Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print June 29, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042929
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BIOLOGY OF REPRODUCTION 73, 881–890 (2005)
DOI: 10.1095/biolreprod.105.042929
© 2005 by the Society for the Study of Reproduction, Inc.

Shedding of the Germinal Angiotensin I-Converting Enzyme (gACE) Involves a Serine Protease and Is Activated by Epididymal Fluid1

Véronique Thimon 3, Sonia Métayer 3, Maya Belghazi 4, Françoise Dacheux 3, Jean-Louis Dacheux 3, and Jean-Luc Gatti 2, 3

Gamètes Mâles et Fertilité3 Service de Spectrométrie de Masse pour la Protéomique,4 UMR 6175 INRA-CNRS-Université de Tours-Haras Nationaux, Station de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Nouzilly, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present report describes how the soluble germinal angiotensin I-converting enzyme (gACE) appears in the epididymal fluid, where it has been identified in some laboratory rodents and domestic ungulates. We showed that this gACE results from an active proteolytic process that releases the enzyme's extracellular domain from sperm in a precise spatiotemporal location during epididymal transit and that this process involves serine protease activity. Using polyclonal antibodies against the C-terminal intracellular sequence of ACE, a fragment of approximately 10 kDa was detected on the sperm extract only in the epididymal region, where the gACE release occurs. The fluid enzyme was purified, and the cleavage site was determined by mass spectrometry to be between Arg622 and Leu623 of the mature sheep gACE sequence (equivalent to Arg627 and Arg1203 of the human mature gACE and somatic ACE sequences, respectively). Thereafter, the C-terminal Arg was removed, leaving Ala621 as a C-terminal. Using an in vitro assay, gACE cleavage from sperm was strongly increased by the presence of epididymal fluid from the release zone, and this increase was inhibited specifically by the serine protease-inhibitor AEBSF but not by para-aminobenzamidine. None of the other inhibitors tested, such as metallo- or cystein-protease inhibitors, had a similar effect on release. It was also found that this process did not involve changes in gACE phosphorylation.

epididymis, gamete biology, male reproductive tract, sperm, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The posttesticular processes involved in mammalian sperm developing the capacity to fertilize an ovum have not been resolved, but convincing evidence indicates that this process includes changes in the sperm surface that result from the various interactions with the epithelium lining the extratesticular duct system and with proteins secreted in the lumen of this duct. The present study examines how the germinal isoform of angiotensin I-converting enzyme (gACE) is released into the lumen of the duct.

A number of different cell surface proteins, such as membrane-anchored enzymes, receptors, and adhesion molecules, are targets of specific proteolytic processing that releases their extracellular domain and may function as a posttranslational switch for their biological activity [1–4]. The process, referred to as protein ectodomain shedding, occurs in different types of cells and tissues, and the secretases or sheddases involved are mainly members of the ADAM metalloprotease family [3, 5].

Angiotensin I-converting enzyme is a key factor in the renin-angiotensin system that is involved in the homeostatic regulation of blood pressure, and it is used clinically to control human hypertension by inhibiting this enzyme [6– 8]. This type-I ectoprotein peptidase converts angiotensin I into the active vasopressor angiotensin II and inactivates the vasodilator bradykinin [6]. Two different isoforms of ACE have been found in male mammals: a somatic ACE (sACE), which is expressed in numerous tissues (vascular endothelium, renal tubule, and intestinal epithelium), and gACE (also known as testicular ACE), which is present on the plasma membrane of testicular sperm [9–11]. In addition to these membrane-associated forms, sACE also exists as a soluble protein that can be isolated from blood [8, 12]. Various mouse knockout models for ACE have been used to demonstrate the roles of the different somatic forms in regulating blood pressure and tissue development [13–18]. They also have demonstrated an important role of gACE in fertility, because male ACE knockout animals have shown low fertility linked to a decrease in the ability of their spermatozoa to travel through the female genital tract and bind to the zona pellucida. It has been suggested that this effect results from the recently discovered glycosyl phosphatidylIinositol (GPI)-anchored protein releasing activity of this enzyme [13–19]. Furthermore, it has been demonstrated in vitro that in different cell culture systems overexpressing gACE or sACE, the extracellular domain of the protein can be released from the cell surface by a proteolytic process. It also has been shown that phorbol ester increased this release and that ACE phosphorylation regulates the process [20–22]. The cleavage involves a cell membrane-associated ACE sheddase (or ACE secretase) that was first described as a metalloprotease activated by phorbol ester, but it recently has been suggested that a serine protease could be involved as well [4, 23–25].

We have shown previously that gACE, which normally is located at the membrane surface of testicular sperm [11, 26], is released in vivo during the transit of the sperm in the epididymis, a long and convoluted duct that connects the testis to the vas deferens, where sperm gain their fertilizing ability [27, 28]. This release occurs in a specific area of the duct (the caput epididymidis), and the gACE released subsequently remains present and active in the epididymal fluid and in the seminal plasma after ejaculation [29–31]. Observed in different mammalian species from cattle to rodents, this release suggests a well-conserved physiological mechanism.

We demonstrate here that gACE cleavage results from a specific proteolytic process and occurs in vivo at the same sequence site as that for gACE and sACE in cell models. Our findings support the involvement of a serine protease in this process, and they show that in contrast to cell culture models, the release of gACE from the sperm cell surface is not correlated with changes in its phosphorylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Fluids and Sperm

Experiments on animals were conducted according to the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction. Epididymides and testes were removed from adult Ile-de-France rams or Large-White boars either by castration of anesthetized animals or after slaughter. Luminal fluids were collected from the testis and different zones of the epididymis (caput, zones 0–3/4; corpus, zones 4–6/7; cauda, zones 7–9) as previously described [32–34]. Spermatozoa were separated from the fluid by centrifugation (10 min, 1500 x g), suspended in PBS solution, and washed twice (10 min, 1500 x g). The plasma components from the testicular and epididymal fluids were centrifuged twice at 15 000 x g for 5 min and kept at –20°C. Sperm were layered on a discontinuous PBS-Percoll gradient (50% and 90% layers) and then centrifuged (10 min, 3000 x g). Spermatozoa were taken at the 90% interface and washed in PBS. Sperm were extracted by mixing the pellet with an equivalent volume of sample buffer. The mixture was centrifuged at 15 000 x g for 10 min. ß-Mercaptoethanol was added to the supernatant before heating (5 min at 90°C). The samples were then used directly or stored at –20°C.

Gel Electrophoresis and Protein Blotting

The methods for preparing gels and samples were as described previously [33]. The proteins were transferred to nitrocellulose (0.8 mA/cm2 for 2 h) for immunodetection. The membranes were blocked for 1 h with Tris-buffered saline supplemented with 0.5% (w/v) Tween 20 (TBST) and 5% (w/v) dried skimmed milk or 0.5% BSA. Two types of antibodies were used: a rabbit polyclonal antibody prepared against purified soluble gACE (Ac699) that recognized both gACE and sACE [31] and rabbit polyclonal antibodies directed against the highly conserved, final 20 amino acids of the intracellular C-terminal sequence (28C8 and 28D8; a gift from Dr. François Alhenc-Gelas, U652 INSERM, Paris, France) [35], or Ac2005 that was prepared in our facilities against the same conserved C-terminal sequence. These antibodies showed the same specificity and are referred to as anti-CT throughout the text. The mouse monoclonal antiphosphotyrosine and the antiphosphoserine antibodies were commercially available (from Santa Cruz Biotechnology, Santa Cruz, CA, and Sigma, St. Quentin Fallavier, France, respectively). Western blots were incubated (with agitation for 2 h at 37°C) with antibodies (dilution at 1:5000 for 28C8, Ac2005, and Ac699 and 1:2500 for antiphosphotyrosine and antiphosphoserine). Blots were washed with the same buffer and then incubated with a goat anti-mouse or anti-rabbit antibody conjugated with peroxidase (dilution 1:5000 in TBST with 5% milk) for 1 h at 37°C. After three washes with TBST, the peroxidase was detected with a chemoluminescent substrate and visualized by a fluorescent imager. When needed, the digital images of the Western blots were analyzed using computer software (1D Elite; Amersham Pharmacia, St. Quentin en Yveline, France).

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction

Total RNAs were prepared from frozen samples (200 mg) of liver, kidney, lung, heart, testis, and specified zones of the epididymis (RNAble Technique; Eurobio, les Ullis, France). The reverse transcriptase assay was performed on 3 µg of total RNA using the Superscript Reverse Transcriptase H (Invitrogen, Cergy Pontoise, France) and oligo(dt) primers. The gACE was sequenced by successive polymerase chain reactions (PCRs) with 30 pmol of specific primers obtained from a partial sequence published for sheep ACE [36] (forward, 1135-GCCTGGGACTTCTTCA-1150; reverse, 1650-AAAGCTCACGAAGTACCTGA-1631; numbered as shown in Fig. 1) and primers deduced from the human testicular sequence [37] that were highly homologous with bovine [38] and murine somatic sequences (forward A, 1-ATGGGCCAGGGTTGG-15; reverse A, 624-CCAGCTCTTC1CACGCCCAGG-605; forward B, 605-CCTGGGCGTGGAAGAGCTGG-624; reverse B, 946-AGGGTGCCA-938; forward C, 794-CGCTCTACCTGAACCTGCAT-813; reverse C, 1190-TTCACTGAGGTGCACTGCTT-1171; forward D, 1619-GTGTGCCTTATGTCAGGTACT-1639; reverse D, 2172-GGAGTGTCTCAGCTCCACCTC-2152). The PCR was performed for 35 cycles at the temperature specified for the primer set and with the final 5-min elongation step at 72°C. Aliquots (5 µl) of each reaction mixture were analyzed on 1.5% ethidium bromide-stained agarose gel. The PCR amplicons were either sent directly for sequencing or sequenced after amplification and clonal selection in PCR2.1 vector (Invitrogen). Specific primers were designed for the gACE study in the different tissues (gACE forward, 105-GACCACCAGCCAGGGGAC-122; gACE reverse, 308-AACTTGCTGTTGTCCGTGCT-289), with the forward primer being situated in the specific gACE N-terminal region. ß-Actin primers (forward, 5'-GGACTTCGAGCAGGAGATGG-3'; reverse, 5'-GCACCGTGTTGGCGTAGAGG-3') were used as controls for the PCR and to equilibrate the quantity of mRNA.



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  		FIG. 1. Nucleotide and predicted amino acid sequences of the cDNA that encodes ram gACE (GenBank accession no. AJ920032). The underlined sequences are those previously obtained by Edman sequencing; the boxed amino acid indicates where the mature sequence begins

Northern Blot Analysis

For each sample, 20 µg of total RNA were separated by electrophoresis on 1% agarose formaldehyde gel. The RNA was transferred to a nylon membrane (Hybond N+; Amersham) by overnight capillary blotting in 20x SSC (single-strength: 0.15 M sodium chloride and 0.015 M sodium citrate) and cross-linked by exposure for 30 sec under ultraviolet light. The membrane was stored at room temperature until prehybridization. A cDNA probe for the ram gACE was obtained by reverse transcription-PCR using the primers specific for ovine gACE. The cDNA probe was then labeled with [32P]dCTP (Megaprime II; Amersham). Hybridization was performed using a 32P-labeled probe. The membrane was prehybridized with 100 µg/ml of salmon sperm DNA (2 h at 42°C), and hybridization was performed overnight at 42°C. The membrane was then washed once in 1x SSC with 0.5% SDS (20 min at room temperature) and then three times in 0.2x SSC with 0.5% SDS (20 min at 68°C). The ACE transcript was visualized after exposure on a radioactivity-sensitive screen or film.

Purification of the Soluble Form of gACE

Ram gACE was purified from luminal fluid from the caput epididymidis (zone 2) and cauda epididymidis (zone 9) as described previously [31]. Briefly, the fluids were first separated by gel filtration, and the fractions containing the gACE were then pooled, dialyzed, loaded on an anion-exchange column, and eluted with a linear NaCl gradient. The gACE-positive fractions were pooled, dialyzed, and loaded on a C18 reverse-phase column using a water/acetonitrile/0.1% trifluoroacetic gradient. Each step of purification was controlled by electrophoresis and Western blot analysis using the rabbit polyclonal antibody against the ram gACE (Ac699). The purest fraction from the reverse phase was used for analysis by mass spectrometry.

The purified gACE from epididymal zones 2 and 9 were reduced and alkylated with iodoacetamide and then incubated overnight at 37°C in a microtube with 12.5 ng/µl of endo-lysine C (sequencing grade; Roche, Meillan, France). The solution was then dried, reconstituted with 0.1% formic acid, and sonicated for 10 min. The peptides generated were either analyzed directly by MALDI (M@LDI-L/R; Waters Micromass, Manchester, UK) or sequenced by nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS; Q-TOF-Global equipped with a nano-electro spray ionization source; Waters Micromass) in automatic mode. The peptides were loaded on a C18 column (Nano Ease Atlantis dC18, 3 µm, 75 µm I.D., 150 mm length,Waters) and eluted with a 5–60% linear gradient at a flow rate of 180 nl/min for 30 min (buffer A, water/acetonitrile [98: 2, v/v] and 0.1% formic acid; buffer B, water/acetonitrile [20:80, v/v] and 0.1% formic acid). The peptide masses and sequences obtained were blasted manually against the translated sequence obtained for ram gACE.

In Vitro Assay for gACE Released from the Sperm Membrane

Spermatozoa from testes and epididymal zones 0 and 1 (retaining their membrane gACE) were washed twice with PBS, resuspended in PBS+ (PBS supplemented with 2 mM lactate, 1 mM pyruvate, 1 mM glucose, and 4 µl/ml of gentamicin), and then distributed in microtubes (108 spermatozoa/ml) before incubation at 37°C. Where indicated, this buffer was supplemented with protease inhibitors, fluid from epididymal zone 2 (final concentration, 5 mg/ml), or with pervanadate (final concentration, 2 mM pervanadate; 50 mM pervanadate stock solution was prepared by mixing equal volumes of 100 mM solution of H2O2 with a 100 mM solution of sodium orthovanadate). At indicated times, aliquots were taken, and the spermatozoa were separated from the medium by centrifugation (15 000 x g, 10 min, 4°C). The supernatant was mixed with reducing buffer, and the sperm pellet was treated as described above except for the orthovanadate assays. For those assays, the washed sperm pellet was extracted using PBS supplemented with 1% SDS, protease-inhibitor cocktail (1:100, v/v; Sigma), and 2 mM pervanadate before centrifugation (15 000 x g, 5 min). The supernatant was mixed with reducing buffer as described above.

Immunoprecipitation of Membrane gACE and Its Proteolytic Fragment

Sperm from epididymal zones 0/1, 2, and 9 were centrifuged (1500 x g, 5 min), washed twice in PBS+, and counted. A pellet containing 107 spermatozoa was then extracted with 100 µl of 0.5% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) in PBS containing protease- and phosphatase-inhibitor cocktails (1:100, v/v). After centrifugation (15 000 x g, 10 min), the supernatant was mixed with the same volume of the same buffer containing 4% (v/v) of the anti-C-terminal (anti-Cter) antibody without CHAPS. After 2 h, Protein A sepharose beads were added (final concentration, 4%, v/v) for two more hours. The beads were separated by centrifugation (1500 x g, 30 sec) and washed three times with PBS inhibitor. The beads were mixed with Laemmli buffer supplemented with phosphatase inhibitors, then boiled and centrifuged (15 000 x g, 5 min) before loading onto a gel or storage at –20°C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Determining Sequences of Ovine ACE

Previously, we have shown that gACE, a 94- to 110-kDa protein, appeared in the fluid from the caput epididymis in rodents and various domestic mammals and that this protein originated from the sperm cell surface. To ascertain that all ACE in epididymal fluid derives from the sperm, we analyzed the distribution of sACE and gACE in tissues (including zones of the epididymis) for which no data were available. The complete sequence of ram gACE cDNA was determined from total mRNA extracted from the ram testis, and a partial specific cDNA sequence was determined for the N-domain of sheep sACE from the kidney (Figs. 1 and 2A).



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  		FIG. 2. A) Nucleotide and predicted amino acid sequences of the partial cDNA that encodes sheep sACE (GenBank accession no. AJ920033). B) Boxshade sequence comparison of the ovine gACE and sACE deduced amino sequences, the N-domain (sACE-Nt) and C-domain (sACE-Ct) of bovine sACE (GenBank accession no. 1919242A), and the human gACE and sACE (GenBank accession no. NP_690044 and NP_000780, respectively). The sequences are arranged according to the percentage of similarity with the sACE sequence

Figure 1 shows that the cDNA coding for ram gACE comprises 2172 bases representing 724 amino acid residues. The mature N-terminal sequence, previously determined by Edman sequencing, starts at Glu29, leaving a mature, germinal-specific N-terminal sequence of 31 amino acids before the common C-terminal domain with sACE (compared to sACE from bovine). All other amino acid sequences previously obtained by N-terminal sequencing of peptides from epididymal fluid were retrieved (Fig. 1, underlined sequence).

The sheep gACE showed 75% homology with human, rabbit, and mouse gACE and 65% and 90% homology with the N-terminal and C-terminal domains of bovine sACE, respectively (Fig. 2B). The sACE sequence showed more than 95% homology with the sACE N-terminal domain in humans, cattle, mice, rabbits, and other mammals (Fig. 2B). Comparison between the sheep amino acid sequences showed only 76% homology (65% similarity) between gACE and the partial sequence obtained for the sACE N-domain (Fig. 2B).

These results also indicate that the gACE and sACE primers were specific and could be used to differentiate these isoforms by PCR on total mRNA from different tissues (testis, kidney, liver, heart, and epididymis) (Fig. 3). The gACE primers gave the 203-base pair (bp) amplicon size was expected only in the testis (Fig. 3, A and B), whereas the sACE primers gave the 515-bp amplicon in all the tissues examined, including the testis and all zones of the epididymis (Fig. 3, A and B). However, a higher level of mRNA was present in the caput region (zone 1) than in the rest of the epididymis. The PCR results were confirmed further by Northern blot analysis using the 203-bp gACE amplicon as a probe: One mRNA band was observed in the testis extract but was not present in the epididymis (Fig. 3C) or the other tissues that were probed (kidney, liver, and heart; not shown).



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  		FIG. 3. A) Complementary DNA was obtained by PCR for gACE only in the testis (T), whereas sACE also was amplified in the different tissues (kidneys [K], heart [H], and lung [L]). Actin PCR was used to equilibrate cDNA of the different tissues. B) PCR using cDNA prepared from the testis and the different epididymal zones showed the expression of gACE in the testis, whereas sACE mRNA was found in the testis and all zones of the epididymis. Actin PCR was used to equilibrate cDNA of the different tissues. C) Total RNA from the testis and different epididymal zones was hybridized with a cDNA probe of the gACE. A transcript was observed only in the testis. A cDNA probe against the 18S mRNA was used to visualize mRNA quantities

In Vivo gACE Is Released from Ram and Boar Sperm by Proteolytic Cleavage

Extracts of ram sperm and the luminal fluids obtained from the testis and all zones of the epididymis were probed with polyclonal antibodies. The sperm extracts were probed against the C-terminal intracellular sequence, and the epididymal fluid gACE was probed against polyclonal Ac699.

In the epididymal fluids from zones 1/2 through 9, strong reactivity was found at approximately 94 kDa (Fig. 4), which is in agreement with our previous findings [31]. A reactive band at approximately 10 kDa was present in sperm extracts, mainly for samples from zones 1/2 and 3, with decreasing intensity in zones 4–9 (Fig. 4). No reactivity for this fragment could be seen in sperm extracts from epididymal zone 0 or the testis or in the epididymal fluid. Similar results were obtained using the same antibodies on samples from the boar testis and epididymis (Fig. 4). Because the observed 10-kDa fragment (representing the trans-membrane and intracellular domains of the gACE) could only have originated from the cell surface gACE, this suggests that it is generated via active proteolytic cleavage at the membrane level during the release of gACE in the fluid.



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  		FIG. 4. Western blots of ram and boar epididymal fluid and sperm extracts from the testis (T) and different zones of the epididymis (zones 0– 9) probed with the antibody against the gACE extracellular domain (Ac699) or the anti-Cter intracellular region of gACE (C-ter) showing the correlation between gACE release and appearance of the 10-kDa fragment

Determination of gACE Sequence Cleavage Site in the Ram

Mass spectrometry was carried out on soluble gACE peptides purified from epididymal zones 2 and 8/9 by high-performance liquid chromatography after digestion with endoproteinase Lys-C of the final reverse-phase purified proteins. Analysis showed that several of the peptides could not be explained by the theoretical lysine-cleavage pattern that is expected from the deduced amino acid sequence. These fragments were further sequenced by LC-MS/MS. Amino acid sequences and fragmentation patterns were obtained for three fragments: m/z 1689.91 in epididymal zone 2 (Fig. 5A), and m/z 1533.81 and m/z 1555.78 in zones 2 and 9 (Fig. 5, A and B). The m/z 1689.91 fragmentation gave the sequence shown in Figure 5C, which made it possible to deduce the complete fragment sequence LGWPQYNWTPNSAR (theoretical m/z 1689.81). Similarly, the fragment m/z 1555.78 was deduced to be a sodium adduct of the peptide LGWPQYNWTPNSA (theoretical m/ z 1533.71), which corresponds to the fragment found at m/ z 1533.81 on the spectrum (Fig. 5, C and D). Because the yield of the m/z 1689.91 fragment was greater for gACE from zone 2 than for gACE from zone 8/9, the first cleavage was considered to occur after the Arg622 (corresponding to Arg627 and Arg1203 of mature human gACE and sACE, respectively) and was followed by the removal of this amino acid, leaving Ala621 as the last amino acid (Fig. 5D).



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  		FIG. 5. Mass spectrometry of gACE endo-lysine-generated fragments from gACE purified from zone 2 (A) and zone 9 (B). The LC-MS-MS sequencing of the fragment of m/z 1555.78 from zone 9 also is shown (C). The successive cleavage sequence sites were then deduced from these data (D)

Analysis of the other peptides that were purified also showed two new alternate N-terminals for gACE, with both being acetylated. The first was Ac-TTSQATTSNLVSDEAEARK (m/z 2049.9), and the second was Ac-SQATTSNLVSDEAEARK (m/z 1847.8).

Release of gACE from Ram Sperm In Vitro

The mechanism of gACE shedding was characterized further by determining its dependence on external factors. Ram sperm were obtained from the testis, rete testis, and zones 0 and 1 of the epididymis (where all the gACE is still on the sperm membrane) and then incubated under different media conditions. Three hours of incubation in PBS+ (see Materials and Methods) produced a large increase in the 10-kDa immunoreactive C-terminal fragment on sperm extracts (Fig. 6). This increase was correlated with the increase of immunoreactive gACE protein in the medium (Fig. 6A), and this soluble enzyme retained its activity (not shown). On the other hand, similar results were obtained when trypsin, a serine protease reported to release the extracellular domain of ACE, was included in the medium and also when the sperm were frozen for 2 h at –20°C, thawed, and incubated for 1 h in PBS (not shown). This last result led us to verify sperm integrity during the incubation period, and we observed that the number of dead sperm (as determined by propidium iodide staining) increased by more than 20–25% after 3 h. Indeed, the dead sperm were shown to be the source of the increased concentration of free and active acrosin, a sperm acrosomal serine protease, in the incubation medium (determined both by Western blot analysis and zymography; not shown). A potent acrosin inhibitor, para-aminobenzamidine (PABA) [39, 40], was therefore included in incubation medium. This treatment inhibited acrosin release and activation (not shown), and it reduced gACE shedding by more than 60% (Fig. 6B). When a second serine protease inhibitor (AEBSF) was included, almost 80% of the release was inhibited (Fig. 6B).



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  		FIG. 6. A) Sperm from the testis and from zones 0 and 1 were incubated in PBS+, PBS+ supplemented with 5 mM PABA, or PBS+/PABA supplemented with 2 mM of AEBSF for different times (0–3 h). The supernatant (super) and the sperm pellet extract (spz) were blotted and probed with the Ac699 antibody (super) or the anti-Cter antibody, respectively. Sperm extract from zone 2 was used as a control. B) Western blots of sperm extract were analyzed, and signal values obtained for the C-terminal fragment were reported as a percentage of cleavage, using the highest value (obtained at 3 h in PBS+) as 100% (maximum cleavage). Results were obtained from four separate experiments and are presented as the mean ± SEM. C) Sperm extract obtained after incubation in PBS+/PABA and PBS+/PABA/AEBSF supplemented with 5 mg/ml of proteins from zone 2 (FZ2). D) Results from the experiments shown in C reported as a percentage of the maximum signal obtained in PBS+/PABA/FZ2. Results were obtained from three separate experiments and are presented as the mean ± SEM

When sperm were incubated with luminal fluid from the rete testis and zones 2 and 9 of the epididymis in the presence of PABA to avoid artefactual release, only the fluid from zone 2 showed a strong increase in the specific fragment as compared to PBS+/PABA (Fig. 6 and not shown). Similar results were obtained even with longer incubation times (16 and 24 h), and of the different classes of inhibitor tested (1 µg/ml of aprotinin, 5 mM PMSF, 0.5 mM leupeptin, 0.2 mM antipaïn, 0.3 mg/ml of trypsin-chimotrypsin inhibitor, 3 mM cystein protease inhibitor [E-64], 0.3 mM bestatin, 0.14 mM pepstatin, and 1 mM thiorphan), only AEBSF (2–5 mM) produced a significant and repeatable decrease in this response (~50%) (Fig. 6D). In some experiments, we found that when including EDTA (5 mM) or 1,10-phenantrolin (5 mM), both metalloprotease inhibitors produced an increased intensity of the fragment, but not of the released protein, suggesting that degradation of the fragment was inhibited once it was formed.

Role of Phosphorylations in gACE Shedding

Because reports on somatic cells indicate that phosphorylation modulates the shedding of ACE from cell cultures [20, 21], the role of phosphorylation in the shedding of gACE was examined in spermatozoa. However, we found that the addition of pervanadate (a phosphatase inhibitor and activator of different kinases), which increased shedding in the in vitro model [21], had virtually no effect on gACE shedding from sperm. Inclusion of up to 2 mM pervanadate in the sperm assay had almost no effect on the shedding of gACE, as shown by Western blot analysis with the anti-Cter antibody, and no increase was noticeable in tyrosine phosphorylation at the molecular range of the intracellular fragment (Fig. 7A) or of the shed enzyme (not shown).



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  		FIG. 7. A) Western blots of sperm extracts obtained under the different medium conditions described and probed either with the ACE anti-Cter antibody (Cter) or with the antiphosphotyrosine antibody. B, C: Sperm from zones 0/1, 2, and 9 were extracted with CHAPS and immunoprecipitated with gACE Ac699 antibody (B) or with anti-Cter antibody (C). The immunoprecipitate was stained with Coomassie blue (CBB) and then transferred and probed with anti-Cter, antiphosphoserine (P-ser), or antiphosphotyrosine (P-tyr)

Possible changes in endogenous phosphorylation of gACE in sperm during epididymal transit also were examined. Sperm taken from zones 1, 2, and 9 of the epididymis were extracted with a mild detergent, and the extract was immunoprecipitated with the anti-Cter antibody. However, immunoprecipitates probed with antiphosphoserine and antiphosphotyrosine antibodies showed no specific reaction with the complete membrane gACE or with the intracellular fragment (Fig. 7B), whereas both antibodies recognized a number of different proteins in the initial extract. The lack of reaction was not a result of the absence of immunoprecipitation of intact gACE or its fragment, as shown when the same blot was probed with the anti-Cter antibody (Fig. 7B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The ACE gene encodes two related but structurally different isoforms at different body locations. Both isoforms derive from the same gene, but gACE is translated by a tissue-specific promoter situated in the 12th intron of the sACE gene, which is active only in male haploid germ cells [11, 26, 41]. The sACE isoform is a protein of approximately 150 kDa that consists of two redundant domains (N- and C-domains), each including a zinc-binding catalytic site (His-Glu-X-X-His). Because of its alternative initiation site, gACE is of 90–110 kDa, representing only the C-terminal domain of sACE, including the hydrophobic membrane anchor and the short cytoplasmic tail, and it shows a specific N-terminal, consisting of 60 amino acids. Both domains of sACE and gACE cleave angiotensin I, bradykinin, and different bioactive peptides in vitro and show quite similar enzymatic characteristics, although they may have different functions in vivo [30, 42, 43]. The sequence data obtained for ovine gACE and sACE were in agreement with this description and indicate, to our knowledge for the first time, that at least three different N-terminals (one free and two acetylated) coexist for the gACE-released protein. Because of their posttranslational modifications, the two acetyl-N-terminals have not been described previously. The exact role of this common posttranslational modification remains unknown, but it may relate to the stability, physiological function, and degradation of proteins [44].

It is difficult to differentiate between sACE and gACE because C- and N-domains are duplicated sequences and because the C-domain sequence is common between sACE and gACE [10, 30]. Our previous studies showed that ACE is absent in the epididymal fluid from azoospermic animals and indicated that the ACE in epididymal luminal fluid derives from sperm gACE [29, 31]. In the present study, we provide definitive proof that all ACE in epididymal luminal fluid derives from gACE released from sperm during transit through the caput epididymidis. First, using PCR and Northern blot analysis, we showed that there is no mRNA for gACE and, therefore, no gACE protein expressed in the epididymis. Moreover, except in the very proximal zones, the quantity of sACE mRNA was very low in the epididymis, and the protein was only just visible on Western blots prepared from extracts of the caput epididymidis of the ram. This is in agreement with findings for the mouse and rat [29] (not shown). Second, all the amino acid sequences that we have determined, both in the present and a previous study [19], for purified ACE from epididymal fluid fell within the sequence obtained for gACE, including the specific N-terminal sequence, which was clearly different from that of the purified renal ovine sACE, LDPALQPGXF-, obtained by Edman sequencing (unpublished data). Third, we showed that the release of gACE from sperm results from an active proteolytic process, leaving a transient C-terminal fragment of less than 10 kDa in the sperm. We also have determined that this cleavage results from the action of a protease in a region of the gACE between Arg622 and Leu623, leaving a 74-amino acid fragment inserted in the plasma membrane, which is a size in agreement with that observed on SDS-PAGE. The role of a protease in shedding was demonstrated further by in vitro experiments showing that the fluid from zone 2 of the epididymis (where release occurs in vivo) stimulated the cleavage and that a specific serine protease inhibitor, AEBSF, could partially inhibit the process. Altogether, this demonstrates that gACE shedding is an active process that occurs in a specific region of the epididymis and might involve a specific serine protease.

It has been shown that the cell-expressed sACE and gACE are cleaved after the Arg1203 and Arg 627 residue, respectively, at a site identical to that reported in the present study [22, 24]. This cleavage site also was found on soluble human and porcine sACE purified from physiological fluids (blood plasma and seminal plasma), and the authors suggested that the last Arg residue also may be removed after release [24]. This is very similar to what we observed with the released gACE during its transport in epididymal luminal fluid. Earlier studies performed in vitro on cell-culture systems that overexpress gACE or sACE and with a kidney membrane cell-free assay concluded that the ACE sheddase was a metalloprotease, mainly on the basis of inhibition obtained with metalloprotease and specific hydroxamate-based inhibitory compounds [4, 45, 46]. Moreover, the various secretases reported in the literature to date are mainly protease members of the ADAM family of metalloproteases, although to our knowledge, none of the ADAMs tested has been shown to be involved in ACE shedding [5, 47]. In our in vitro assay, we did not test the specific hydroxamate-inhibitory compounds, but we did test several metalloprotease inhibitors, including 1,10-phenantroline, thiorphan containing a thiol amido acid that selectively binds to the active site of zinc metalloproteases and blocks their activity, and EDTA. Even at high doses, none of these inhibitors had an effect on release, although EDTA and phenantrolin have been reported to inhibit the secretase from the kidney microvillar membrane [23]. In contrast, we found that AEBSF, an irreversible serine protease inhibitor, was able to strongly and specifically reduce the shedding. Various reports have challenged the idea of a sole metalloprotease as an ACE sheddase, because under certain conditions, such cleavage was not inhibited by the metalloprotease inhibitors but, rather, by the serine protease inhibitor [21, 48]. Moreover, the site of cleavage after an Arg is a classical site for serine protease, and it has been shown that the ACE shedding could be mimicked by the action of trypsin in the in vitro models or by trypsin or acrosin in the sperm model described here. These findings and our present results thus suggest that different types of proteases may play the role of sheddase, according to the cell line or the tissue.

The results obtained in our in vitro system with the fluid of zone 2 may suggest that the sheddase is secreted or released into the epididymal fluid in this precise region of the epididymis or that a cascade of protease activation involving a specific serine protease in this epididymal fluid affects the activation of the sperm membrane sheddase. An alternative interpretation is that a protease inhibitor is removed from the fluid, facilitating the sheddase activation (or the protease cascade that leads to its activation). Different types of protease have been described in epididymal fluid, and reports have appeared of sperm surface transformations involving proteolytic removal of all, or part, of a protein, including fertilin and cyritestin, which are two ADAM members involved with ensuring the fertility of sperm [49, 50]. In this respect, a serine protease has been reported to be involved in the epididymal processing of fertilin ß and the pH20–2B1 sperm surface protein [51, 52].

Our findings also demonstrate that gACE shedding from sperm is not linked to changes in phosphorylation before or during the process, either on the external domain or in the intracellular-transmembrane fragment. Phosphorylation of the extracellular ACE domain has been reported, but such phosphorylation is intracellular, occurring before ACE is targeted to the plasma membrane [21], which is a process that could occur only during spermatogenesis and not after sperm leave the testis. In contrast, intracytoplasmic phosphorylation of the ACE tail can, apparently, inhibit shedding, suggesting a regulatory mechanism for the quantity of ACE at the membrane level [20]. Again, the absence of intracytoplasmic phosphorylation of sperm gACE during transit, and in the presence or absence of pervanadate, suggests that this mechanism is not involved in gACE shedding from sperm.

Surprisingly, the ACE knockout mouse phenotype demonstrated the importance of gACE in male reproduction. Male ACE knockout mice show renal defects, spontaneous hypotension, and considerably lowered fertility, although they apparently have normal spermatozoa [8, 17, 18, 53]. It was initially suggested that this infertility resulted mainly from impairment of the sperm's ability to travel through the female genital tract and bind to the egg investments. The low fertility is solely related to the absence of gACE expression on sperm, as shown by the restoration of fertility when gACE is reintroduced and expressed in germ cells of a male ACE-KO [54]. Because all our findings indicate that the sperm cell surface gACE is rapidly removed during epididymal transit, this protein may not be involved directly in interactions between sperm and cells in the female tract or in primary sperm-egg binding. A new enzymatic activity has been reported recently for ACE: cleavage of the GPI-anchored protein at the level of the GPI tail under certain conditions [19]. These authors also suggested that the lowered fertility of gACE knockout mice might result from lack of processing of some sperm GPI proteins by gACE either during or after the acrosome reaction. On the other hand, the absence of gACE at an early stage in the development of sperm membrane domains also could impair the positioning or processing of other surface components, as shown for the interdependence of fertilin {alpha}, ß, and cyritestin in their respective knockouts [50, 55]. Alternatively, the absence of a large quantity of this enzyme at a specific level of the epididymis may affect the function of epithelial cells and result in subtle modifications in epididymal maturation. Clearly, more work is needed to resolve the role of ACE in male fertility.

In conclusion, we have demonstrated that gACE is shed from the sperm surface by an active proteolytic process involving serine protease activity. This processing occurs in the same sequence pattern as that reported both in vitro and in vivo for ACE. Because sACE cleavage is difficult to study in vivo, the epididymis (a closed environment) provides a good alternative to the cell-culture model for determining the mechanism of gACE release into the fluid and for exploring the nature of the ACE sheddase involved in this important physiological regulatory mechanism. In addition, these results may help us to understand the mechanisms involved in the reduction of fertility in the gACE knockout mouse, a finding that highlights the importance of gACE in male fertility.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Russell Jones for suggestions on the manuscript, Dr. François Alhenc-Gelas for the gift of the C-terminal antibody and valuable discussions, and Dr. Joelle Dupont for advice on molecular biology techniques. The authors would also like to thank Celine Boursier and Guillaume Tsikis for technical assistance. The authors gratefully acknowledge the help of the staff at the experimental facilities of the Physiologie de la Reproduction et des Corportements.


   FOOTNOTES
 
1 Supported by grants from the Region Centre and the GIS-GENANIMAL. Back

2 Correspondence: Jean-Luc Gatti, Equipe Gamètes mâles et fertilité, UMR 6175 INRA-CNRS-Université de Tours-Haras Nationaux, Station de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Nouzilly, France. FAX: 33 247 42 77 43; gatti{at}tours.inra.fr Back

Received: 18 April 2005.

First decision: 11 May 2005.

Accepted: 22 June 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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