BIOLOGY OF REPRODUCTION (2014) 90(6):125, 1–7 Published online before print 30 April 2014. DOI 10.1095/biolreprod.113.114827

Human Sperm Devoid of Germinal Angiotensin-Converting Enzyme Is Responsible for Total Fertilization Failure and Lower Fertilization Rates by Conventional In Vitro Fertilization1 Le-Jun Li,3 Feng-Bin Zhang,3 Shu-Yuan Liu,3 Yong-Hong Tian,3 Fang Le,3 Li-Ya Wang,3 Hang-Ying Lou,3 Xiang-Rong Xu,3 He-Feng Huang,3 and Fan Jin2,3,4 3Department

of Reproductive Endocrinology, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 4Key Laboratory of Reproductive Genetics, National Ministry of Education (Zhejiang University), Women’s Reproductive Health Laboratory of Zhejiang Province, Hangzhou, Zhejiang, China

In conventional in vitro fertilization (IVF), complete failure of fertilization occurs in 5% to 15% of treatments. Although the causes may be unclear, sperm defects appear to be the major contributor. However, a convincing test is not yet available that can predict the risk of fertilization failure. In this study, we found that germinal angiotensin-converting enzyme (gACE) (also called testicular ACE) was undetectable in sperm from patients who had total fertilization failure (TFF) and lower fertilization rates (LFRs) by IVF based on Western blot and indirect immunofluorescence analyses. Additionally, almost all of the patients without gACE on sperm (23 of 25) manifested a TT genotype of the rs4316 single-nucleotide polymorphism of ACE. Overall, our results indicate that the absence of gACE expression is responsible for TFF and LFRs by IVF. The rs4316 polymorphism of ACE might be associated with infertility in those patients. We conclude that sperm lacking gACE may be recognized before commencing IVF and that the patients may be directed instead to consider intracytoplasmic sperm injection. conventional in vitro fertilization, germinal angiotensin-converting enzyme, lower fertilization rates, sperm, total fertilization failure

INTRODUCTION In most assisted reproductive technology programs, 60% to 70% of retrieved oocytes will successfully fertilize. In most conventional in vitro fertilization (IVF) programs, total fertilization failure (TFF) occurs among 5% to 15% and lower fertilization rates (LFRs) (defined as ,25% fertilization) occur among 20%, with a recurrent failure rate of approximately 30% to 67% [1–3]. Intracytoplasmic sperm injection overcomes previous fertilization failure experienced with IVF [4, 5], but this method requires the reinvestment of time, trust, and 1 Supported

by grants 81370760, 2014CB943302, LZ13H040001, 2012CB944901, 81200475, and 30901493 from the National Natural Science Foundation of China, National Basic Research Programs of China, Zhejiang Provincial Natural Science Foundation of China, and Zhejiang Provincial Department of Education Foundation of China. 2Correspondence: Fan Jin, Department of Reproductive Endocrinology, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310006, China. E-mail: [email protected]

MATERIALS AND METHODS Patient Selection This study was conducted among patients enrolled in the assisted reproduction program at Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China, from April 2011 to May 2013. The inclusion criteria for the female partner were the following as discussed by Yoon et al. [16]: 1) age 38 yr or younger and 2) more than three retrieved matured oocytes (metaphase II [MII]). Any immature, deformed, or postmatured oocytes or oocytes with certain types of abnormalities were

Received: 10 October 2013. First decision: 6 November 2013. Accepted: 19 March 2014. Ó 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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money. Therefore, diagnosing the causes of fertilization failure in IVF is important. However, a convincing test is not yet available that can predict the risk of fertilization failure. The major causes of TFF or LFRs during IVF appear to be defective sperm-zona pellucida binding and penetration, which are mainly due to abnormalities of the sperm and not the oocytes [6]. Experimental results obtained from gene-manipulated animals suggest that many factors thought to be highly important for sperm-zona pellucida binding and penetration turned out to be nonessential (i.e., PH-20 [7], CD46 [8], and acrosin [9]). However, several genes that had never been considered to be involved in fertilization were found to be essential, which was confirmed by a gene-disrupted animal model [10, 11]. Angiotensin-converting enzyme (ACE) is a ubiquitous membrane ectoprotein found in mammalian tissues. Two isoenzymes of ACE exist in human tissues: one is somatic ACE (sACE), while the second is germinal ACE (gACE [also called testicular ACE]), found exclusively in male germinal cells after meiosis [12]. The two isoforms of ACE are encoded by a single gene on 17q23 [13]. The germinal isoform is transcribed by an alternative promoter within intron 12 only during spermatogenesis. Only gACE contains the translated exon 13, leading to a unique N-terminal sequence of 67 amino acids [14]. Germinal ACE knockout in mice did not influence sperm number, morphology, or motility, but it did cause a defect in sperm binding to the zona pellucida of the oocyte [15]. Because this phenotype resembles some cases of human TFF, it is possible that aberrant expression of human gACE protein on sperm underlies failed fertilization in some idiopathic patients. This study aimed to assess the determinants of gACE in patients with TFF and LFRs by IVF and to establish their predictive value for those patients. Moreover, we also searched for potential mutations in the alternative promoter, unique Nterminal sequence and the active site of gACE in patients to determine whether any potential changes detected could correlate with aberrant expression of gACE protein on sperm.

ABSTRACT

LI ET AL. TABLE 1. Clinical characteristics of patients included in the study.* Characteristic Age of male participants (yr) Age of female participants (yr) Infertility duration (yr) No. of retrieved MII oocytes Fertilization rate (%) Sperm concentration (3106/ml) Sperm progressive motility (%)

TFF (n ¼ 40)

LFR (n ¼ 50)

31.70 30.03 4.55 9.83

6 4.01 6 3.45 6 3.00 6 4.71 0 64.80 6 24.77a 33.80 6 6.51d

32.30 30.22 4.35 10.90 17.0 61.62 35.00

6 6 6 6 6 6 6

5.03 4.25 3.52 6.23 6.2 20.10b 6.49e

NFR (n ¼ 50) 33.00 30.78 3.89 11.80 85.4 76.74 38.40

6 6 6 6 6 6 6

4.29 3.42 2.59 5.26 13.0 26.83c 8.81f

P value 0.3928 0.6021 0.5614 0.24 ,0.0001 0.0054 0.0089

* Data are expressed as mean 6 SD. a P ¼ 0.0327 (TFF vs. NFR). b P ¼ 0.5030 (TFF vs. LFR). c P ¼ 0.0019 (LFR vs. NFR). d P ¼ 0.0068 (TFF vs. NFR). e P ¼ 0.3823 (TFF vs. LFR). f P ¼ 0.0294 (LFR vs. NFR). anate-labeled mouse anti-goat IgG secondary antibody (MultiSciences Biotech Co., Ltd.) for 1 h at 378C in the dark, and later again washed in PBS three times. The nuclei were subsequently counterstained by overlaying 4 0 ,6diamidino-2-phenylindole. The negative control samples were prepared by replacing the primary antibody with normal goat serum.

Amplification of the Twelfth Intron of the ACE Gene and the Coding Region for the Catalytic Domain of Human gACE by PCR The DNA from the sperm was extracted by a modified guanidinium thiocyanate method [20]. The twelfth intron of the ACE gene and the catalytic domain of human gACE were amplified by PCR. The sequence of primers for amplification of the restriction fragment length polymorphism (RFLP) was forward 5 0 -GGTAAAGCCCTGAGTGAGGATGG-3 0 and reverse 5 0 CACCTGGGATGTCCGGTCATAT-3 0 (amplicon size, 760 base pair [bp]). The sequence of primers for amplification of the catalytic domain was forward 5 0 -CACTGTTCCCTTATGCCCAG-3 0 and reverse 5 0 -AAGCTCAC CACCCTTTCTTG-3 0 (amplicon size, 353 bp). The PCR conditions were as follows: 1 ll of each primer (10 lmol/L), 2.5 ll of 1.25 mM deoxynucleotide triphosphates, 0.5 U of TaKaRa Ex Taq Hot Start polymerase (Takara Bio Inc., Dalian, China), 2.5 ll of 103 Taq buffer, and 1 ll of DNA in a final volume of 25 ll. The PCR products were sequenced directly and were confirmed by cloning into a pMD19-T vector using a TA cloning kit (Takara Bio Inc.) and then sequenced.

SDS-PAGE and Immunoblot Analysis After the completion of IVF, the remaining sperm of patients were washed in PBS and stored as previously described by Goodrich et al. [17]. In brief, the sperm samples were obtained from patients by masturbation following 3 to 7 days of sexual abstinence and then were allowed to liquefy for 30 to 60 min at room temperature before processing. Semen analysis was performed according to recommendations by the World Health Organization [18] for count and motility. All of the experiments were performed using motile sperm isolated by density gradients (Isolate; Irvine Scientific, Santa Ana, CA) according to the manufacturer’s instructions. The sperm isolated by this method were collected and washed in PBS three times. The sperm were resuspended in frozen storage buffer (50 mM HEPES buffer [pH 7.5], 10 mM sodium chloride, 5 mM magnesium acetate, and 25% glycerol) and stored at 808C. After storage, the samples were thawed, washed twice in 12 ml of PBS, and resuspended in a somatic cell lysis buffer (0.1% SDS and 0.5% Triton X-100 in distilled water). The testicular tissue specimens (the testes of normal spermatogenesis, maturation arrest [MA] at the spermatocyte stage, and Sertoli cell-only syndrome [SCOS]) were mechanically homogenized. They were homogenized in ristocetin-induced platelet agglutination buffer using 5-mm stainless steel beads (Qiagen, Hilden, Germany) with a Qiagen Tissue Lyser II set at 30 Hz for three sessions of 30-sec duration before protein extraction. The SDS-PAGE was performed according to the method by Laemmli [19]. Protein extracts from the sperm were prepared by boiling the cells in sample buffer with b-mercaptoethanol and subsequently centrifuging at 12 000 3 g to yield a supernatant fraction. Next, 10 lg of the protein from the sperm was loaded on each gel and electrotransferred to polyvinylidene fluoride membranes using standard procedures. Germinal ACE was detected using goat polyclonal antibody (1:1000 dilution, sc-12187; Santa Cruz Biotechnology, Santa Cruz, CA) and epitope mapping at the C-terminus of ACE of human origin, followed by mouse anti-goat secondary antibody (MultiSciences Biotech Co., Ltd. Beijing, China). Horseradish peroxidase-coupled secondary antibodies were detected using luminal chemiluminescent substrate (Biological Industries, Kibbutz Beit Haemek, Israel). Quantified on band volume with respect to alpha-tubulin (1:1000; Sigma, St. Louis, MO), the expression was assessed using ImageQuant TL7.0 software (GE Healthcare, Salt Lake City, UT).

RFLP Detection of the rs4316 Polymorphism To facilitate rapid screening of additional patients and controls, we designed a strategy to PCR amplify and detect the polymorphism with a restriction endonuclease (PCR-RFLP) (Supplemental Figure S1; all Supplemental Data are available online at www.biolreprod.org) [21]. Substitution of C by T in this position would destroy a restriction enzyme cleavage site for BseYI. Accordingly, after digestion with BseYI, the fragments of the amplicon with C in this position will have the 48-bp, 177-bp, 181-bp, and 354-bp fragments where the amplicon with T will have the 177-bp, 181-bp, and 402-bp fragments.

Statistical Analysis ANOVA and Student t-test were used for comparisons of continuous variables, and chi-square test was used for categorical variables. HardyWeinberg equilibrium was tested for using Pearson chi-square test with 1 df in order to compare the observed genotype frequencies with the expected genotype frequencies among study subjects. P , 0.05 was considered significant. All tests were two-sided. Statistical analyses were performed using SPSS software (version 18.0; SPSS Corp., Chicago, IL).

RESULTS

Indirect Immunofluorescence

Clinical Characteristics of Infertile Patients

The sperm were suspended in PBS and smeared onto a slide coated with poly-L-lysine, permeabilized with 0.1% Triton X-100, and blocked in PBS/10% bovine fetal serum (v/v). The slides were incubated with anti-gACE (1:100 dilution, sc-12187; Santa Cruz Biotechnology) overnight at 48C. The slides were later washed in PBS three times, incubated with fluorescein isothiocy-

Clinical characteristics for the IVF cycle are summarized in Table 1 and in Supplemental Table S1. No statistically differences in age, infertility duration, and number of retrieved 2

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excluded from this study. Other inclusion criteria were semen characteristics of greater than 20% progressive motility and of more than 20 3 106 total sperm. Sperm samples from the individuals were divided into three groups based on the following experimental design as discussed by Yoon et al. [16]: a TFF group with fertilization failure of all of the available oocytes, an LFR group with fertilization rates below 25%, and an NFR group with fertilization rates above 60%. Fertilization was assessed by the presence of two pronuclei (2PN) and two polar bodies, defining the fertilization rate with the following equation: (Number of 2PN / Number of MII Oocytes) 3 100. The institutional review board at Women’s Hospital approved the study. All participants provided written informed consent.

gACE AS A MARKER TO PREDICT FERTILIZATION FAILURE

RFLP Detection of the rs4316 Polymorphism in Patients and Control Groups

FIG. 1. Representative gels for gACE protein levels in testes and sperm of infertile patients. Normal indicates normal spermatogenesis.

MII oocytes were noted among the three groups. When the conventional parameters of semen quality were compared, a significant difference was detected (P , 0.05) (Table 1). The sperm concentration and progressive motility before sperm preparation were higher in the NFR group than in the TFF group and LFR group (P , 0.05) (Table 1). There was no significant difference between the TFF group and the LFR group (P . 0.05) (Table 1).

Mutation Analyses of the Catalytic Domain of the Human gACE Gene

Expression of gACE in Human Testis and Sperm

Germinal ACE has only a single catalytic domain containing the HEXXH consensus site motif. Thus, we designed a PCR sequencing method to complete mutation analyses of this motif. Only one patient whose sperm had normal gACE expression in the TFF group demonstrated a change in the 1005th codon in the form of a heterozygous novel mutation (AGT . TGT) (Fig. 6). This is a heterozygous substitution (pSer1005Cys) that converts the codon for serine to the codon for cysteine.

As shown in Figure 1, sACE was found in the testes of patients with SCOS, MA, and normal spermatogenesis, but it was not observed in sperm. However, gACE was found in the testes with normal spermatogenesis and sperm but not in the testes of patients with SCOS and MA. Indirect immunofluorescence of viable spermatozoa showed that gACE was localized on the postacrosomal area, neck, and midpiece of morphologically normal cells as reported previously by Nikolaeva et al. [22].

DISCUSSION

Differential Expression of gACE in Patients

Failed fertilization may result from impaired spermatozoa, oocyte deficiencies, or defects in the in vitro sperm or oocyte medium. Most cases of failed fertilization in IVF are believed to relate to male factor deficiencies. Supporting this result is the observation that failed fertilized oocytes reincubated with donor spermatozoa usually fertilize [2, 6]. Our results confirm previous findings [24, 25] and indicate a correlation among the sperm concentration, progressive motility, and fertilization rate in IVF. This indicates that semen samples differ even when they have been classified within the range of being normal according to routine standards in the laboratory, suggesting at least in part an andrological factor [25]. In addition to no clear cutoff value for the sperm concentration, sperm progressive

Western blot analysis (Fig. 2) demonstrated that gACE protein expression was significantly down-regulated by approximately fourfold in 38% (15 of 40) of the TFF patients and in 20% (10 of 50) of the LFR patients compared with others. Indirect immunofluorescence studies (Fig. 3) confirmed that gACE expression on sperm was either absent or demonstrated a paucity of staining in those patients. Western blot analysis (Fig. 4) also demonstrated statistically differences in relative gACE protein expression among the three groups (P , 0.05). Compared with the NFR group, relative gACE protein expression was reduced in the TFF group (P , 0.05) and in the LFR group (P , 0.05).

FIG. 2. Western blot analysis of gACE on sperm with different fertilization rates. Fertilization rate (FR) indicates fertilized oocytes/matured MII oocytes.

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Mutation screening of the twelfth intron of the ACE gene identified polymorphism rs4316 in nine patients whose sperm only weakly expressed gACE in the TFF group and in nine controls in the NFR group. All nine patients in the TFF group had the TT genotype of this polymorphism. No TT genotype was detected in any of the nine controls in the NFR group; only six controls had the CT genotype. To allow for rapid screening of additional patients and controls, we designed a PCR-RFLP method to genotype the rs4316 polymorphism (Fig. 5). Hardy-Weinberg equilibrium was observed among genotypes in the NFR group. Nonsignificant differences were detected in the genotype frequency among the three groups (P . 0.05), but compared with the NFR group, the TT genotype frequency increased in the combined TFF and LFR groups (P , 0.05) (Table 2). The C allele frequency in the LFR group (26%) was markedly lower compared with that in the NFR group (43%) (P , 0.05) and was lower compared with the 49.49% frequency reported in the dbSNP-National Center for Biotechnology Information public database (http://www.ncbi.nlm.nih.gov/SNP/) [23]. However, nonsignificant differences were detected in the TFF group compared with the NFR group (P . 0.05) (Table 2).

LI ET AL.

fertilization failure is observed in 5% to 15% of cases in IVF programs, and LFRs are seen in 20% of couples with normal sperm counts undergoing IVF, with a substantial recurrent failure rate of 30% to 67% being observed [1, 3, 5, 31]. Thus, the evidence suggests that TFF and LFRs are usually not random and reflect a fertilization disorder. Our data show that

motility could define fertilization failure [25]. This indicates a weakness of conventional criteria in semen analysis whereby semen parameters alone do not allow accurate prediction of fertilization failure. Moreover, the possibility of failed fertilization in an IVF cycle is loosely correlated with the number of available oocytes and with female age [2]. In this study, we sought to associate failed fertilization with a possible inability of these patients’ sperm to express gACE, which is exclusively expressed by developing sperm and is involved in sperm-egg binding. The functions of the ACE isozymes in male reproduction have been investigated using knockout experiments [15, 26]. ACE was important for achieving IVF, and the spermatozoa from mice lacking both of the ACE isozymes showed defects in transport within the oviducts and in binding to the zona pellucida. Moreover, sperm-specific expression of the testicular isozyme showed that the fertility phenotype is solely associated with gACE [27]. Expression of gACE exclusively in sperm restored Ace/ male fertility. Furthermore, previous studies [28, 29] have demonstrated that ACE activity is associated with human male fertility. Despite the importance of gACE for male fertility, the association of human fertilization failure in IVF with a possible inability of these patients’ sperm to express gACE has been rarely investigated. The present results of Western blot analysis confirm previous findings shown by immunohistochemistry [30] and indicate that gACE protein is expressed exclusively in developing spermatids and mature sperm, which were not found in SCOS and MA testes but were found in normal spermatogenesis testes. Consistent with previous observations [22], gACE was found to be located on the postacrosomal area, neck, and midpiece of the human sperm in the present study. Importantly, Western blot and indirect immunofluorescence analyses indicated an absence of gACE in the sperm of the patients who had TFF. Total

FIG. 4. Statistical analysis of relative gACE protein expression by Western blot analysis. Quantity One software (Bio-Rad Laboratories, Inc., Hercules CA) was used to quantify the signal intensity of the bands. The gACE signal in each lane was normalized to alpha-tubulin in the same lane before comparison. Data are expressed as mean 6 SEM. *P , 0.05. NFR, normal fertilization rates.

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FIG. 3. Indirect immunofluorescence analysis of gACE on sperm of infertile patients. On sperm of TFF patients, the expression of gACE was absent, or there was a paucity of staining. Expression of gACE was detected by goat anti-gACE polyclonal antibody and subsequently fluorescein-labeled mouse antigoat IgG (green). A 4 0 ,6-diamidino-2-phenylindole mounting medium was used for cell nuclei staining. Original magnification 3400.

gACE AS A MARKER TO PREDICT FERTILIZATION FAILURE TABLE 2. Genotype and allele frequencies of the rs4316 polymorphism in patients. Variable Genotype (%) TT CT CC Allele frequency (%) T C

TFF (n ¼ 40)

LFR (n ¼ 50)

NFR (n ¼ 50)

23 (57.5) 10 (25.0) 7 (17.5)

30 (60.0) 14 (28.0) 6 (12.0)

17 (34.0) 23 (46.0) 10 (20.0)

56 (70.0) 24 (30.0)

74 (74.0) 26 (26.0)

57 (57.0) 43 (43.0)

P value 0.070a 0.064b 0.754c 0.034d 0.030e 0.073f 0.552g

a

v2 ¼ 8.661, P ¼ 0.070 (comparisons of the three groups). v2 ¼ 5.508, P ¼ 0.064 (TFF vs. NFR). c v2 ¼ 0.564, P ¼ 0.754 (TFF vs. LFR). d v2 ¼ 6.785, P ¼ 0.034 . a ¼ 0.017 (LFR vs. NFR); v2 ¼ 8.165, P ¼ 0.017 (TFF þ NFR vs. NFR). e v2 ¼ 7.036, P ¼ 0.030 (comparisons of the three groups). f v2 ¼ 3.214, P ¼ 0.073 (TFF vs. NFR). g v2 ¼ 0.354, P ¼ 0.552 (TFF vs. LFR); v2 ¼ 6.395, P ¼ 0.011 , a ¼ 0.017 (LFR vs. NFR). b

FIG. 5. Detection of the rs4316 polymorphism of ACE. A) Chromatograms corresponding to TT genotype, TC genotype, and CC genotype. The corresponding amino acids are shown. B) Design of the PCR-RFLP strategy to genotype the rs4316 polymorphism. A schematic representation of the PCR amplification product and the BseYI sites present in the T allele and in the C allele is shown. C) The results of the electrophoretic separation after BseYI digestion of the PCR products corresponding to the three possible genotypes of the polymorphism and without BseYI digestion are shown. M, DNA molecular marker DL1000.

38% of the patients with TFF and 20% of the patients with LFRs had no gACE on sperm. This finding implies that male factor deficiencies in failed fertilization are mainly caused by a lack of gACE on their sperm. We also found that 23 of 25 patients without gACE on their sperm (15 patients in the TFF group and eight patients in the LFR group) had a TT genotype of the rs4316 single-nucleotide polymorphism (SNP) of ACE. The transcription of gACE occurs via a testis-specific promoter located within intron 12 of the sACE gene. In addition, the germinal isoform possesses a specific N-terminal-transcribed sequence that is absent in the sACE cDNA [32, 33]. The remainder of the molecule is identical to the carboxyl half of sACE. Mice lacking sACE have low blood pressure and develop severe kidney pathology [26]. However, the patients herein without gACE had no apparent cardiovascular problems or kidney problems; thus, we screened the promoter and the specific N-terminal sequence of the gACE gene. We detected no promoter polymorphic variation of gACE, but we did detect a TT genotype of the rs4316 SNP in patients without gACE. Using PCR-RFLP, we

FIG. 6. A heterozygous substitution of the catalytic domain of the gACE gene was confirmed by DNA sequencing. The corresponding amino acids (AAs) and the site of the zinc-binding motif HEMGH active site are shown. The arrow points to a heterozygous TA substitution.

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determined a significantly increased risk of the T allele frequency in the LFR group compared with the NFR group but not in the TFF group. However, the phenomenon that almost all of the patients (23 of 25) without gACE on their sperm manifested the TT genotype must be taken under consideration, and studies with larger sample sizes are needed to assess any potential connection. Meanwhile, a potential explanation for a possible association between the polymorphism and fertilization failure is that the region influences alternative splicing. A reasonable hypothesis is that this polymorphism could result in changes in the expression of gACE, leading to fertilization failure. Because a portion of TFF patients express normal gACE in their sperm, we also focused on whether any change exists in the active site of gACE in these patients. Angiotensinconverting enzyme is composed of two homologous catalytic domains, the N-domain and the C-domain. Each domain contains an active site with a zinc-binding motif (HEMGH) in dipeptidase activity that is necessary and sufficient for fertilization [34]. Germinal ACE, roughly half as large, is composed of a unique amino terminus and the C-terminal domain of sACE [35–38]. The data in mice with point mutations by homologous recombination into genomic DNA to convert the HEMGH motif to KEMGK are consistent with the results of ACE-null mice [39]. Furthermore, the mutations do not seem to affect sperm glycosyl phosphatidylino sitol-ase (GPIase) activity; however, those male mice are almost

LI ET AL.

infertile, and binding of the mutant sperm to eggs was nearly abolished [34]. Recent data suggest that the removal of GPIanchored protein TEX101 by ACE was essential to produce fertile spermatozoa [40]. In this study, a novel heterozygous mutation (pSer1005Cys) near the zinc-binding motif HEMGH was identified. Similar to that in mutant mice, this genetic change had no effect on sperm number and morphology or on the expression levels of gACE as measured by Western blot analysis and indirect immunofluorescence (data not shown). These data suggest a crucial role of gACE dipeptidase activity in reproduction. In conclusion, TFF and LFRs are functionally associated with reduced or absent expression of gACE in the sperm of these patients. Thus, gACE may be used as a marker to predict fertilization failure. The TT genotype of the rs4316 polymorphism may be a high-risk factor. Studies are needed to examine the prevalence of the homozygous genotype change among larger cohorts of fertilization failure and in other cases of undiagnosed male infertility.

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