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Bioscience Education and Research Center,6 Akita University, Akita City, Akita 010-8543, Japan
Laboratory of Animal Experiments for Regeneration,4 Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
CREST Programme,5 Japan Science and Technology Society, Sakyo-ku, Kyoto 606-8507, Japan
ABSTRACT
The angiotensin-converting enzyme (ACE) plays a crucial role in male fertilization and is a key regulator of blood pressure. Testicular ACE (tACE), the germinal specific isozyme expressed on different promoters, exclusively carries out the role of ACE in fertility, although the site and mode of action are not well known. To investigate the contribution of tACE in fertilization, we produced transgenic mouse lines carrying a dipeptidase-inactivated mutant. Although the transgenic mice showed normal blood pressure, kidney morphology, and fertility, reduced fertilization was observed after in vitro fertilization (IVF). The sperm-zona pellucida (ZP) binding was exclusively impaired in these lines in a manner similar to that observed in an Ace knockout mouse. The dipeptidase activity was reduced in epididymal ingredients but not in the testis. Furthermore, direct application of mutant protein did not suppress sperm-ZP binding of intact sperm during IVF, implying that the dipeptidase-inactivated mutant affects sperm modification in the epididymis for ZP binding. Our results indicate that the dipeptidase-inactivated tACE acts in vivo, suggesting that tACE contributes to fertilization as a dipeptidase at least in the epididymis.
angiotensin-converting enzyme, dipeptidase, epididymis, fertilization, in vitro fertilization, sperm, testis, transgenic mouse
Angiotensin-converting enzyme (ACE, CD143) is a well-characterized dipeptidyl carboxypeptidase that regulates the bioactivity of circulating peptides, such as angiotensin I and bradykinin [1–3]. Peptides such as these that regulate blood pressure and induce inflammation are natively catalyzed by the somatic isozyme, somatic ACE (sACE), in peripheral tissues and blood. Somatic ACE is produced by various tissues, including the vascular endothelium, epididymal epithelium, proximal tubules of the kidney, small intestinal epithelium, alveolar macrophages, and neuronal cells of the basal ganglia. It is also broadly distributed in body fluids in a soluble form, suggesting the wider involvement of this enzyme in multiple biologic processes [4, 5].
Somatic ACE contains two homologous catalytic domains, both of which contain the zinc-binding motif, HEXXH, and a downstream E residue [6], and removal of zinc from the enzyme moiety by chelation completely abolishes the enzyme's dipeptidase activity [7]. Somatic ACE also contains a chloride ion-binding motif near the catalytic center [8], and chloride binding at this site upregulates the catalytic activity of the enzyme [2, 3]. This indicates that the chloride density of body fluids would directly influence ACE dipeptidase activity.
In contrast to sACE, little is known about the function of the testicular isozyme, tACE. It is encoded by the second half of the Ace locus, the expression of which is exclusively driven by a testis-specific promoter located in intron 12 of the gene [9, 10]. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of sACE and shows similar catalytic activity in vitro [11]. Gene knockout experiments in mice have revealed a crucial role for ACE in male fertilization, with the sperm of mutant mice showing impaired uterotubular migration and ability to bind the zona pellucida (ZP) of the egg [12–14]. These defects were rescued by introducing tACE, but not sACE, to the germ cells, implicating tACE as crucial for establishing normal male fertility [15, 16]. Although the phenotypes are quite clear, the active site through which tACE elicits this function and the underlying molecular mechanisms remain unclear.
We recently identified another enzymatic activity for ACE, the GPI-anchored protein-releasing activity (GPIase) [17]. Although the as-yet uncharacterized catalytic site for this activity is distinct from that governing the dipeptidase activity, the sperm-ZP binding insufficiency of Ace knockout sperm can be rescued by a dipeptidase-inactivated tACE, implicating the GPIase activity as crucial for the function of the enzyme in the sperm.
However, evidence exists that the dipeptidase activity of ACE is still involved in male fertility; when the active center of tACE was selectively inactivated in mice, male mice developed impaired fertility similar to the Ace knockout [18]. The mutant protein was readily detected in the sperm of these mice, arguing that ACE acts in fertilization via its dipeptidase activity but not by GPIase action.
Moreover, knockout mice for well-known ACE substrates, such as angiotensinogen and bradykinin, never show impaired male fertility, suggesting substrates specific for tACE [19, 20].
The above background prompted us to investigate further the biologic activity of tACE in vivo using a transgenic approach to introduce dipeptidase-inactivated tACE. Transgenic mice carrying a mutant protein showed varying degrees of reduced male fertility similar in mechanism to the Ace knockout mouse. The reduced dipeptidase activity in the epididymal ingredient, but not in the testis, of transgenic mice carrying mutant tACE suggested that tACE acts as a dipeptidase in the sperm on epididymal transit.
The cDNA encoding tACE was prepared according to the method described previously, with slight modification [17]. Briefly, mouse testis cDNA was produced by reverse transcription (RT), and the tACE cDNA was amplified by PCR using primer pairs 5'-TGAATTCCACCATGGGCCAAGGTTGGGCTACTCCAGG-3' and 5'-CGAATTCTTATGGGACACTCCTCTGC-3'. This cDNA encodes the full-length ACE-T protein. A peptidase-inactivated mutant with amino acids His413 and His417 replaced by Lys (designated UK) was synthesized by site-directed mutagenesis using 5'-CATGGAGGACTTGGTGATAGCGCACAAGGAAATGGGCAAGATCCAGT ATTTCATGC-3' as a mutation primer. The mutant and wild-type constructs were ligated between the CAAG promoter in a strong and broad driving element containing the cytomegalovirus immediate-early enhancer and chicken beta-actin promoter, as well as the rabbit beta-globin polyA+ signal (Fig. 1) [21].
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Development of Transgenic Mice and Animal Maintenance
The CAAG-tAce (wild-type tACE, designated WT) was injected into one-cell embryos of 129B6F1 genetic background, and transgenic lines were maintained by mating with C57BL/6J mice (more than five generations). The transgenic mouse line was named Tg (CAAG-tAce) 1Gnk (MGI: 3714633). The CAAG-tAce*H413K*H417K (designated UK) transgene was injected into C57BL/6J one-cell embryos, and transgenic lines were maintained with the isogenic background. The transgenic mouse lines were named Tg (CAAG-tAce*H413K*H417K) 1
3Gnk (MGI: 3714634). Transgenic founders were obtained by screening siblings by PCR using tail DNA as a template and primers 5'-TGAATTCCACCATGGGCCAAGGTTGGGCTACTCCAGG-3' and 5'-CAACACTTGGGCTGTCCGGTT-3'. Ace knockout mice (JR#002679) were purchased from the Jackson Laboratory and were screened by PCR using primers 5'-CTTGACGAGTTCTTCTGAGG-3', 5'-AGAAAAGCACGGAGGTATCC-3', and 5'-ACTGCCCGGTCCAGGTTCTG-3'. All animal experiments were performed with the approval of the Animal Experiment Committees of the Institute for Frontier Medical Sciences and Kyoto University.
Tissues and ingredients of cauda epididymis, containing both sperm and fluid, were homogenized on ice in RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA) containing a Complete protease inhibitor cocktail tablet (Roche, Mannheim, Germany). The homogenates were centrifuged at 25 000 x g, the supernatants were collected, and protein estimations were carried out. In the next step, 10 µg protein per sample or 10 µl serum was subjected to SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. The efficiency of the transfer was checked by staining the membranes with Coomassie Brilliant Blue (Fluka) after immunoblotting. The membranes were probed with a mouse monoclonal antibody against ACE, 1D5 [22], or a mouse monoclonal antibody against ADAM2 (Chemicon International Inc., Temecula, CA), and were detected using the ECL Plus system (GE Healthcare). We evaluated the quantity of ACE and ADAM2 in testes and epididymal ingredients by measuring the density of the bands detected on immunoblotting using a densitometer.
Histology and Immunohistochemistry
Excised tissues were fixed in 10% neutral buffered formalin and processed into paraffin for sectioning (5 µm thickness). The sections were stained with hematoxylin and eosin and observed under a brightfield light microscope. For detection of ACE proteins, the sections were incubated with a rat monoclonal antibody against mouse ACE, H7 [23] and were processed through the ENVISION-plus system (DakoCytomation, Glostrup, Denmark), followed by counterstaining with hematoxylin.
Measurement of Arterial Blood Pressure
Tail arterial pressure was measured in conscious mice using an automated tail cuff system (BP-98A, Softron, Tokyo, Japan). Values are mean ± SD compared with nontransgenic (NTg) controls by Student t-test.
In Vitro Fertilization (IVF) Assay
Adult C57BL/6J females (more than 10 wk old) were superovulated by injecting 6.7 IU equine chorionic gonadotropin (Teikoku Zoki, Tokyo, Japan) followed 48 h later with 6.7 IU human chorionic gonadotropin (Teikoku Zoki). Ovulated eggs surrounded by cumulus mass were collected from the oviducts 16 h after the second injection. Eggs with cumuli were incubated in 300 µl HTF medium (101.61 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.4 mM KH2PO4, 6.42 mM CaCl2, 25 mM NaHCO3, 2.77 mM glucose, 3.4 µg/ml sodium lactate, 0.34 mM sodium pyruvate, 0.2 mM penicillin G sodium salt, 0.03 mM streptomycin, 4 mg/ml BSA, and 0.4 µl of 0.5% phenol red) overlaid with mineral oil. Sperm from the cauda epididymis were preincubated in 200 µl HTF and then added to the egg drop at a final concentration of approximately 1.0 x 105 sperm/ml. Eggs were washed with modified Whitten medium (mWM; 109.51 mM NaCl, 4.78 mM KCl, 1.19 mM MgSO4, 1.19 mM KH2PO4, 22.62 mM NaHCO3, 5.55 mM glucose, 1.49 mM calcium lactate, 0.23 mM sodium pyruvate, 19.1 µg/ml EDTA, 10 µM ß-mercaptoethanol, 0.2 mM penicillin G sodium salt, 0.03 mM streptomycin, 3 mg/ml BSA, and 0.2 µl of 0.5% phenol red) after 7-h contact with the sperm, and they then were incubated in fresh mWM for another 16 h. For quantification of fertilization, the total numbers of embryos and numbers of two-cell embryos in a population were determined to generate the percent fertilization value: (%) fertilization = (number of two-cell embryos/number of total embryos) x 100. Values are mean ± SD. Differences from NTg controls were assessed by Student t-test. A P value < 0.01 was considered significant.
Adult BDF1 females (more than 10 wk old) were superovulated, and sperm were incubated as described above. The collected eggs were removed from cumulus cells by incubation with 175 U/ml bovine testicular hyaluronidase (Sigma Chemical Co., St. Louis, MO) for 5 min. The incubated sperm were placed at a concentration of approximately 5.0 x 104 sperm/ml with cumulus-free eggs in 300 µl HTF for 2 h.
Eggs were not washed at all. This is because we cannot distinguish loose binding, which might indicate sperm just going to bind to the ZP with biologic significance, between nonspecific binding. For each experiment, the same treatments were always performed in parallel on Ace knockout sperm as a control. The degree of Ace knockout sperm binding was similar to the results shown in the literature [12–14]. Eggs were then fixed with 2% neutral buffered formalin, stained with Hoechst 33258 (Molecular Probes, Eugene, OR), and visualized under a fluorescent microscope. The number of sperm was counted at a focal plane that showed the widest diameter of the eggs. Values are expressed as mean ± SD and compared with NTg controls in experiment 1 and UK-2 in experiment 2 by Student t-test.
To examine the effect of ACE treatment on intact sperm (Ace+/+ and Ace+/– genotypes), the incubated sperm were placed at a concentration of approximately 1.0 x 105 sperm/ml in close contact with cumulus-free eggs for 5 h in the presence of 0.1 µM recombinant tACE protein prepared as described previously [17]. Triplicate experiments were performed with similar results, and a representative example was depicted. Values are expressed as mean ± SD and compared with the vehicle (buffer only) by Student t-test.
Ace transgenic mice were mated with acrosin-GFP transgenic mice [24] to produce double transgenic mice. In these mice, green fluorescent protein (GFP) is dispersed from the sperm acrosome upon the acrosomal reaction, resulting in loss of fluorescence. Sperm from the cauda epididymis were collected and treated with calcium ionophore A23187 in TYH medium [25] to induce the acrosomal reaction under two conditions: treatment with 10 µM after 2 h of incubation, or treatment with 20 µM without incubation. Then, the treated sperm were fixed with 2% neutral buffered formalin, spread on glass microscope slides, and observed under a fluorescent microscope. Fifty sperm were observed for the presence of GFP fluorescence, and GFP– sperm (acrosomal reacted) were counted. The percent acrosomal reaction value was calculated using the formula: (%) acrosomal reaction = (number of GFP– sperm/50) x 100.
Adult BDF1 females (more than 10 wk old) were superovulated, and sperm were incubated as described above. Zona pellucida of collected eggs were removed by incubation in acid Tyrode solution (Sigma) for 1 min. Incubated sperm were placed at a concentration of approximately 5.0 x 104 sperm/ml in close contact with zona-free eggs in 300 µl HTF for 3 h. Eggs were then fixed with Kalnoa solution (methanol:acetic acid = 3:1 [v/v]), stained with Hoechst 33258, and visualized by fluorescence microscopy. The number of pronuclei in the eggs was counted. The percent fusion value was calculated by the formula: (%) fusion = (number of 
2 pronuclei embryos/number of total embryos) x 100.
GPIase activity was examined by a placental alkaline phosphatase (PLAP) conversion assay on Triton X-114 partition, as described previously [17]. Briefly, testis and epididymal ingredients were solubulized in a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, and Complete protease inhibitor (Boehringer Mannheim, Mannheim, Germany), and lysates were centrifuged at 25 000 x g. The collected supernatants were 10-fold diluted with a buffer containing 20 mM Tris, pH 7.5, and 150 mM NaCl. These samples were mixed with 0.3 IU/ml of detergent-soluble PLAP in a buffer containing 100 mM 2-(N-Morpholino)ethanesulfonic acid, pH 6.0, and 5 mM CaCl2, and reaction was performed for 60 min at 37°C. We stopped the reaction by adding Triton X-114 at a final concentration of 2%, followed by microcentrifugation at 25°C. The water-soluble phase was collected, and PLAP activity was measured by alkaline phosphatase detection kit (Nacalai Tesque, Kyoto, Japan). Values are expressed as mean ± SD and compared with NTg controls by Student t-test.
Testis and epididymal ingredients were solubilized in a buffer containing 50 mM Tris, pH 8.0; 150 mM NaCl; 1% Triton X-100; and Complete protease inhibitor (Boehringer Mannheim), and lysates were centrifuged at 25 000 x g. The ACE peptidase activity was measured by colorimetry, as described previously [26], using p-hydroxyhippuryl-L-histidyl-L-leucine (pHHHL) as substrate. Values are expressed as mean ± SD and were compared with NTg controls by Student t-test.
Development of Transgenic Mice
To clarify the function of ACE in vivo, we initially induced amino acid substitutions in the core sequence of the tACE active site to disrupt its dipeptidase activity (Fig. 1). In a previous study, we reported that the UK mutant absolutely lacked dipeptidase activity but conserved GPIase activity in vitro [17]. The DNA constructs encoding this mutant as well as wild-type protein were ligated downstream of the CAAG promoter, a strong and broad driving element [21], and expressed in mice to develop transgenic animals. Six transgenic founders were raised after screening 36 newborn mice for WT transgenesis (16.7%), and 6 founders were produced from 22 newborns transgenic for UK (27.2%). All male transgenic founders were fertile. A total of 3 of 4 WT founders and 5 of 5 UK founders transmitted their respective transgenes to descendents.
To investigate the function of ACE in male reproduction, we selected transgenic lines for transgene expression in the testis: one line from WT and three lines from UK mice. Because the monoclonal antibody we used here could not distinguish mutant protein from the wild type, we selected transgenic lines showing tACE expression higher than NTg. The degree of transgene expression differed among the UK lines, with the highest in line 1, a moderate level in line 3, and the lowest in line 2 (Fig. 2A). In all lines, both the male and female transgenic mice showed normal in vivo fertility comparable with C57BL/6J NTg (six to seven newborns per litter) and germline transmission in a Mendelian fashion (Table 1 and data not shown). This observation indicates that the uterotubular migration of the sperm was not impaired in the male Ace transgenic mice, whereas it was present in Ace knockout mice.
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Then, to clarify whether the impairment in sperm-ZP binding was related to mutant protein expression in the sperm, we examined epididymal ingredients collected from the cauda epididymis. These samples contained both sperm and epididymal fluid. We detected transgenic proteins in the epididymal ingredient of WT, UK1, and UK3 lines (Fig. 2B), the magnitude of which seemed to correlate with epididymal tissue expression (Fig. 3A), implying that a large portion of transgenic protein in the epididymal ingredient originated from the epididymis. Thus, mutant ACE proteins derived from the epididymis seem to play some roles on sperm during epididymal transit.
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Expression of Transgene in Various Organs
Expression of the respective transgenes was examined in various organs by immunoblotting. The transgenic protein could be distinguished from endogenous sACE by the difference in molecular size. As shown in Figure 3A, all transgenic proteins were highly expressed in heart, lung, and reproductive tracts, including the prostate, seminal vesicle, and epididymis, with the exception of the UK2 line. The transgenic protein was broadly expressed in the UK3 line, and blood levels of the proteins were present in varying amounts in all WT and UK lines (Fig. 3B).
Normal Blood Pressure and Kidney Morphogenesis in Transgenic Mice
Ace knockout mice show low blood pressure and have abnormal kidney morphogenesis as well as male infertility [12–14]. In contrast, the Ace transgenic mice in the present study were normotensive and had normal kidney morphogenesis, indicating that neither WT nor mutant ACE had any effect on these somatic tissues, even when overexpressed (Fig. 4 and Table 2).
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Reduction of IVF and Impairment of Sperm-ZP Binding in Mutant ACE Transgenic Mice
Next, we examined the sperm functions of transgenic mice. The sperm morphology, number of epididymal sperm, and motility were normal (G. Kondoh, unpublished data). Another phenotype of the male Ace knockout mice showed insufficient sperm-ZP binding, with associated impairment of IVF success. Testing of our transgenic lines for IVF efficiency showed about a 30% considerable reduction in the UK3 line (Fig. 5). The IVF rate of the WT mice, which had considerable exogenous ACE expression in both the testis and epididymal ingredients (Fig. 2), was comparable to that of NTg controls, suggesting that overexpression of ACE protein has no influence on IVF rates. Subsequently, we examined sperm-ZP binding in all transgenic lines and found it to be halved in both UK1 and UK3 lines expressing mutant protein in both the testis and epididymis (Table 3). Other sperm functions, such as acrosomal reaction and fusion, were examined, but the results were comparable to those for NTg controls (Tables 4 and 5).
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Considered together, these findings indicate that impaired sperm-ZP binding caused a reduction in IVF success and that the phenomenon seems to be a hypomorph of Ace knockout sperm.
Localization of Transgenic Proteins in the Testis
The testicular localization of transgenic proteins was then assessed using immunohistochemistry. As shown in Figure 6e, tACE was highly expressed in spermatids in the seminiferous tubules of NTg controls, as reported previously [23, 27, 28]. In the transgenic mice, tACE in WT and UK2 lines was localized to similar areas of the testes as in control mice (Fig. 6, a and c). Lines UK1 and UK3 showed ectopic tACE expression in spermatogonia, in addition to the normal spermatid expression (Fig. 6, b and d). As shown in Figure 2A, testicular tACE in UK1 and UK3 was double that in NTg control, which might reflect this ectopic expression. Since the spermatogonial immunostaining of tACE was more prominent in UK1, but a low IVF rate was seen in UK3, the ectopic expression of tACE seems to have no influence on sperm function.
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GPIase Activity in Transgenic Testis and Epididymal Ingredient
To clarify whether both or either activity of ACE is involved in sperm-ZP binding, GPIase activity was first examined in transgenic testis and epididymal ingredient. Testicular tissues and epididymal ingredients, which contained both sperm and fluid, were solublized and examined for GPIase activity by PLAP conversion assay on Triton X-114 partition. We observed elevation of GPIase activity in both the testis and epididymis of WT mice compared with NTg control, whereas not in all UK lines (Table 6). Although the UK protein exhibits a GPIase activity in vitro [17], it seemed lacking GPIase activity in vivo and had no effect on IVF success or sperm-ZP binding.
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Dipeptidase Activity in Transgenic Testis and Epididymal Ingredient
Using pHHHL as substrate, we used the colorimetric assay to assess dipeptidase activities of the same tissues. As indicated in Table 7, dipeptidase activity in the testis of transgenic lines was similar to that of the NTg control. In contrast, the activity was valuable among each animal in the epididymal ingredient but was considerably elevated in WT and reduced in UK1 and UK3 lines (Fig. 7). That the amount of mutant protein in UK1 is less than endogenous expression (Fig. 2B), suggesting that a small amount of mutant protein is sufficient for suppressing dipeptidase activity on the sperm. These observations suggest that the dipeptidase-inactivated tACE acts in vivo, at least in the epididymis, like a dominant-negative molecule.
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The Mutant ACE Protein Does Not Suppress Sperm-ZP Binding in Intact Sperm
To examine the possible suppressive role of mutant ACE in sperm on binding to ZP, intact sperm from NTg mice (Ace+/+) were treated with recombinant ACE proteins, and the effects on sperm-ZP binding were examined. We also used Ace+/– sperm, which show normal fertility but have only 50% of control levels of ACE protein expression. As indicated in Table 8, not all recombinant proteins suppressed sperm-ZP binding of both Ace+/+ and Ace+/– sperm. Moreover, tACE protein stimulated the binding of Ace+/– sperm to ZP, which was similar to our previous observations with the Ace knockout sperm [17]. These observations support the notion that the mutant ACE played a suppressive role on the sperm at epididymal transit but not at sperm-egg assembly.
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We demonstrated previously that ACE possesses GPIase activity and contributes to male fertilization [17]. To investigate the function of dipeptidase-inactivated ACE in vivo, we generated transgenic mice carrying a tACE mutant. Although the mutant proteins were broadly distributed in these mice, they had suppressive effects specifically on male fertility but not on somatic functions, such as blood pressure regulation or kidney morphogenesis. Although the dipeptidase characteristics of tACE and sACE are similar in vitro [11], the mutant tACE seemed to affect endogenous tACE specifically but not sACE in mice. Previous studies proposed functional differences of these isozymes in vivo [15, 16]. Our results provide support to this notion. The findings presented here also indicated that the mutant tACE proteins did not have nonspecific actions in other tissues by ectopic overexpression.
Functional differences between two isoforms might also exist in the GPIase activity. Leisle et al. argued that ACE does not possess considerable GPIase activity [29]. They used multiple species of sACE, but no tACE, in their studies and could not detect apparent GPIase action. In contrast, we used tACE in our previous studies and found considerable GPIase activity [17]. Therefore, we compared two isoforms in term of GPIase activity and found a weaker activity for sACE than tACE (G. Kondoh, unpublished data). Thus, the controversy could be explained by the difference in the isoform used.
It is also reported that male fertility depends on the dipeptidase activity of tACE. When the Ace gene was mutated similarly to our UK mutation in mice, male mice had impaired fertility synonymous to Ace knockout [18]. The mutant protein was readily detected in the sperm of these mice, arguing that ACE acts in fertilization via its dipetidase activity but not by GPIase action. In the present study, the UK protein lacked GPIase activity in vivo; therefore, the possibility of GPIase action rescuing sperm-ZP binding insufficiency caused by dipeptidase inactivation could not be excluded. Indeed, another mutant tACE as well as wild-type protein rescued sperm-ZP binding insufficiency of Ace knockout sperm in vitro [17].
Transgenic males carrying the mutant ACE had exclusively impaired sperm-ZP binding, as did the Ace knockout mouse. This result also showed that the mutant molecule is a useful tool for analyzing the dipeptidase activity of tACE in male fertilization. However, none of the transgenic lines tested here showed abnormal fertility in vivo, suggesting that the mutant tACE protein did not impair the uterotubular migration of the sperm, a prominent phenotype of Ace knockout mice. This may be explained by two possibilities: 1) the amount of mutant proteins expressed was not sufficient to induce suppression of the process, and/or 2) this process is governed by other ACE functions, such as the GPIase activity.
Testicular ACE may also contribute to sperm-ZP binding more directly via GPIase activity. Recently, the membrane localization of ADAM3, a key sperm-ZP binding factor, was found to be altered in Ace knockout sperm [22]. The authors showed that tACE protein translocated to a lipid raft-positive membrane fraction and was degraded when localized in nonraft fraction, suggesting that the enzyme was active in the raft, where GPI-anchored proteins accumulate. In Ace knockout sperm, ADAM3 is absent from the lipid raft-positive fraction, and both Ace and Adam3 knockout sperm exhibited similar impairment of sperm-ZP binding [30]. Since ADAM3 possesses an adhesion protein motif, it has been proposed as a downstream player of sperm-ZP binding with its membrane localization governed by tACE, possibly via the ACE GPIase activity. Thus, tACE may act as a dipeptidase in sperm modification in the epididymis, as well as a GPIase in mature sperms on binding to ZP, contributing to fertilization in a dual mode.
It has been proposed that the state of tACE in the sperm is dramatically modulated during epididymal transit. Testicular ACE is shed from sperm surface, and the biochemical properties of released enzyme have been characterized [31, 32]. The role of tACE in the epididymis was also implied [33], although its unique substrates and functions have not yet been clarified. Based on the observations described in the present study, we postulate that mutant tACE is functional, suggesting that it affects sperm in the epididymis, resulting in sperm-ZP binding insufficiency, similarly to a dominant-negative molecule. More recently, a tACE-related protein, which lacks dipeptidase sequences, was discovered by proteomics analysis of the mouse sperm membrane [34]. Disruption of a similar gene in Caenorhabditis elegans resulted in failure of cell fusion [35], implying the biologic significance of the dipeptidase-inactive counterpart of ACE. Our results propose that such molecule may regulate tACE function in vivo.
ACKNOWLEDGMENTS
We thank A. Murakami, S. Kitano, H. Takemura, K. Hirota, and T. Matsushita for technical assistance, M. Okabe for providing anti-ACE antibody and a acrosin-GFP transgenic mouse, and S. Danilov for anti-ACE antibody.
FOOTNOTES
1Supported by grants from the Uehara Memorial Foundation and the Ministry of Education, Science, Sports, and Culture of Japan. 
3These authors contributed equally to this work.
Correspondence: 2Gen Kondoh, Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Sciences, Kyoto University, 53 Syogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. FAX: 81 75 751 4860; e-mail: kondohg{at}frontier.kyoto-u.ac.jp
Received: 8 January 2007.
First decision: 14 February 2007.
Accepted: 17 July 2007.
REFERENCES
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  G. Kondoh, H. Watanabe, Y. Tashima, Y. Maeda, and T. Kinoshita Testicular Angiotensin-Converting Enzyme with Different Glycan Modification: Characterization on Glycosylphosphatidylinositol-Anchored Protein Releasing and Dipeptidase Activities J. Biochem., January 1, 2009; 145(1): 115 - 121. [Abstract] [Full Text] [PDF]
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