and acrosome reaction, and these events are necessary for the fertilization of the egg [
Costa and Thundathil [
Gur et al. [
tACE can be found in the sperm of Nelore bulls. The cryopreservation process leads to a decrease in the intensity of the bands of this enzyme, and the inactivation of tACE reduces the ability of sperm to undergo the acrosome reaction.
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (Processo: 470561/2012-2).
None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
Almeida, R.G., Faria, F.J.C., Furtado, F.H.G., Santos, E.F. and Costa, D.S. (2019) Characterization of Testicular Angiotensin-Converting Enzyme before and after Semen Cryopreservation and in the Acrosome Reaction of Spermatozoids of Nelore Bulls. Agricultural Sciences, 10, 518-529. https://doi.org/10.4236/as.2019.104041