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Articles |
a Institute of Human Genetics, University of Göttingen, D-37073 Göttingen, Germany
b Institute of Genetics, University of Düsseldorf, D-40225 Düsseldorf, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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isintegrin and 
etalloprotease) family of proteins, which are proposed to be involved in cell-cell adhesion through binding to integrin receptors. Several lines of evidence support a role of cyritestin and other members of this protein family in the fusion of sperm and the egg plasma membrane. In an effort to elucidate the physiological function of cyritestin, we have disrupted its locus by homologous recombination. Male homozygous null mutants are infertile, even though spermatogenesis, mating, and migration of sperm from the uterus into the oviduct are normal. In vitro experiments showed that infertility is due to the inability of the cyritestin-deficient sperm to bind to the zona pellucida. However, after removal of the zona pellucida, sperm-egg membrane fusion monitored by the presence of pronuclei and generation of 2- and 4-cell embryos did not reveal any differences from the wild-type situation. These results demonstrate that cyritestin is crucial in the fertilization process at the level of the sperm-zona pellucida interaction.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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isintegrin and etalloprotease domain. The presence of a disintegrin domain similar to that of soluble snake venom toxins led to the suggestion that the members of the ADAM family are involved in cell-cell adhesion through binding to integrins [5–8]. Several ADAM members including fertilin [9] and cyritestin [10, 11] have been identified in male germ cells and proposed to participate in the binding and/or fusion between sperm and the egg plasma membrane. Cyritestin is the product of the Cyrn gene on mouse chromosome 8 [12]. Experiments using cyritestin antisera have demonstrated that the protein is localized in the acrosomal region of spermatids and spermatozoa, and that it undergoes posttranslational modification after incorporation into the acrosomal membrane [13]. The N-terminal part including the metalloprotease-like domain is removed during spermiogenesis by processing of the precursor protein to a major form. Mature cyritestin contains a domain with homology to a family of integrin ligands known as disintegrin. These data suggested that cyritestin plays a role in sperm function rather than in testicular germ cell maturation. Support for this hypothesis came from in vitro fertilization experiments with synthetic peptides derived from the disintegrin domain of cyritestin. Such peptides interfered with successful fertilization at lower concentrations than did peptides corresponding to fertilin ß, thus identifying cyritestin as one of the major candidates involved in sperm-egg interaction events at the level of egg plasma membrane [10, 11]. To determine whether cyritestin indeed plays a role in sperm-egg interaction, we have disrupted its locus and examined the fertility of mice lacking cyritestin protein.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An 11-kilobase (kb) SalI-BamHI genomic fragment (SalI is from the polylinker of the phage clone) containing exons 2, 3, and 4 of the Cyrn gene was isolated from a J1 129/Sv mouse genomic library (Stratagene Fix II vector). This fragment was subcloned into SalI-BamHI restricted pBluescript vector (Stratagene, La Jolla, CA). The 3-kb SalI-AvaI genomic fragment containing exon 2 was subcloned into SpeI restricted pBluescript vector and then digested with XbaI and BamHI. The isolated 3-kb insert including exon 2 was used as the 5' homologous arm and inserted into XbaI-BamHI-digested pPNT vector [14] to produce clone 1. A 5.5-kb genomic fragment containing exon 4 was subcloned into EcoRI-BamHI restricted pBluescript vector and subsequently digested with XhoI and NotI. The isolated 5.5-kb fragment was then inserted into XhoI-NotI-digested clone 1. The resulting 15.5-kb targeting vector (Fig. 1A) was linearized with NotI and used for transfection of embryonic stem (ES) cells. The relevant genomic sequence data of the Cyrn gene are available as EMBL entries AJ010688 to AJ010696.
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ES Cell Culture and Generation of Chimeric Mice
The ES cell line R1 was cultured as described [15]. Confluent plates were washed in PBS buffer and trypsinized, and the cells were suspended in the same buffer at 2 x 107 cells/ml. Aliquots of this cell suspension were mixed with 50 µg linearized targeting vector and electroporated at 250 V and 500 µF using a Bio-Rad (Richmond, CA) Gene Pulser apparatus. Cells were plated onto nonselective medium in the presence of G418-resistant embryonic mouse fibroblasts. Thirty-six hours later, selection was applied using medium containing G418 at 350 µg/ml and ganciclovir at 2 µM. After 10 days of selection, individual drug-resistant clones were picked into 24-well trays. Three days later, individual recombinant ES clones were replicated into 24-well trays for freezing and isolation of DNA. Chimeric mice from ES cells carrying the disrupted Cyrn allele were generated by aggregating 10–15 compact ES cells and two 2.5-day-old embryos of the CD1 mouse strain as described [16]. Male and female chimeras were mated with CD1 to identify ES cell-derived offspring. The heterozygous offspring were identified by Southern blot analysis and bred to obtain homozygous mice.
DNA Analysis
Genomic DNA was extracted from ES cells [17] and mouse tails [18], digested with ApaI, electrophoresed, and blotted onto Hybond N+ membranes (Amersham, Piscataway, NJ). The blots were hybridized with a 32P-labeled 1.5-kb EcoR1 fragment (P1 in Fig. 1A). Absence of additional random integration of the targeting construct was checked by rehybridization with a neomycin phosphotransferase II DNA fragment. Hybridization was carried out at 65°C overnight in the following solution: 6-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 5-strength Denhardt's solution (single-strength Denhardt's solution is 1% BSA, 1% polyvinylpyrrolidone, and 1% Ficoll 400), 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA. Filters were washed twice at 65°C to final stringency of 0.2-strength SSC, 0.1% SDS.
RNA Blot Hybridization
Total RNA was extracted from testis using the RNA Now Kit (ITC Biotechnologies, Heidelberg, Germany) according to the manufacturer's recommendation. The RNA was size-fractionated by electrophoresis on a 1% agarose gel containing formaldehyde, and blotted. The membrane was hybridized with the random-primed Cyrn cDNA fragments [6] under the same conditions as used for Southern blot analysis. Rehybridization was carried out using a human elongation factor-2 DNA probe [19].
Reverse-Transcriptase (RT)-Polymerase Chain Reaction (PCR)
Total testicular RNA (10 µg) was annealed with a reverse oligonucleotide primer R4: 5'-TGACTGGCTGCTGTTACTACTG-3' (Fig. 1A). Complementary DNA synthesis was performed using 200 U of Superscript reverse transcriptase (GIBCO/BRL, Gaithersburg, MD) and 40 U of ribonuclease inhibitor (RNasin; Boehringer-Mannheim, Indianapolis, IN) according to the manufacturer's recommendation in a final volume of 20 µl. PCR was carried out with 5 µl of synthesized cDNA, 10 pmol each of reverse primer 4R, forward primer 4F (5'-ATGGCTGCTGCCTTATTCCTA-3' (Fig. 1A), and 3 U of Taq polymerase. Cycling conditions were 30 sec at 94°C, 30 sec at 58°C, and 30 sec at 68°C. The amplified fragments were purified and directly sequenced with the primer 4F according to the Taq Dye Terminator protocol (Applied Biosystems, Foster City, CA).
Western Blot Analysis
SDS (SDS-PAGE) was performed as described [20]. Testes were homogenized in 10 vol of NP-TMN buffer (50 mM Tris-HCl, 2 mM MgCl2, 1% NP-40, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml pepstatin A, 17.4 µg/ml PMSF, pH 7.4). Homogenates were adjusted to a final protein concentration of 10 µg/µl. Twenty micrograms of each homogenate was subjected to SDS-PAGE, and the gels were blotted to nitrocellulose membranes (Sartorius Corp., Göttingen, Germany) as described [13]. Cyritestin was probed with anti-750, a rabbit antibody directed against the cytoplasmic domain of cyritestin [13, 21]. Neomycin phosphotransferase II was detected with a commercially available antibody (5 Prime
3 Prime, Boulder, CO).
Fertility Test
To examine the fertility of Cyrn-deficient males, 25 sexually mature Cyrn-/- male mice were mated, each with 4 females (100 females total), for 8 wk. Females were checked for the presence of vaginal plugs and/or pregnancy. Furthermore, 8-wk-old CD1 females were superovulated by i.p. injections of 5 U eCG followed by 5 U hCG 46–48 h later, and mated with Cyrn+/+ or Cyrn-/- males. Eggs from females having a copulatory plug were isolated. The oviducts were dissected out and flushed in M2 medium (Sigma, St. Louis, MO). The eggs were treated with M2 containing hyaluronidase (300 µg/ml) to remove the cumulus, washed in M2, and then maintained in M16 (Sigma) for 2–6 h for assessment of the presence of male and female pronuclei. The eggs were then cultured in M16 covered with mineral oil to check them for progressive development over 3 days.
Binding and In Vitro Fertilization Assays
Sexually mature male mice (wild-type and homozygous for the targeted mutation) were used for the experiments. Female CD1 mice were superovulated. Oocytes were collected 10–12 h after hCG administration, and cumulus cells were removed. Zonae pellucidae were isolated by forcing oocytes through a micropipette with a 30- to 50-µm internal diameter and were washed through drops of fresh fertilization medium [18]. Epididymal sperm (105–106), capacitated for 1.5 h, were added to the washed zonae pellucidae, zona-free oocytes, and intact oocytes in 400-µl drops of fertilization medium and incubated for 1 h (binding assay) and 6 h (in vitro fertilization) at 37°C in 5% CO2. Using a large-bore micropipette, zonae pellucidae and eggs were washed in M16 and photographed, and the eggs were then cultured in M16 as above.
Sperm Analysis
Epididymides were collected from 3-mo-old Cyrn+/+ and Cyrn-/- male mice and dissected in Tyrode's medium. Sperm number and motility were determined by light microscopy, and sperm morphology was examined by scanning electron microscopy. To assay the acrosome reaction, epididymal sperm were capacitated for 1.5 h in Tyrode's medium and then incubated for 5 min at 37°C in 5% CO2 incubator in Tyrode's medium plus the calcium ionophore A23187 (20 µM; Sigma). To determine the percentage of sperm that had undergone an acrosome reaction, sperm were fixed and stained with Coomassie brilliant blue R250 as previously described [22]. At least 200 sperm from each male were assayed for the presence or absence of the characteristic dark blue acrosomal crescent. To examine sperm transport in the female reproductive tract, males were mated with mature CD1 females. Six hours after mating, uterus and oviducts from females with vaginal plug were flushed with M2 medium, and the number of sperm was counted.
Competition Experiments
Isolated zonae pellucidae from unfertilized and fertilized eggs were incubated for 15 min with fluorescein-labeled cyritestin peptide (Cyripep457-F: R-K-S-K-D-Q-C-D-F-P-E-F; 60 µM). The peptide was conjugated with fluorescein as described by Linder and Heinlein [10]. After incubation, zonae were washed three times in M2 medium, fixed in 2.5% glutaraldehyde, and photographed. Competition experiments were performed with unlabeled Cyripep457 and an unspecific peptide YS15 (P-A-Q-N-T-G-H-S-R-G-H-E-S-S-M), respectively.
To test the effect of the cyritestin peptide (Cyripep457) on the sperm-egg binding, isolated zonae pellucidae and intact unfertilized eggs were preincubated with Cyripep457 (60 µM in M16) for 30 min at 37°C in 5% CO2. For control, the same experiment was performed using the unspecific peptide YS15. The binding of epididymal sperm to peptide-treated zonae and eggs was carried out as described in the section Binding and In Vitro Fertilization Assays. Sperm binding was scored under the light microscope, and the mean number of sperm bound to 10 zonae or eggs was determined.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mouse cDNA for cyritestin was used to screen a mouse genomic 
phage library derived from strain 129/Sv. One of the isolated phages contained the 5' region of the gene as determined by the presence of the sequences for exons 2–4. The exon positions were identified by restriction enzyme site mapping and Southern blot analysis. The Cyrn targeting vector was achieved by substituting exon 3 of the gene with the neomycin phosphotransferase II (neo) in reverse orientation relative to the Cyrn transcription unit (Fig. 1A). Thus, transcripts made from the targeted allele and spliced from exon 2 to exon 4 would contain an in-frame stop codon and encode a protein of 81 amino acids. The targeting vector was linearized and used to transfect R1 ES cells. ES cells containing the disrupted allele were selected using Southern blot analysis (data not shown).
Two independent ES cell lines were used to develop chimeric mice that transmitted the mutant Cyrn allele to their offspring. Heterozygous males and females were viable and fertile and were intercrossed to produce F2 Cyrn-/- homozygotes.
Screening for the mutation in the progeny was carried out by Southern blot analysis with the 3' probe (Fig. 1B). The ratio of the three genotypes in the F2 generation was not significantly different from the expected values for Mendelian transmission of the two alleles. Homozygous Cyrn-/- mice were viable and developed normally. Northern blot analysis of RNA derived from testes showed a reduced level of Cyrn transcript in testicular RNA of Cyrn-/ - mice (Fig. 1C). To confirm the deletion of exon 3 in the targeted Cyrn transcript, we performed RT-PCR on testicular RNA with primers located in exons 2 and 4 (Fig. 1A). The predicted splice event from exon 2 to 4 produced a smaller RT-PCR product from the mutant allele (Fig. 1D). Sequence analysis confirmed the absence of exon 3 from Cyrn transcripts in testicular RNA of Cyrn-/- mice. Western blot analysis with an antibody against the cytoplasmic domain of cyritestin revealed that the antibody reacted with the expected 110-kDa cyritestin precursor in testicular extracts of Cyrn+/+ and Cyrn+/- mice. No cyritestin protein was detectable in testicular extracts from Cyrn-/- mice (Fig. 1E).
Cyrn Homozygous Mutant Male Mice Were Infertile
Breeding of Cyrn-/- females with wild-type males revealed that the females were fertile and produced normal-sized litters. However, all male Cyrn-/- mice were infertile despite normal sexual behavior towards female mice and production of copulation plugs. Even though 25 Cyrn-/- males were each mated with 4 wild-type females for 2 mo, no pregnancy was obtained (Table 1).
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The infertility of Cyrn-/- males led us to investigate more closely whether spermatogenesis, sperm motility, and the transport of the spermatozoa through the female reproductive tract were disturbed. As judged by testicular histology, spermatogenesis was normal (data not shown). The number and motility of epididymal sperm were similar to those of wild-type litter mates (Table 2). The mean number of sperm recovered from the uterus and oviduct of females inseminated by Cyrn-/- males did not significantly differ from that counted from females inseminated by age-matched wild-type males (Table 2). These data indicate that the infertility of the Cyrn-/- males was not due to a defect in sperm motility or sperm transport through the female reproductive tract. In addition, there was no significant difference in assays of the sperm acrosome reaction between Cyrn-/- sperm and wild-type sperm.
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Cyrn Was Required for Sperm-Egg Binding
To further analyze the role of cyritestin on sperm competence for fertilization in vivo, we collected eggs from wild-type females after mating with Cyrn-/- or wild-type males. Eighty-five percent of eggs recovered from females inseminated by wild-type males had pronuclei, and 60% developed into 4- to 8-cell stages after further culture. In contrast, all 245 eggs that were examined from females inseminated by Cyrn-/- males lacked pronuclei and failed to develop further. These results revealed that the infertility of the Cyrn-/- males was due to failure of sperm to fertilize eggs.
To address the question whether the infertility of Cyrn-/- sperm in vivo is due to their failure to bind and penetrate the zona pellucida or due to an impairment in fusing with egg plasma membrane, we performed in vitro fertilization experiments. As shown in Figure 2, A and B, incubation of Cyrn-/- sperm with zona-intact eggs did not lead to any binding and fertilization, indicating that the lack of cyritestin had significantly altered the capacity of sperm to adhere to and fertilize zona-intact eggs. This result was further substantiated by incubating isolated zonae pellucidae with Cyrn-/- sperm, which were found to be incapable of adhering to the zonae. In contrast, the rate of in vitro fertilization in assays with zona-free eggs was similar with Cyrn-/- or wild-type sperm, suggesting that the lack of cyritestin did not prevent the fusion of sperm and the egg plasma membrane. Moreover, zona-free eggs fertilized by Cyrn-/- sperm developed normally to 2- and 4-cell stages. This led us to suggest that cyritestin is essential for sperm-zona pellucida binding but not required for sperm-egg plasma membrane interaction and early embryonic development.
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Inhibition of Sperm-Egg Binding by Cyritestin Peptide In Vitro
The cross-talk of the members of the ADAM family with their ligands has been proposed to be mediated by their disintegrin domains. To determine whether the disintegrin loop sequence of cyritestin can interact with a ligand located in the zona pellucida, experiments were performed using a fluorescence-labeled synthetic peptide, Cyripep457F, and unlabeled peptides as competitors. The labeled peptide comprising the central part of the disintegrin loop of cyritestin [6] was found to bind to isolated zonae pellucidae from unfertilized and fertilized eggs. The binding could be prevented by addition of unlabeled competitor peptide of the same sequence (Fig. 3A). In contrast, competition with an unlabeled, unspecific peptide (YS15) had no effect on Cyripep457 binding (data not shown). It thus appeared that part of the cyritestin molecule might act as a ligand for receptors in the zona pellucida and that lack of this ligand could be sufficient to prevent gamete interaction.
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To determine the effect of Cyripep457 on sperm-egg binding, isolated zonae pellucidae from unfertilized eggs and intact unfertilized eggs were preincubated with the cyritestin peptide or the unspecific peptide YS15 before incubation with wild-type sperm (Fig. 3B). The Cyripep457 was found to drastically reduce the sperm binding to the zona pellucida. The mean number of sperm bound to Cyripep457-treated zonae and eggs was reduced to 25% and 28% that of sperm bound to untreated zonae and eggs. The mean number of sperm bound to YS15-treated zonae and eggs was slightly reduced, to 84% and 88%, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another member of the ADAM family suspected to play a role during fertilization is cyritestin (ADAM3). As in the case of fertilin ß, synthetic competitor peptides comprising the central disintegrin-loop sequence of the protein led to a decreased in vitro fertilization rate [10, 11]. In fact, the corresponding cyritestin peptide was an even better inhibitor than the one from fertilin ß. To confirm this suggested role of cyritestin during fertilization, we have generated and analyzed homozygous mice with a disrupted Cyrn gene. All cyritestin-deficient male mice were infertile, and this infertility was due to the failure of mutant sperm to bind to the zona pellucida. In contrast, interaction with the egg plasma membrane was apparently unaffected. From these data, one must assume that cyritestin is involved in the interaction of sperm and the zona pellucida. To gain some evidence for this role, we incubated a fluorescent synthetic peptide comprising the central disintegrin domain sequence with isolated zonae pellucidae from unfertilized and fertilized eggs. Competition experiments indicated that specific binding of the peptide occurred. Furthermore, sperm binding to zonae pellucidae of unfertilized eggs as well as to intact unfertilized eggs was found to be drastically reduced by preincubation with the cyritestin peptide. Thus, the zona appears to contain receptors for the cyritestin disintegrin loop. According to the results of Wassarman [4], the carbohydrate moiety of ZP3 (specifically terminal galactose) is the receptor on the zona pellucida for sperm. It remains to be determined whether cyritestin specifically interacts with carbohydrates of ZP3.
Several different sperm surface proteins have been proposed to function as zona pellucida adhesion molecules [1–4]. This function has been shown only by targeted disruption for fertilin ß [27] and cyritestin (this work). The failure of Cyrn-/- and fertilin-/- sperm to bind to the zona pellucida suggests that the lack of one of these proteins cannot be compensated for by other sperm surface proteins. Thus, we assume that sperm-egg interaction requires a network of binding molecules.
The inactivation of the fertilin ß gene in male mice resulted not only in reduced sperm-plasma membrane fusion and failure of sperm to bind to zona pellucida but also in reduced migration of sperm from the uterus into the oviduct [27]. Therefore, it can be suggested that fertilin ß acts at different levels of reproduction. In cyritestin-deficient male mice, the number of spermatozoa reaching the oviduct was found to be similar to that of wild-type mice. Thus, it can be assumed that cyritestin acts only at the level of sperm-zona pellucida interaction.
Cyritestin (ADAM3) has also been described in the human, with two related gene loci, one probably representing an inactive pseudogene [28, 29]. Nevertheless, in many infertile couples the binding of sperm to the egg in therapeutic in vitro fertilization procedures is disturbed. According to the results presented in this paper, mutations in the ADAM3 gene could well be responsible for some cases of human male infertility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Wolfgang Engel, Institute of Human Genetics Gosslerstrasse 12d, D-37073 Göttingen, Germany. FAX: 49 551 399303; wengel{at}gwdg.de
Accepted: July 20, 1999.
Received: April 22, 1999.
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N. Inoue, M. Ikawa, T. Nakanishi, M. Matsumoto, M. Nomura, T. Seya, and M. Okabe Disruption of Mouse CD46 Causes an Accelerated Spontaneous Acrosome Reaction in Sperm Mol. Cell. Biol., April 1, 2003; 23(7): 2614 - 2622. [Abstract] [Full Text] [PDF]
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 E. O. Weinberg, M. Mirotsou, J. Gannon, V. J. Dzau, R. T. Lee, and R. E. Pratt Sex dependence and temporal dependence of the left ventricular genomic response to pressure overload Physiol Genomics, January 15, 2003; 12(2): 113 - 127. [Abstract] [Full Text] [PDF]
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P. Talbot, B. D. Shur, and D. G. Myles Cell Adhesion and Fertilization: Steps in Oocyte Transport, Sperm-Zona Pellucida Interactions, and Sperm-Egg Fusion Biol Reprod, January 1, 2003; 68(1): 1 - 9. [Abstract] [Full Text] [PDF]
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 J. Frayne, E. A.C. Hurd, and L. Hall Human tMDC III: a sperm protein with a potential role in oocyte recognition Mol. Hum. Reprod., September 1, 2002; 8(9): 817 - 822. [Abstract] [Full Text] [PDF]
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 P. Primakoff and D. G. Myles Penetration, Adhesion, and Fusion in Mammalian Sperm-Egg Interaction Science, June 21, 2002; 296(5576): 2183 - 2185. [Abstract] [Full Text] |
