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a URA 1291 INRA-CNRS, Institut National de la Recherche Agronomique, Station de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Monnaie, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 94-kDa protein was purified from ram cauda epididymal fluid, and a rabbit polyclonal antiserum was obtained. This antiserum showed that membranes of testicular sperm and sperm from the initial caput were positive for the presence of an immunologically related antigen. The protein was immunolocalized mainly on the flagellar intermediate piece, whereas in some corpus and caudal sperm, only the apical ridge of the acrosomal vesicle was labeled.
The purified protein was microsequenced: its N-terminal was not found in the sequence database, but its tryptic fragments matched the sequence of the angiotensin I-converting enzyme (ACE). Indeed, the purified 94-kDa protein exhibited a carboxypeptidase activity inhibited by specific blockers of ACE. All the soluble seminal plasma ACE activity in the ram was attributable to the 94-kDa epididymal fluid ACE. The polyclonal antiserum also showed that a soluble form of ACE appeared specifically in the caput epididymal fluid of the boar, stallion, and bull. This soluble form was responsible for all the ACE activity observed in the fluid from the distal caput to the cauda epididymidis in these species.
Our results strongly suggest that the epididymal fluid ACE derives from the germinal form of ACE that is liberated from the testicular sperm in a specific epididymal area.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During epididymal transit, an extensive remodeling of the sperm plasma membrane surface takes place in response to changes in the fluid. The sperm membrane is constituted of several well-defined domains established during spermatogenesis, and a redistribution of testicular sperm membrane proteins among these domains occurs during transit [1, 8]. Interactions between the sperm surface and the proteins of the luminal fluid also include integration of some of the secreted epididymal proteins [1, 2, 9, 10]. Finally, some of the surface compounds disappear, but the nature of these proteins and the mechanism of their removal remain to be elucidated [6, 9–11].
Using two-dimensional gel electrophoresis, we have recently compared the proteins of the luminal fluid of various regions of the ram and boar epididymis with 35S-labeled proteins secreted by tissue from the same area in order to increase our understanding of the proteins that make up the epididymal fluid [3, 7, 12]. These studies have revealed that some compounds present in the fluid do not result from epididymal secretory activity. A protein of about 105 kDa appears in the fluid of the caput region in the boar and ram [6, 7]. Studies of variations in sperm surface proteins have also been performed in the ram [6, 9, 11]. These have shown that a compound of 115 kDa that is present on testicular sperm, and also on sperm from region 1 of the epididymis, is not found on the sperm membrane in the other regions [6, 11]. A similar sperm membrane compound has also been described in the boar [10]. These findings suggest a possible sperm origin of the 105-kDa fluid compound. We have therefore characterized this compound in order to ascertain its origin and obtain further information on its possible role in the sperm maturation process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epididymides and testes were surgically removed by castration or were obtained from freshly killed adult Ile de France rams, Large White and Meishan boars, Selle Français stallions, and Holstein bulls. Testicular and epididymal fluids were collected as previously described [13]. Spermatozoa were separated from the fluid (10 min, 10 000 x g), resuspended in PBS solution, and washed by two cycles of centrifugation (10 min, 500 x g). After dilution at about 108 spermatozoa/ml, sperm were deposed on a discontinuous PBS-Percoll gradient (40% and 90% layers) and centrifuged (10 min, 400 x g). Spermatozoa taken at the 90% interface were then washed twice, and the membrane was extracted by mixing the pellet with an equivalent volume of PBS containing 3% (w:v) N-octyl-ß-D-glucopyranoside and the following protease inhibitors: 0.5 mM EDTA; 2 mM para-aminobenzamidine; 50 µg/ml N
-p-tosyl-L-lysine chloromethyl ketone; 10 µg/ml each of antipain, leupeptin, bestatin, E64, and pepstatin A. PMSF (0.5 mM) was added just before extraction. The mixture was left to stand at 4°C for 10–15 min; it was then centrifuged at 18 000 x g for 10 min. The supernatant was carefully removed and centrifuged again. The second supernatant was used directly or stored at -20°C. To measure the angiotensin I-converting enzyme (ACE) activity of the membrane extract, sperm were treated as described, but the extraction mixture was used without protease inhibitors. Activity was measured immediately after the second centrifugation.
Ram spermatogenesis was stopped by a 2°C elevation of the scrotum temperature using an insulated bag [14]. The bag was maintained in place continuously, and the animals were kept in a room with controlled light cycle and temperature (20–25°C). The decrease in sperm production was ascertained by the total number of sperm in ejaculates. After collection, seminal plasma was separated from sperm and/or cellular debris by centrifugation (two cycles at 18 000 x g, 10 min, 4°C), and the last supernatant was stored at -20°C until use.
Gel Electrophoresis and Protein Blotting
Sample preparation and methods for isoelectric focalization have previously been described [7]. Semi-dry transfer of proteins to nitrocellulose was performed over 2 h at 0.8 mA/cm2. The blots were blocked with 20 mM Tris-HCl and 150 mM NaCl, pH 7.3 (TBS), supplemented with 0.5% Tween 20 and 5% goat serum. The second antibody was a goat anti-rabbit antibody conjugated with peroxidase (Institut Pasteur, Paris, France; dilution 1:2000 to 1:5000), and the peroxidase was revealed with 4-chloro--naphthol.
Purification of the 94-kDa Protein
The 94-kDa protein in the caudal fluid was purified by successive column chromatography using an HPLC system (Prosys; Biosepra, Villeneuve-la-Garenne, France). The first stage was a gel filtration column (Hiload 16/60, Superdex 200; Pharmacia, St. Quentin en Yveline, France), used with a flow of 0.7 ml/min and 0.5 M NaCl, 50 mM Tris-HCl (pH 7.5) buffered solution. The 94-kDa protein was obtained at a retention time corresponding to about 90–100 kDa, indicating that the protein was monomeric, as confirmed by gel electrophoresis under nonreducing conditions. The 94-kDa fractions were pooled and dialyzed against a mixture of Tris-HCl/Tris-base (50 mM) at pH 8.5 before concentration on a pressurized ultrafiltration device (Minicell; Amicon, Epernon, France) fitted with a 10-kDa cutoff membrane. The concentrated solution was loaded onto an ion-exchange chromatography column (Q-Hyper-D, 10 x 150 mm; Biosepra) equilibrated with the same buffer. Elution was started by a step at 10 mM NaCl (1 ml/min) for 5 min; then a 10–200 mM NaCl linear gradient was applied for 40 min (1 ml/min). The protein was desorbed at about 50–75 mM NaCl. The 94-kDa fractions were dialyzed against deionized water, vacuum dried, and dissolved in a mixture of 10% acetonitrile-0.1% trifluoroacetic acid (TFA) in deionized water. The reverse-phase column (C18, 5 µm, 300 A, 3.9 x 150 mm; Waters, St Quentin en Yveline, France) was subjected to a 10–100% linear gradient (0.5 ml/min) with buffer A (deionized water-0.1% TFA) and buffer B (90% acetonitrile-10% deionized water-0.1% TFA). One main peak was obtained at about 40–45% buffer B. The purity of the protein was assessed by one- and two-dimensional gel electrophoresis (not shown). The purified protein represented about 0.5% of the starting material (w:w).
N-Terminal Amino Acid Sequence Analysis
The N-terminal amino acid sequences of the protein and of its fragments separated by reverse-phase HPLC after tryptic digestion (trypsin sequencing grade from Sigma Chemical Co., St. Louis, MO; 1:100, w:w) were obtained on a Porton sequencer (Model LF3000; Beckman, Palo Alto, CA) using reagents and methods recommended by the manufacturer. Sequence homology research in data banks was performed using BLAST and FASTA software [15].
Antibody Production and Immunolocalization
Before the first injection, serum from different rabbits was tested on fluids from various epididymal regions by Western blotting. One presenting no reactions was chosen for immunization with the purified 94-kDa protein. Four injections were performed, each of about 100 µg of protein. The initial injection included Freund's complete adjuvant; 3 wk later, the first boost, including incomplete adjuvant, was given, followed by two more boosts at intervals of 15 days. The last serum was used for the experiments; it was diluted 1:10 000 with the ram fluids and 1:5000 with the ram membrane extracts and the boar, horse, and bull fluids.
Immunolocalization was performed on Percoll-washed spermatozoa from the same animal. The cells were incubated in TBS-10% goat serum and then with the anti-94-kDa polyclonal antibody (dilution 1:100). The incubated sperm were washed carefully by two centrifugations and resuspended in TBS-goat serum; they were then incubated for 30 min with a goat-anti-rabbit antibody labeled with fluorescein isothiocyanate (1:100 dilution; Institut Pasteur). Labeling was photographed under a fluorescence microscope using 400 ASA black and white film; all the fluorescent micrographs were photographed using the same exposure time. The preimmune serum and the second antibody alone were tested under the same conditions, and no reaction was observed with spermatozoa from the various zones.
ACE Activity Measurement
ACE activity was measured as described previously [16] using furanacryloyl-L-phenylalanylglycylglycine as substrate (FAPGG; Sigma). The activity of the 94-kDa protein was measured on the Q-hyperD fractions, which contained mainly the 94-kDa protein. Captopril and the ACE-inhibitory peptide P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro (both from Sigma) were dissolved in ethanol-dimethyl sulfoxide (50%-50%) at a concentration of 10-3 M.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis by SDS-PAGE of perfused fluid from the ram testis and from various epididymal zones (Fig. 1) showed that a protein of about 105 kDa appeared in the fluid of the caput epididymal region (Fig. 2A); after increasing in intensity through several zones, this protein either changed in molecular mass or was replaced by a compound with a lower molecular mass of 94 kDa in the cauda epididymidis. Concomitantly, a compound of about 105 kDa decreased in intensity on the sperm plasma membrane as visualized on Coomassie blue-stained gel (Fig. 2B).
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This protein was totally absent from two-dimensional gel electrophoresis of the epididymal fluid from azoospermic rams (Fig. 3) after a period of moderate heating of the scrotum [14]. The same result was also observed from a vas efferens-ligatured animal in which sperm transit was stopped (not shown).
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These observations suggested a possible sperm origin for the 105- to 94-kDa epididymal fluid protein.
Immunocharacterization of the Protein
The 94-kDa protein was purified from ram cauda epididymal fluid proteins and used to obtain a monospecific rabbit polyclonal antibody (see Materials and Methods). The specificity of this anti-94-kDa protein antibody was tested on a nitrocellulose replica of a two-dimensional gel electrophoresis of the ram cauda epididymal fluid (Fig. 4). Only the spots forming the 94-kDa protein reacted with the antibody.
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The polyclonal antibody did not react with any proteins in the rete testis fluid or the fluid of region 0 of the epididymis (Fig. 5A). The reaction with the protein was very faint in the epididymal fluid from region 1 but clearly visible in the fluids from zone 2 to the cauda epididymidis.
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The antibody reacted with a protein of about 105 kDa in spermatozoa membrane extracts from the testis and all regions of the epididymis (Fig. 5B), showing that the two compounds were immunorelated. It is of note that there was a decrease in reaction intensity in membrane extracts from regions past the zone where the protein appeared in the fluid.
The antibody was also used to demonstrate that the apparent molecular mass of the protein changed from 105 kDa to 94 kDa during its passage from caput to corpus epididymidis (Fig. 6). The antibody reaction showed that the membrane and fluid proteins from zone 2 presented a slight difference in molecular mass and that the main shift in molecular mass to 94 kDa occurred between zone 2 and zone 4.
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The anti-94-kDa polyclonal antibody produced intense immunolabeling on the intermediate piece of testicular sperm (Fig. 7, panel T) and on epididymal sperm from zone 0 (Fig. 7, panel 0) and zone 1 (not shown). A diffuse immunoreaction was also observed on the acrosomal cap. With sperm from zone 2 (Fig. 7, panel 2) and zone 3, the reaction on the intermediate piece was greatly decreased. After zone 3, only some sperm showed a positive reaction, which was restricted to the ridge of the acrosome (e.g., zone 6: Fig. 7, panel 6). This was unchanged in the subsequent zones (e.g., zone 9: Fig. 7, panel 9) and also in ejaculated sperm (not shown).
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Biochemical Characterization
The ram cauda 94-kDa protein obtained from reverse-phase HPLC was subjected to N-terminal sequencing. Among the 24 successive amino acids obtained, 5 could not be identified (Fig. 8). This N-terminal sequence did not match any protein sequence in the databases. The protein was then submitted to tryptic cleavage, and four of the main peaks separated by reverse-phase chromatography were sequenced (Fig. 8): one of them showed 2 simultaneous sequences with an equivalent level of amino acids, indicating the presence of two fragments (peak 51). Sequences from these tryptic fragments were strongly homologous with the ACE sequence, and the best fit was found with the Bos taurus [17] protein (Fig. 8). This similarity also allowed matching of the amino acids of the two peptides of peak 51 with ACE sequences (Fig. 8).
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The ACE activity of the ram cauda 94-kDa protein was tested using FAPPG as substrate [16]. Carboxypeptidase activity of about 20 µmol FAPGG converted per minute per milligram of protein was found within the 94-kDa peak fractions obtained by ion-exchange chromatography. This activity was inhibited by captopril (IC50 = 4.6 x 10-9 M) and by the peptide P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro (IC50 = 7 x 10-8 M), both of which are specific ACE inhibitors. These values were in good agreement with those previously published for ACE inhibition [18].
No ACE activity was found in the ram testicular fluid or the epididymal fluid from zone 0, where the 105- to 94-kDa protein was absent (Fig. 5C). Very low activity appeared between zones 0 and 1; a considerable increase occurred in the distal caput (zone 2 and 3), followed by a decrease in the corpus. The activity then slightly increased in the cauda fluid.
Because it was not possible to measure activity using intact sperm with the FAPGG method, the ACE activity was estimated on membrane extracts from sperm of the various epididymal zones. We observed a diminution during caput transit; then the activity remained almost constant until the end of the epididymis (Fig. 5C). Fluid and membrane extract activities were totally inhibited by captopril.
The Soluble Fluid Form of ACE Was Also Present in the Boar, Stallion, and Bull Epididymis
The presence of free ACE in the epididymal fluids of boars, stallions, and bulls was researched by immunoblotting with the ram anti-94-kDa protein antibody and by measuring the fluid carboxypeptidase activity. Immunoreactive bands at the expected molecular size were found in the fluids of these three species, with epididymal distribution similar to that observed in the ram (Fig. 9). In the boar, the protein was first weakly labeled on the blot at about 105 kDa in zone 1–2, and a shift in its molecular mass occurred in zone 3. A slight shift was also visible with the horse protein between zones 2 and 4. In the bull, two highly reactive bands were observed from the caput epididymidis: one at about 94 kDa and the other at a slightly higher molecular mass. Several other minor reactive bands were visible thereafter between the two main proteins.
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The FAPPG-degrading activity of epididymal fluids from the boar, horse, and bull paralleled the presence of the 94-kDa protein in the fluid (Fig. 9). We also observed that the ACE activity in the fluids was lower in these species than in the ram.
Relationship between Ram Seminal Plasma ACE and the Soluble Epididymal Form
The proportion of ACE activity due to the epididymal enzyme in ram seminal plasma was analyzed using a temporarily azoospermic animal. The variations in ACE activity always followed the number of sperm during the decreased sperm phase as well as during the increased sperm phase when scrotal heating was stopped. No activity could be measured when the sperm concentrations were lower than 106 sperm/ml (Fig. 10). The variations in activity in the seminal plasma were closely related to the presence of the 94-kDa protein. For example, when no enzyme activity was observed, no immunoreaction could be found in the seminal plasma on blotting even when a sensitive chemoluminescent method was used (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ACE is a ubiquitous membrane ectoprotein found in mammalian tissues [19, 20]. Two forms of this enzyme are known: one, of 150–180 kDa, is restricted to somatic tissues, including the epididymal epithelium and the prostate, while the second, of 90–110 kDa, is expressed exclusively in a stage-specific manner by haploid germ cells [19–21]. The somatic form is constituted of two homologous catalytic domains, named the N- and the C-terminal domains. The testicular form is restricted to the C-terminal domain, generated by the activity of a testis-specific promoter situated within the 12th intron of the somatic gene. This germinal form is characterized by a species-specific 65-amino acid N-terminal [19]. The C-terminal domains of both forms of the enzyme contain a hydrophobic amino acid sequence that allows cellular membrane insertion. It has been shown in somatic tissues (such as the lungs and kidneys) that the cell-bound ACE can become a free fluid enzyme after cleavage of this 7.5-kDa anchor [22].
Significant ACE activity has been reported in the genital tract of mammals, particularly in the epididymis [23–26]. Most of these studies have been based on enzyme activity measurements, and those concerning the epididymis are confusing, mainly because of the use of tissue extracts that included potential activity from the epithelium (and the surrounding tissues), blood and lymph, sperm cells, and epididymal fluid. Decrease in epididymal ACE activity was observed in rats after castration and ligature of efferent ducts, suggesting that the possible origin of ACE was germ cells; but the possible presence of a free form derived from the somatic ACE and transported by the testicular fluid could not be excluded in this experiment [27, 28].
Our findings and previous reports from the literature lead us to strongly suggest that the ACE present in the epididymal fluid is derived from the germinal cell membrane form and is released from the moment when sperm pass through the caput epididymidis. This membrane origin is supported by our immunochemistry results showing the disappearance of the 105- to 94-kDa compound from the sperm membrane when the protein appears in the fluid. It was previously reported that an iodinated compound of about 115 kDa of ram testicular and epididymal zone 1 sperm membrane disappeared from the sperm in zone 2 [5, 11]. The 105- to 94-kDa fluid protein reported here could therefore represent this disappearing sperm membrane compound. We also observed that the 105- to 94-kDa protein was not secreted by the epithelium, since it was absent from autoradiography of 35S-labeled proteins liberated into the fluid [12]. Moreover, because the 94-kDa protein had a specific N-terminal and a molecular mass within the range of the germinal ACE (110 kDa), this strongly suggests that it could not be derived from the somatic ACE present on epididymal epithelial cells (see also the previous paragraph). We also observed that the 105- to 94-kDa compound is recognized by an antibody directed against testicular sperm membrane with exactly the same epididymal distribution as obtained with the anti-94-kDa polyclonal antibody (unpublished results). Finally, each treatment that eliminated the sperm from the epididymal duct (such as scrotal heating, ligature of the vas efferens, or castration and testosterone supplementation [unpublished results]) resulted in the disappearance of this protein from the fluid.
When the protein is liberated in the epididymal fluid, there is a slight difference between its molecular mass and that of the sperm membrane protein; this suggests the loss of the plasma membrane anchor [22], which is in agreement with our assertion of a sperm origin (see Fig. 6). Thereafter, once the protein is in the fluid, the molecular mass further decreases, from about 105 to 94 kDa in the caput-corpus junction fluid. This shift could result from a change in the glycosylation state: the testicular enzyme is highly glycosylated [29], and our observations showed that the protein is polymorphic (see Figs. 3 and 4) and that it exhibits a change in molecular mass after glycosidase treatment (unpublished results). Partial proteolysis could also affect the molecular mass of the enzyme, but only further studies of the N- and C-terminal sequences from the 105- and 94-kDa forms will provide an answer. Both hypotheses remain therefore to be tested. It is of note that the molecular mass of the protein remaining on the sperm did not change throughout the epididymis.
In some cases, an immunoreactive compound migrating at less than 10 kDa from the main protein was observed in the fluid and on the membrane. The N-terminal of this protein was sequenced in the caudal fluid and was exactly the same as the N-terminal of the 94-kDa protein. This protein could represent a degradation product or a different posttransductional modification of the protein. These changes in the molecular mass of the protein or in the ratio between the quantity of this protein and the total proteins could be at the origin of the differences in ACE activity observed in the fluid throughout the epididymis.
The present results clearly establish that a soluble form of ACE exists in the epididymis of at least four different mammalian species (ram, boar, stallion, and bull), although slight differences in the molecular form and activity were seen. We have also observed an immunoreaction with a 94-kDa protein from the caudal fluid of rats with our anti-94-kDa polyclonal antibody (unpublished results). Furthermore, our study in the ram also showed that all the seminal plasma activity is apparently due to this epididymal free form.
Liberation of epididymal sperm ACE in the various domestic species studied occurred in a localized area, suggesting the need for a specific environment. ACE release in somatic tissue could be due to a cell plasma membrane-linked metalloprotease, named secretase (or sheddase), but this enzyme has yet to be characterized [22]. In the reproductive tract, proteolytic processing of several sperm membrane proteins during epididymal transit has already been described, and the role of the sperm intracellular proteases in this process has been suggested [30–34]. However, this concerns only posttesticular protein redistribution, where the degraded compounds remain inserted in the membrane and no massive release from the sperm surface has been described. Proteolytic enzymes are present in the testicular and epididymal fluids: for example, procathepsin L, a lysosomal precursor of a highly active protease, is secreted in the boar caput epididymidis [35] at the location where the ACE proteolytic process occurs. This enzyme has also been reported to be present in the acrosome of guinea pig spermatozoa [36]. However, in fluids in which proteolytic enzymes are detected, powerful inhibitors, such as 2-macroglobulin and cystatin C, are also found [37]. A disequilibrium between an enzyme and its inhibitors could be at the origin of in situ proteolytic activity.
The angiotensin-converting enzyme plays a critical role in blood pressure regulation via the degradation of the vasodilator bradykinin and the formation of angiotensin II. Angiotensin II can act on different types of receptors, and one of its main effects is the regulation of fluid and electrolyte homeostasis [28, 29]. Indeed, in vitro studies have shown that angiotensin II activates the ionic transport (particularly that of chloride ions) of cultured rat cauda epididymal cells [28, 29]. In the epididymis, angiotensin II and its receptors are immunolocalized mainly in the basal cells of the epididymal epithelium, with an increase from the caput to the cauda [28, 38]. In vivo, the main reabsorption of water and ions from the testis occurs in the efferent ducts [1, 2, 5] where no soluble ACE can be detected in the luminal fluid, but ACE activity on the sperm membrane might act in these regions to produce angiotensin II in the lumen. In vitro studies have also shown that the epithelium produces angiotensin I and transforms it into angiotensin II without the need for exogenous ACE, in agreement with the presence of somatic ACE on these cells [19, 21, 28]. Thus, in view of the regulation of ionic transport by the epithelium, the apparently large quantity of the enzyme liberated in the caput luminal fluid is certainly more than is needed for the formation of angiotensin II.
This is also sustained by the results of several groups that obtained ACE-deficient transgenic mice in which both somatic and germinal ACE were absent [39–41]. These animals had lowered blood pressure, and the males had impaired fertility although they had normal motile sperm. Such results indicate that sperm production is possible without ACE but that the sperm lack an unidentified differentiation step that prevents them from being fertile. The precise stage at which the absence of ACE interferes with fertility has not been determined, but it has been demonstrated that these sperm are unable to go through the female genital tract and then to bind to and fertilize the egg [40,41]. A role for ACE has been suggested in the capacitation and acrosome reaction processes [26, 27, 42], and the angiotensin AT1 receptor was immunolocalized on the tail of rat and human sperm [43]. Meanwhile, it has recently been reported that angiotensinogen-deficient mice had normal fertility, although they did not produce angiotensin I, and hence angiotensin II—demonstrating that angiotensin II is not necessary for the formation and activity of the epididymis and sperm in this species [41, 44]. All these results suggest that ACE is necessary for fertility but apparently not for its angiotensin II formation activity. Since ACE also has a number of other potential substrates (such as bradykinin and LHRH), it may have a carboxypeptidase activity toward other epididymal peptide(s) involved in sperm maturation.
Not all the testicular sperm ACE was released during transit; in our study ACE activity was found in membrane extracts of sperm from the corpus and the cauda epididymidis and also on ejaculated sperm. The remaining enzyme was immunolocalized on the acrosomal region of some of the ram sperm, and it has recently been shown in ejaculated horse and human sperm that the enzyme is situated within the acrosomal vesicle [45, 46]. This internal localization is supported by studies in which intact ejaculated boar and ram sperm activity in the degradation of bradykinin was very low [47]. This intraacrosomal location could also explain why the remaining sperm protein could not be iodinated after the caput epididymidis [6, 9]. Finally, the acrosomal location certainly protects the protein from proteolytic release in the epididymis and may explain its possible role in postejaculatory events [39–41].
In conclusion, we demonstrated biochemically that, like somatic membrane-bound ACE, germ cell membrane-bound ACE can be released in vivo in a soluble active form and that this release occurs in a very precise epididymal area. The important role played by this enzyme in reproductive function and in sperm physiology, and the mechanism of its epididymal release, remain to be clarified. This study also demonstrates that testicular sperm can be the vehicle for active compounds that can be released in a specific area of the reproductive tract.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 33 247 427 743; gatti{at}tours.inra.fr
Accepted: November 30, 1998.
Received: March 20, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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