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Research Article |
Genome Information Research Center,6 Pharmaceutical Sciences,7 Research Institute for Microbial Diseases,8 Osaka University, Osaka, 565-0871, Japan
ABSTRACT
Male mice deficient for the calmegin (Clgn) or the angiotensin-converting enzyme (Ace) gene show impaired sperm migration into the oviduct and loss of sperm-zona pellucida binding ability in vitro. Since CLGN is a molecular chaperone for membrane transport of target proteins and ACE is a membrane protein, we looked for ACE on the sperm membranes from Clgn–/– mice. ACE was present and showed normal activity, indicating that CLGN is not involved in transporting ACE to the sperm membranes. The ablation of the Adam2 and Adam3 genes generated animals whose sperm did not bind the zona pellucida, which led us to examine the presence of ADAM2 and ADAM3 in Clgn–/– and Ace–/– sperm. ADAM3 was absent from Clgn–/– sperm. In the Ace–/– mice, while ADAM2 was found normally in the sperm, ADAM3 disappeared from the Triton X-114 detergent-enriched phase after phase separation, which suggests that ACE is involved in distributing ADAM3 to a location where it can participate in sperm-zona pellucida binding. This diminished amount of ADAM3 in the Triton X-114 detergent-enriched phase may explain the inability of Clgn–/– and Ace–/– sperm to bind to the zona pellucida.
gamete biology, in vitro fertilization, male sexual function, sperm capacitation
INTRODUCTION
Mammalian fertilization is accomplished through complex processes that involve cell–matrix and cell–cell interactions. The zona pellucida (ZP) is an extracellular matrix that serves as a barrier to protect the egg from physiological damage [1] and to block polyspermic fertilization. Based on biochemical analyses, different sperm proteins have been postulated to play roles in ZP binding, penetration and fusion. However, when genes, such as Acr (acrosin) [2], B4galt1 [3], Spam1 (previously known as PH-20) [4], Mfge8 (previously known as SED1) [5], and Cd46 [6] are ablated, the predicted phenotypes do not appear, which suggests that the various models used to explain fertilization need to be redefined. Among the various proteins that have been reported to have critical roles in fertilization, only the A Disintegrin And Metalloprotease 2 (Adam2) gene (previously known as fertilin-ß) has been shown to be essential [7]. Even in this case, the observed phenotype, i.e., loss of ZP-binding ability, is different from that of the predicted phenotype, i.e., a role in sperm-egg fusion. Additional proteins that may play roles in sperm-ZP binding have emerged over the years. For example, we have shown that the calmegin (Clgn) gene is essential for sperm binding to the ZP [8]. Other genes, such as Adam1a and Adam3, as well as the angiotensin-converting enzyme (Ace), have also emerged as essential factors for sperm-ZP binding [9–11].
It is intriguing that the disruption of certain genes, e.g., Clgn, Ace, Adam1a, Adam2, and Adam3, results in a similar sperm phenotype, i.e., failure of sperm to bind to the ZP. Since CLGN functions as a testis-specific molecular chaperone, we previously examined the possibility of ADAM2 misfolding in sperm from Clgn–/– mice [12]. In wild-type sperm, both ADAM1 and ADAM2 bind CLGN and this interaction probably leads to the correct tertiary folding of these proteins. However, in the absence of CLGN, the ADAM1/ADAM2 heterodimer disappears from testicular extracts. Subsequently, it has been shown that the Adam1 gene comprises two similar genes, Adam1a and Adam1b, and that only ADAM1b is present in sperm, where it forms a heterodimer with ADAM2 [13, 14]. It is clear that when the ADAM1b/ADAM2 heterodimer fails to assemble in the testis, ADAM2 disappears from the sperm of the Clgn–/– male mouse. These results helped to clarify why Clgn–/– and Adam2–/– mice share a similar phenotype, i.e., the sperm cannot bind to the ZP because they lack ADAM2.
To continue our studies towards deciphering the role of candidate genes in sperm-ZP binding, we turned our attention to the angiotensin-converting enzyme. Somatic ACE (sACE) is a zinc metalloprotease that is involved in blood pressure regulation by cleaving bioactive peptides, such as angiotensin I and bradykinin. Testicular ACE (tACE) has also been described, and is generated by alternative promoter usage [15]. Both isoforms have similar enzymatic activities. Ablation of both sACE and tACE results in males with reduced fertility [16]. Males that lack only sACE are fertile, which indicates that tACE is responsible for the infertility [17, 18]. We now report a role of tACE in the localization of ADAM3 so that it can participate in sperm-zona pellucida binding.
MATERIALS AND METHODS
Animals
Clgn+/– and Clgn–/– male mice were obtained by mating Clgn–/– females with Clgn+/– males. The Ace–/– mice were originally produced by Krege et al. [9]. Mice heterozygous for alterations in exon 14 of the Ace gene, which contains critical amino acids for sACE and tACE functions [15], were purchased from the Jackson Laboratory (Bar Harbor, ME). Ace+/– and Ace–/– male mice were obtained by mating Ace+/– females with Ace+/– males. All of the experiments were performed with the approval of the Animal Care and Use Committee of Osaka University.
Antibodies
Affinity-purified rabbit anti-ADAM1a (1aCysE), anti-ADAM1b (1bCysE), and anti-SPAM1 (previously known as PH-20) [4, 13] antibodies, which were produced by immunizing with recombinant proteins that correspond to a unique region within each protein, were kind gifts from Drs. Hitoshi Nishimura and Tadashi Baba (University of Tsukuba, Ibaraki, Japan). Monoclonal antibodies (Mabs) against mouse ADAM2 (fertilin-ß; 9D2.2) and ADAM3 (cyritestin; 7C1.2) were purchased from Chemicon International Inc. (Temecula, CA). Rabbit anti-SPAM1 antiserum [19] was a kind gift from Dr. Paul Primakoff (University of California, Davis, CA). Affinity-purified antibodies against ADAM2, IZUMO1, CD46, and CLGN were obtained as described previously [6, 8, 12, 20]. The rat anti-mouse sperm tail antigen Mab (#124) was generated in our laboratory and was used to examine the separation of sperm heads and tails.
To produce monoclonal antibodies against ACE, we prepared an amino-terminal recombinant tACE protein (AA #31–140). A DNA fragment that contained 110 amino acids starting from the N-terminus of the mature form of tACE (without the signal peptide) was obtained by PCR-amplification using a forward primer that corresponds to nucleotides 89–113 (5'-TTCCATGGCCACTGACCACGTGACAGCCAA-3') and a reverse primer that corresponds to nucleotides 399–420 (5'-ACTCGAGAGAGTTTTGAAAGTTGCTCAC-3'). The DNA was ligated into the NcoI and XhoI restriction sites of pET28b (Novagen, Madison, WI) and transformed. The recombinant tACE protein was expressed as a fusion protein with the 6xHis tag at the C-terminus, and purified by Ni-NTA resin affinity column chromatography (Qiagen, Hilden, Germany). Monoclonal antibodies against ACE were generated by MBL (Nagoya, Japan). Two 7-week-old, female Ace–/– mice were immunized i.p. with 50 µg of recombinant protein emulsified in complete Freunds adjuvant (Day 0) or incomplete Freunds adjuvant (Days 7, 14, 21, and 28). Spleen cells were harvested three days after the fifth immunization and fused with mouse myeloma cells using polyethylene glycol. Wells that contained hybridomas were screened by an enzyme-linked immunosorbent assay (ELISA) for recombinant tACE protein. Positive clones were grown, and their supernatants were used for further screening. The supernatant from one clone, 1D5, was used for immunoblot analysis.
Fractionation of Sperm Heads and Tails
Sperm from the cauda epididymis and vas deferens were collected in TBS. Sperm heads and tails were separated by mild sonication on ice (five times with flash set at output level 1) using the Ultrasonic Disruptor UD-201 (Tomy Digital Biology, Tokyo, Japan), followed by the addition of an equal amount of 1.8 M sucrose. The 2-ml sample was layered on a discontinuous sucrose gradient composed of 2 ml of 0.9 M sucrose, 1 ml of 2.05 M sucrose, and 1 ml of 2.2 M sucrose; all of the sucrose solutions contained 10 mM Tris-HCl (pH 7.5) and 0.15 M NaCl. The gradients were centrifuged at 100 000 x g for 12 h in a SW50.1 rotor (Beckman Coulter, Tokyo, Japan). Sperm tails banded at the 0.9 M/2.05 M interface, and the sperm heads were found in the pellet. Both fractions were resuspended in TBS and pelleted by centrifugation at 1000 x g for 10 min. The samples were subjected to immunoblot analysis, as described below.
Phase Separation of Sperm Triton X-114 Extracts
Cauda epididymal and vas deferens sperm were collected into PBS and centrifuged at 1000 x g for 10 min at 4°C. The sperm pellets were suspended in PBS that contained 1% Triton X-114 [21], 10 mM benzamidine, 0.5 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. The sperm suspensions were placed on ice for 20 min with occasional vortexing. After centrifuging at 15 700 x g for 20 min at 4°C, the supernatants were collected, and layered onto 6% sucrose/PBS in microtubes. After incubation at 37°C for 15 min, the tubes were centrifuged at 1000 x g for 15 min at room temperature, to separate the Triton X-114 extract into the detergent-depleted phase, sucrose phase, and detergent-enriched phase. The separated detergent-depleted and detergent-enriched phases were mixed with SDS-sample buffer, boiled, and subjected to SDS-PAGE and immunoblot analysis.
Immunoblot Analysis
Sperm from the cauda epididymis and vas deferens of sexually mature male mice were collected and incubated in lysis buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, 1% Triton X-100, 10 mM benzamidine, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A) for 20 min on ice with occasional vortexing. The testis and kidney were excised, minced, and homogenized in lysis buffer, and then placed on ice for 1 h. The sperm and tissue extracts were pelleted at 15 700 x g for 5 min at 4°C, and the supernatants were collected. The protein concentration in each homogenate was determined using the Coomassie protein assay reagent (Pierce, Rockford, IL). Proteins were separated by SDS-PAGE under reducing conditions (CLGN, ADAM2, ADAM3, ACE, SPAM1, and #124) or non-reducing conditions (ADAM1b, IZUMO1, and CD46), and were transferred electrophoretically to PVDF membranes (Immobilon-P; Millipore Corp., Bedford, MA). After blocking in TBS-T buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween-20] that contained 5% skim milk, the blots were incubated with primary antibody overnight at 4°C, and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The detection of immunoreactive bands was performed using the enhanced chemiluminescence (ECL) Western blotting detection kit (GE Healthcare Bio-Sciences). Signal intensities were determined using the Scion Image software (Scion Corp., Frederick, MD).
Immunoprecipitation
Testes were collected and homogenized in lysis buffer using the Potter-Elvehjem homogenizer with 10 strokes at 1000 rpm. The homogenates were placed on ice for 1 h with occasionally vortexing, and then centrifuged at 15 700 x g for 5 min at 4°C. The supernatants were collected and precleared by the addition of protein A-Sepharose (GE Healthcare Bio-Sciences) overnight at 4°C. After centrifugation, antibodies were added to the supernatants, and the solutions were incubated with rocking for 3 h at 4°C. Protein A–Sepharose was then added to each tube, and incubation was allowed to continue for 2 h at 4°C. After washing four times with lysis buffer, the final pellet was resuspended in 8 M urea and SDS–PAGE loading buffer, boiled for 3 min, and the proteins were separated by SDS–PAGE under reducing conditions.
Measurement of ACE Activity
Testis and sperm homogenates were prepared as described above. The protein concentration of each homogenate was adjusted to 0.2 mg/ml with lysis buffer, and 100 µl of each sample was applied to a MicroSpinTM G-25 column (GE Healthcare Bio-Sciences), to remove residual salt. The activity of ACE in each homogenate was measured using the Angiotensin-I Converting Enzyme Activity Assay Kit (Yagai Research Center, Yamagata, Japan) according to the manufacturers protocol.
Sperm-ZP Binding Assay
The in vitro assays for sperm binding to zona pellucida were performed with eggs from which the cumulus cells had been removed by treatment with bovine testicular hyaluronidase (175 U/ml; Sigma) for 5 min. In brief, cumulus-free eggs from female B6D2F1 mice (>2 months old; Japan SLC, Inc., Shizuoka, Japan) were placed in a 200-µl drop of modified Krebs-Ringer bicarbonate solution (TYH medium) that contained glucose, sodium pyruvate, bovine serum albumin, and antibiotics [22]. An aliquot of capacitated sperm (2 x 105 sperm/ml) from Clgn–/– or Ace–/– males and their wild-type littermates was inseminated and the mixture were incubated for 30 min at 37°C under 5% CO2 in air. Eggs were fixed and the bound sperm were observed with an IX-70 fluorescent microscope (Olympus) after Hoechst 33258 staining.
RESULTS
ADAMs Are Present in Clgn-Deficient Mouse Testes But Not in Sperm
Since disruption of the genes that encode Adam1a, Adam2, and Adam3 results in a male sterility phenotype that is similar to that seen for Clgn–/– animals [7, 8, 10, 11], we analyzed whether these ADAMs are present in the testis and sperm of CLGN-null mice. When a testicular extract from Clgn+/– mice was immunoprecipitated with anti-CLGN antibody and the resulting precipitate was subjected to immunoblot analysis using antibodies to ADAM1a, ADAM1b, ADAM2, and ADAM3, all of the ADAMs were detected (Fig. 1A). These results indicate that CLGN interacts with ADAMs in the testis. As expected, the ADAMs in the testes of Clgn–/– mice were not immunoprecipitated by the anti-CLGN antibody. Even though the ADAMs were present in Clgn–/– males (data not shown), the ADAM1a/ADAM2 and ADAM1b/ADAM2 heterodimers were absent after the testicular extract was immunoprecipitated with the anti-ADAM2 antibody and the precipitate was probed with the various ADAM antibodies, which indicates that CLGN is involved in the formation of these heterodimers (Fig. 1B). In contrast, these heterodimers were observed in the Clgn+/– mice. We also examined the effect of CLGN disruption on ADAM3, as it has been previously reported that the absence of the ADAM1a/ADAM2 heterodimer due to ADAM1a disruption leads to the disappearance of sperm ADAM3 [10, 23]. ADAM3, as well as ADAM1b and ADAM2, were not present in Clgn–/– sperm, which suggests that infertility is due to the disappearance of the ADAMs in this sperm population (Fig. 1C).
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ACE Is Present and Localizes Normally in Clgn–/– Sperm
Since the sperm from Clgn–/– and Ace–/– mice show impaired migration into the oviduct and ZP-binding ability, we tested whether CLGN controls tertiary folding of the ACE protein, as it does for the ADAMs. To generate antibodies specific for ACE, we immunized Ace–/– female mice with the recombinant N-terminal region of the mouse tACE protein. All of the monoclonal antibodies obtained recognized both sACE and tACE and worked well for immunoblotting (but not for immunostaining). Immunoblot analysis of kidney proteins with the anti-ACE antibody detected a band with Mr 158 000, which is consistent with the molecular mass of sACE (Fig. 2A). In testicular and sperm extracts, a band with Mr 112 000 was detected, which is similar to the size calculated for tACE. Both bands disappeared in Ace–/– mice, which indicates that these bands are sACE and tACE, respectively.
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After fractionation of the sperm heads and tails, tACE was found only in the head fraction from wild-type sperm (Fig. 2B). As expected, ADAM3 and IZUMO1 were also found exclusively in sperm heads, while an antigen that was recognized by the anti-sperm tail-specific Mab (#124) was found only in the tails. The Clgn–/– sperm had normal levels of tACE, which indicates that the lack of CLGN has no effect on the appearance of tACE (Fig. 2A). This result is supported by the finding that ACE was not immunoprecipitated from the Clgn+/– testicular extract by the anti-CLGN antibody (data not shown). When tACE peptidase activity was measured, tACE was found to be biologically active in Clgn–/– testis and sperm at levels comparable to those seen in Clgn+/– mice (Fig. 2C). After phase separation of sperm with Triton X-114, the band with Mr 112 000 was detected only in the detergent-enriched phases of the Ace+/– and Clgn+/– sperm (Fig. 2D). The smaller bands in the detergent-depleted phase most likely reflect processed and solubilized forms of tACE. A similar fractionation pattern was found for the tACE of Clgn–/– sperm, which suggests that tACE is not directly involved in sperm-ZP interactions.
ADAMs are Present in the Ace–/– Testis and Sperm
We examined the possibility of aberrant expression of ADAM proteins in Ace–/– mouse sperm, as was noted for Clgn–/– sperm (shown in Fig. 1C) [12]. When the protein extract from Ace–/– testis was immunoprecipitated with the anti-CLGN antibody and the immunoprecipitate was subjected to immunoblot analysis using antibodies to ADAM1a, ADAM1b, ADAM2, and ADAM3, all of these ADAMs were detected (Fig. 3A). The Ace–/– testicular extract was also immunoprecipitated with the anti-ADAM2 antibody, and the precipitants were subjected to immunoblot analysis with the various anti-ADAM antibodies. Both ADAM1a and ADAM1b were detected in this immunoprecipitate, which suggests the formation of ADAM1a/ADAM2 and ADAM1b/ADAM2 heterodimers in the testis (Fig. 3B). Finally, the ADAMs were processed normally in Ace–/– sperm, which is similar to the situation observed in heterozygous sperm (Fig. 3C).
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Aberrant Distribution of ADAM3 after Phase Separation of Ace–/– Sperm
Our results (Fig. 3) indicate that the ADAMs were present and processed normally in Ace–/– testes and sperm. The Triton X-114 phase separation method concentrates proteins into either a detergent-depleted or detergent-enriched phase, depending on the conformation of the protein [24]. When the proteins from Ace–/– sperm were fractionated into detergent-enriched and detergent-depleted phases with Triton X-114, most of the ADAM3 content (80%) disappeared from the detergent-enriched phase, compared to the ADAM3 content of Ace+/– sperm (Fig. 4, A and B). In contrast, both the ADAM1b and ADAM2 levels remained the same in the detergent-enriched phases of the Ace+/– and Ace–/– sperm. We analyzed other sperm membrane proteins using this Triton X-114 phase separation system. Proteins that contain a transmembrane domain, e.g., IZUMO1 and CD46, were distributed in both phases, while the GPI-anchored SPAM1 (PH-20) protein was distributed exclusively in the detergent-enriched phase (Fig. 4A). Of the sperm proteins examined, only ADAM3 decreased in amount in the detergent-enriched phase of the Ace–/– sperm.
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Comparison of Zona Pellucida-Binding Abilities for Clgn and Ace-Null Mouse Sperm
While aberrant subcellular distribution of ADAM3 was observed in both Clgn–/– and Ace–/– sperm, the altered localization was less severe in Ace–/– sperm (Fig. 4). To examine whether the sperm from these null animals differ in their zona pellucida-binding activities, the numbers of sperm bound per cumulus-free egg were determined. Both Clgn–/– and Ace–/– sperm bound eggs poorly, as compared to the wild-type sperm (Fig. 5B). Furthermore, eggs bound significantly lower numbers of Clgn–/– sperm than Ace–/– sperm (P < 0.001). These effects were not caused by differences in sperm motility, which remained normal in both knockout mouse lines [8, 16].
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DISCUSSION
Although a number of proteins have been hypothesized to be essential for various events of fertilization, their actual roles when a particular gene was ablated were not clear [2–6]. The ADAM1b/ADAM2 dimer (fertilin) was initially thought to be involved in sperm-egg fusion [25] but was later postulated to be involved in the sperm–ZP interaction [7, 26]. More recently, ADAM1a/ADAM2 has been hypothesized to have a chaperone activity, serving to direct ADAM3 to the sperm membrane [10, 23]. Given that ablation of the Adam3 gene impairs sperm–ZP binding [11], our findings suggest that ADAM3 acts downstream of the ADAMs network in sperm assembly and functions in sperm–zona pellucida binding.
In studying the aberrant zona pellucida-binding activity of Ace–/– sperm, we examined the integrity of ADAMs on these sperm compared with the Clgn–/– sperm [8]. We have previously reported the disappearance of ADAM2 from Clgn–/– sperm, and in the present study, we found that ADAM3 was also absent in Clgn–/– sperm (Fig. 1C). This loss of ADAM3 was more complete than that reported for Adam2–/– sperm, in which the ADAM3 level decreased to 11% of the wild-type level [27]. In any case, if the loss of ADAM3 is caused by disruption of ADAM2, it was reasonable to assume that the ablation of both Adam2 and Adam3 would lead to a similar phenotype. In the same context, the impaired zona pellucida-binding activity of Clgn–/– sperm could be explained by an initial failure to form the ADAM1a/ADAM2 dimer, which led, as a secondary effect, to the disappearance of ADAM3 from the sperm surface. This scheme may explain why the four different gene knockout mouse lines (Clgn, Adam1a, Adam2, and Adam3) share a similar fertilization phenotype.
The impaired zona pellucida-binding activity of Ace–/– sperm cannot be explained by a similar mechanism, which raises the question as to how the disruption of Ace leads to a failure of sperm binding to the zona pellucida (Fig. 6). Although rodent tACE has been reported to localize to the midpiece of the sperm flagellum and then disappear from mature sperm during passage through the epididymis [28], we show that tACE is localized exclusively to the head of the sperm from the cauda epididymis, which suggests that tACE is involved in sperm-zona pellucida interactions. Recently, the GPIase activity of ACE has been reported to be necessary for the sperm to acquire ZP-binding activity [29]. Kondoh et al. have postulated that some GPI-anchored proteins on the sperm surface play roles in ZP interaction after cleavage or that tACE exposes a ZP-binding factor by shedding GPI-anchored proteins that block binding.
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The appearance and localization of ACE was not affected in Clgn–/– sperm, which suggests that tACE is not a direct mediator of sperm-zona pellucida binding. Rather, we postulate that tACE is involved in distributing ADAM3 to a location in which it can participate in sperm-ZP binding. When ACE was missing, a portion of the ADAM3 content was not distributed in the detergent-enriched fraction of the Ace-deficient sperm (Fig. 6). All of the sperm ADAM3 is located on the plasma membrane [30, 31]. These reports and our findings indicate that some of the membranous ADAM3 content is distributed in the detergent-depleted fraction. Although the amount of ADAM3 in the detergent-enriched phase is small compared to the total amount, the lack of ADAM3 in this phase may have a detrimental effect on sperm function. The manner in which ACE affects ADAM3 distribution in sperm remains unknown, although the appropriate subcellular distribution of ADAM3 appears to be critical for the sperm to develop the ability to bind to the zona pellucida. Our experiments also suggest one possible mechanism for the similar phenotypes observed when the genes for Ace, Clgn, Adam1a, Adam2, and Adam3 were ablated, i.e., the disruption of zona pellucida-binding ability in these mouse lines is mediated entirely through the aberrant distribution of ADAM3. Recently, it has been reported that ADAM3 has zona-binding activity, while ADAM2 and SPAM1 do not have this activity [32]. This report reinforces the idea that ADAM3 is the furthest downstream factor in sperm-zona pellucida binding. Further study is needed to clarify whether ADAM3 participates in a direct or indirect manner in this process.
ACKNOWLEDGMENTS
We are grateful to Dr. Hitoshi Nishimura and Dr. Tadashi Baba (University of Tsukuba) for providing antibodies against SPAM1, ADAM1a, and ADAM1b and for helpful comments. We also thank Dr. Paul Primakoff (University of California, Davis) for providing the antibody against SPAM1.
FOOTNOTES
2 Correspondence: Masaru Okabe, Genome Information Research Center, Osaka University, Yamadaoka 3–1, Suita, Osaka, 565-0871, Japan. FAX: 81 6 6879 8376; okabe{at}gen-info.osaka-u.ac.jp 
3 These authors contributed equally to this work.
5 On sabbatical leave from the Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelphia, PA 19104.
4 Current address: Graduate School of Life and Environmental Sciences, Tsukuba University, Tsukuba Science City, Ibaraki 305-8572, Japan.
1 Supported by grants (11234203 and 00531), a grant-in-aid for Scientific Research, and the 21st Century 200 COE program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by NIH HD06274 (S.B.M.).
Received: 4 April 2006.
First decision: 26 April 2006.
Accepted: 17 July 2006.
REFERENCES
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