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a Center for Recombinant Gamete Contraceptive Vaccinogens and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208
ABSTRACT
Human serum containing sperm-agglutinating antibodies was used to screen a testis cDNA expression library to identify the cognate antigens that may be responsible for this biological effect. The longest positive phage clone (1.9 kb) was sequenced and found to be a testis-specific isoform of calpastatin (tCAST). The testis-specific segment of tCAST is encoded by a single exon within intron 14 of the calpastatin gene. A unique protein isoform is produced that differs in domain structure from the somatic calpastatins (sCAST). Human sCAST most commonly has an N-terminal domain L plus the four functional calpain inhibitory domains. Human tCAST consists of a 40-amino-acid N-terminal T domain plus a part of domain II and all of domains III and IV from the somatic isoform. Our data show that the T domain can target cytosolic localization and membrane association of tCAST, whereas domain I of sCAST exhibits a nuclear localization function. Calpastatin is the endogenous inhibitor of calpain. The calpain/calpastatin system is involved in membrane fusion events for several cell types, and calpain has been localized to the sperm acrosome. We detected tCAST in human sperm and testes extracts by Western blotting with specific antisera. These observations suggest that tCAST may modulate calpain in the calcium-mediated acrosome reaction that is required for fertilization.
calcium, gene regulation, sperm, sperm capacitation/acrosome reaction, spermatid, spermatogenesis
INTRODUCTION
We have isolated several cDNA clones encoding mRNAs specifically expressed in germ cells by screening a human testis 
gt11 expression library with sera containing antisperm antibodies [1]. We report here the discovery of a cDNA with the sequence for a novel isoform of calpastatin present only in testis and sperm.
Calpastatin is the endogenous inhibitor of the ubiquitous neutral cysteine protease calpain (E.C.3.4.22.17). Among its other functions, the calpain/calpastatin system is involved in numerous membrane fusion events, such as neural vesicle exocytosis and platelet and red-cell aggregation [2, 3]. Calpastatin modulation of m-calpain is necessary for myoblast fusion; a decrease of calpastatin accelerates fusion [4, 5]. Calpastatin is upregulated in brain in response to hypoxia, and it is degraded by calpain in response to hypoxic ischemia, thereby serving as a suicide substrate [6]. This is consistent with involvement of the calpain-calpastatin system during the degradative phase of apoptosis in HL-60 cells [7] in which a decrease of calpastatin enhances apoptotic cell death [8]. During programmed cell death, calpastatin is cleaved by caspases [9, 10], thus relieving inhibition of calpain and upregulating its activity.
Multiple forms of calpastatin have been purified from pig heart [11] and been predicted from isolation of rat brain mRNAs [12, 13]. These protein isoforms differ from each other according to their domain structure. We report here that the testis-specific isoform of calpastatin (tCAST) is encoded within the somatic calpastatin (sCAST) gene, and we describe the molecular structure of this protein, cDNA, and gene. Furthermore, we demonstrate differential intracellular targeting signals of the testis and somatic calpastatins resident in their structural domains.
MATERIALS AND METHODS
Constructs
A green fluorescent protein (GFP) reporter plasmid DNA (pEGFP-N2; Clontech, Palo Alto, CA) was used to generate all the GFP fusion proteins by cloning polymerase chain reaction (PCR)-amplified DNA fragments into EcoRI and BamHI sites. Full-length human tCAST cDNA was used as the template to amplify domain T, whereas pTicCS containing sCAST cDNA (a generous gift from Dr. M. Maki) was used to obtain both domains L and I, either combined or separate. Oligonucleotides containing putative nuclear localization sequences (NLS; amino acids 271–286) were subcloned to the N-terminus of GFP, generating NLS-GFP.
Cell Culture and Transient Transfection
HeLa cells were maintained in Dulbecco Modified Eagle Medium supplemented with 10% fetal calf serum. The day before transfection, cells were seeded onto a coverslip and transfected with 2 g of DNA using SuperFect (Qiagen, Valencia, CA). Cells were washed with PBS and fixed in 3.7% formaldehyde 24 h after transfection. The fixed cells were then permeabilized with 0.2% Triton X-100, stained with 4',6-diamidino-2-phenylindole-2HCl (DAPI), mounted in phenylenediamine dihydrochloride solution, and visualized by deconvoluting microscopy.
Sperm Agglutination and Cervical Mucus Penetration Assays
A serum containing antisperm antibodies was obtained from a woman patient diagnosed as being infertile. Positive and negative sperm-agglutinating sera were provided by the Department of Urology, Northwestern University (Evanston, IL). Human semen was obtained from healthy donors and purified by the swim-up technique in modified BWW medium (Irvine Scientific, Irvine, CA).
For sperm agglutination studies, 50 µl of the sperm solution (50 x 106/ml) was added to 100 µl of the test serum diluted in BWW medium, with or without 3% gelatin. The reaction was allowed to proceed for 1 h at 37°C. Solution-phase agglutination reactions were cytocentrifuged at 325 x g for 5 min, stained with Geimsa, and observed at x1000 magnification.
Bovine cervical mucus (Humagen, Charlottesville, VA) was loaded into a Tru-Trax chamber for cervical mucus penetration studies. Approximately 200 µl of the sperm/antiserum mixture described earlier was loaded into the Tru-Trax wells, and the sperm were allowed to migrate for 30 min at room temperature. The distance of the furthest swimming spermatozoon was recorded under x200 magnification.
cDNA Library Screening
The serum was absorbed with Escherichia coli lysate, diluted 1:100 in TTBS (50 mM Tris, 0.15 M sodium chloride, 0.05% Tween-20) and used to screen 5 x 105 plaque-forming units of a human testis cDNA expression library in gt11 [14] according to the method described by Young and Davis [15]. Two positive clones, Y18 and Y19, were plaque purified, and DNA sequencing was performed on a Perkin-Elmer/Applied Biosystems S (Foster City, CA) Prism model 377A automated DNA sequencer.
To confirm the sequence of the 5'-end and around the XhoI site of clone Y19, gel-purified PCR amplimers obtained using the Gene Amp PCR Kit (Perkin Elmer Cetus, Emeryville, CA) were labeled with 32P by the random prime labeling method (Amersham Life Sciences, Inc., Arlington Heights, IL) and used to screen a human testis cDNA library in a -ZAP expression vector (Stratagene, La Jolla, CA). Two positive phage clones were plaque purified and converted to plasmids by in vivo excision using R408 helper phage according to the manufacturer's instructions (Stratagene).
Northern Blot Analysis
Multiple-tissue Northern (MTN) blots containing poly(A)+ mRNA from several human tissues (leukocytes, colon, small intestine, ovary, testis, prostate, thymus, and spleen) were purchased from Clontech and probed with 32P-labeled restriction fragments using hybridization and washing conditions as recommended by the manufacturer (maximum stringency: 0.1x standard saline citrate, 1% SDS, 65°C). Northern blots containing total RNA from various baboon tissues were performed by the Tissue Specificity Core Facility of the Center for Recombinant Gamete Contraceptive Vaccinogens at the University of Virginia (Charlottesville, VA). Both human and baboon blots were stripped in 1% SDS at 95°C for 30 min and then re-exposed to x-ray film for 24 h to ensure complete removal of the bound probe before being reused.
PCR Amplification of Genomic DNA
Oligonucleotide primers were used for PCR amplification of human genomic DNA. Oligonucleotides "somatic exon 14 (sense)" (5'-AGAACCTGAGCTCGACCTC-3') and "testis exon (antisense)" (5'-ATCGTCGACACTGTGGCCGGTGAGCCC-3') were used to amplify the putative intron between exon 14 and the testis-specific exon. Oligonucleotides "testis exon (sense)" (5'-GAAGCAGTTCGTATCTTCC-3') and "somatic exon 15 (antisense)" (5'-GGATGATGAAACAATCCCATC-3') were used to amplify the putative intron between the testis exon and somatic exon 15. These two amplimers were cloned into pBluescript II (Stratagene, La Jolla, CA) and partially sequenced. To complete the characterization and sequencing of this region of the calpastatin gene, this new sequence data was used to design two additional oligonucleotides that flanked the testis exon, "intron 14 (sense)" (5'-TCAGCTCAGGCTGGGGG-3') and "intron 14 (antisense)" (5'-CAACTTTCCCTTCTTCCC-3'), which were used to amplify the testis-specific exon and the adjacent intron sequences. This amplimer was also cloned and sequenced.
Antibody Generation
Female rabbits were immunized with a chimeric peptide containing the putative B-cell epitope specific to tCAST and a promiscuous T-cell epitope from tetanus toxin. The peptide was injected intramuscularly as an emulsion in Montanide ISA 51 (Seppic, Inc., Fairfield, NJ). Rabbit sera were collected after several booster immunizations and then tested for specificity by ELISA and Western blotting. This testis-specific calpastatin chimeric peptide (H-DGERRGAREAVRIFQDQGPSLVDDALINSTKIYSYFPSV-OH) (HTC24–40:TT) was synthesized at Multiple Peptide Systems (under contract N01-HD-5-3231 with the NIH) and made available by the Contraceptive and Reproductive Health Branch, Center for Population Research, NICHD.
Western Blots
Human spermatozoa were washed in 0.01 M PBS (pH 7.4) containing 5 mM EDTA to inhibit calpain activity (Tris/EDTA) and disrupted by three freeze-thaw cycles in 0.05 M Tris-HCl (pH 7.4) containing 1 mM phenylmethyl sulfonyl fluoride (PMSF). Samples were electrophoresed in 12% SDS-PAGE gels and transferred to nitrocellulose membranes, which were then blocked in Tris-buffered saline containing 0.5% Tween 20 (TTBS) for 2 h at room temperature. The filters were incubated with diluted antisera at 4°C overnight, followed by goat antirabbit IgG-horse radish peroxidase (HRP) secondary antibody at room temperature for 1 h, and developed with the Enhanced Chemiluminescent System (Amersham). Autopsy samples of testis and liver were homogenized in 0.05 M Tris-HCl (pH 7.4) containing 1 mM PMSF, centrifuged at 20 000 x g, and the supernatant electrophoresed and blotted as described earlier.
RESULTS
cDNA Library Screening and Northern Blot Analyses
The serum used to screen the cDNA gt11 expression library agglutinated more than 60% of human sperm in a head-to-head orientation at a serum dilution of 1:16 (Fig. 1A). The serum also completely inhibited human sperm penetration of bovine cervical mucus (Fig. 1B).
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Two positive clones, designated Y18 and Y19, were detected by this serum. These clones were of similar size (1.7 and 1.9 kilobases [kb], respectively), cross-hybridized with each other by Southern blot analysis, and had virtually identical restriction patterns with EcoRI, DdeI, and HaeIII (data not shown). However, Y19 contained an internal Xho site, which was absent from clone Y18.
Multiple-tissue Northern blot analyses of human poly(A)+ RNA gave identical patterns with both clones (Fig. 2A). Two mRNAs of 4.6 and 3.0 kb were present in relatively equal amounts in all tissues tested (leukocytes, colon, small intestine, ovary, testis, prostate, thymus, and spleen). A third mRNA of 1.9 kb was present only in testis. A more extensive analysis of 31 baboon tissues (Table 1), including several areas of the brain, confirmed that the 1.9-kb mRNA was unique to the testis (data not shown).
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DNA Sequence Analysis
Clone Y18 and Y19 DNA sequences were obtained by automatic and manual methods. A search of the GenBank sequence database showed that clone Y18 is a truncated version of human calpastatin cDNA [16] corresponding to nucleotides 1090 to 2200 (personal communication with M. Maki; GenBank accession number D16217) plus some additional 3'-untranslated region. Clone Y19 is a testis-specific isoform of calpastatin possessing 186 base pairs (bp) of unique 5'-sequence; the remainder of the transcript is virtually identical to the sequence of calpastatin from nucleotides 1112 to 2486 (personal communication with M. Maki; GenBank accession number D16217). A 3-bp deletion (nucleotide residues 1848–1850) results in a unique XhoI restriction site in the tCAST cDNA. Two additional Y19 clones were isolated from a -ZAP human testis cDNA library to confirm the sequence of the 5'-end and around the XhoI site. Therefore, this deletion does not result from a cloning or PCR artifact. A 135-bp fragment of the 5'-end of the cDNA was used to reprobe the MTN, as shown in Figure 2. This Y19-specific probe hybridized to the smallest (1.9 kb) mRNA present only in the testis lane (Fig. 2B). Thus, clone Y19 encodes a testis-specific isoform of calpastatin from its unique 186-bp sequence at the 5'-end (GenBank accession number U58996).
The deduced amino acid sequence of tCAST is compared to the deduced amino acid sequence of sCAST in Figure 3A. The first 40 amino acids of tCAST are unique. However, beginning at residue 41 of the testis isoform (residue 318 of the somatic isoform), the amino acid sequences are identical, except for the deletion of glutamine 562 of the somatic sequence. The substitution of glutamic acid for glycine at position 592 has been reported previously [17]. The deleted amino terminus of sCAST corresponds to domains L and I and the first exon of domain II [16] (M. Maki, unpublished observations; GenBank accession number D16217; Fig 3B). Thus, tCAST contains three of the four highly conserved motifs (TIPPXYR), which are essential for the inhibitory activity of calpastatin [18, 19].
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Tissue Distribution of Calpastatin
A chimeric peptide containing the sequence of the B-cell epitope of tCAST amino acids 24–40 together with a T-cell epitope of tetanus toxin (TT) [20] was synthesized and used to immunize female rabbits. The rabbit serum contained antibodies that recognized the immunogen and tCAST in human sperm and testis (Fig. 4). No signal was detected in liver extract by the antibody probe. Two molecular size bands were detected in the sperm extract, compared with a single, faster migrating band from testis. The latter probably reflects the tissue degradation in an autopsy specimen. In any case, our antibody detects naturally occurring tCAST in spermatozoa.
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Intracellular Localization Targeted by Domains T, L, and I
We have shown that domain T of murine tCAST is capable of targeting a GFP to membranes (S. Li and E. Goldberg, in preparation). Because domain T of both the human and mouse tCAST is highly conserved, especially the first 15 amino acids, the same region also likely directs the human tCAST to membranes. Indeed, when domain T of human tCAST was fused to GFP (hTE-GFP) (Fig. 5A), the fusion protein was localized to the perinuclear area as aggregates, whereas GFP alone was uniformly distributed in the cell (Fig. 5B). Western blot analysis of a fractionated cell extract confirmed that hTE-GFP was associated with membranes (data not shown). Because tCAST and sCAST differ at their N-terminus and domain T has a targeting function, domains L and I are likely also involved in subcellular localization of sCAST. To address this possibility, we generated a fusion protein between domain L and I and GFP (hLI-GFP). This fusion protein was found to localize predominantly to the nucleus (Fig. 5B). Previous studies [21] suggested that highly basic domain L is likely to contain NLS and to target the protein to the nucleus. Surprisingly, a fusion protein containing domain L and GFP (hL-GFP) was evenly dispersed throughout the cell, indicating that the putative NLS resides in domain I. This was confirmed with a fusion protein of domain I and GFP (hI-GFP) (Fig. 6A), which is targeted to the nucleus (Fig. 6B). A search of the putative NLS within domain I revealed a K277KRKVE motif, which is highly similar to the large T antigen NLS (P126KKKRKVE133) [22]. This sequence is localized to the C-terminal end of domain I and shares no similarity with other CAST domains. Kalderon et al. [22] showed that the mutation Lys128Thr disrupted nuclear targeting. We obtained a similar result from a Lys277Thr construct in which the mutant protein accumulates within the cytoplasm (Fig. 6B). In addition, a peptide with only 16 amino acid residues containing the conserved NLS of domain I was capable of targeting GFP into the nucleus (Fig. 6).
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Characterization of the tCAST Gene
To understand the developmental processes controlling expression of tCAST, we examined its genomic organization. The intron/exon organization of the first 14 exons (domains L, I, and part of II) of sCAST has been reported [21]. These 14 exons encode the first 317 amino acids of the protein and are unique to sCAST; the homology between sCAST and tCAST begins at residue 318 (Fig. 3A). Based on these data, we hypothesized that the testis-specific exons might reside between somatic exons 14 and 15 of the calpastatin gene.
Next, PCR was used to amplify this region of the calpastatin gene in two segments and revealed 800 bp between exon 14 and the testis exon and 2500 bp between the testis exon and exon 15. To determine if the testis-specific portion of the transcript was encoded by one or more exons, two additional oligonucleotides were synthesized based on the new sequence data. Sequence analysis of this PCR fragment revealed the testis sequence to be a single exon. This PCR strategy enabled us to deduce the organization of the gene, as depicted in Figure 7A. The sequence between exon 14 and the testis exon (GenBank accession number U83601; Fig. 7B) contained two cyclic AMP response element (CRE)-like elements, two SP-1 consensus sequences, and a putative TATA box located within 300 bp of the tCAST exon. Experiments are in progress to determine the tCAST transcription start site and whether these sequence motifs function as testis-specific promoter elements regulating tCAST expression.
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DISCUSSION
The results presented here demonstrate that human testis and sperm contain multiple forms of calpastatin, including a novel, testis-specific isoform. The clones Y18 and Y19 were first identified from a cDNA library screen for sperm-specific proteins [1]. Three transcripts were detected in human testes by Northern blot analysis, the smallest of which (1.9 kb) was specific to testis (Fig. 2). DNA sequence analyses indicated that clone Y18 was identical to the published and revised sequence of human calpastatin [16] (personal communication with M. Maki; GenBank accession number D16217). The longest version of clone Y19 contained 186 bp of unique sequence at the 5'-end. The remainder of the molecule was virtually identical to sCAST.
Wei et al. [23] reported that the mRNA for calpastatin is expressed postmeiotically. These authors used a 0.8-kb fragment from the 3'-end of the calpastatin cDNA as a probe, which would detect both the testis and somatic isoforms. As shown by Northern blot analysis (Fig. 2), both sCAST mRNA species and tCAST are abundantly expressed in human testis, although localization to specific cell type has not been accomplished.
Isoforms of calpastatin have been described in several somatic tissues. Geesink et al. [11] purified two forms of 125- and 145-kDa from pig heart and one 125-kDa form from bovine muscle. The latter lacks exon 3, indicating the 125-kDa isoform was the product of an alternate splice of the calpastatin transcript. De Tullio et al. [12] characterized multiple mRNAs for calpastatin in rat brain. Three of the deduced proteins contained an N-terminal domain (domain L) and four inhibitory repeats typical of the calpastatin molecule. The other two are truncated forms, containing the domain L, either free or associated with a single inhibitory repeat. Other differences, because of exon skipping, produce calpastatin forms with different susceptibility to posttranslational modifications. For example, Salamino et al. [24] reported that phosphorylation of active calpastatin promoted a decrease in its inhibitory capacity.
The overall structure of tCAST is strikingly similar to that of another testis-specific isozyme, angiotensin-converting enzyme (ACE). Both proteins are components of a complex and widely distributed proteolytic system. Like tCAST, t-ACE is encoded by a testis-specific mRNA, which is homologous to, but smaller than, the somatic mRNA [25, 26]. The testis isoform of ACE arises from a unique promoter and a single exon residing within intron 12 of the somatic ACE gene [27, 28]. A similar transcriptional strategy generates calspermin from the gene encoding a Ca2+/calmodulin-dependent protein kinase IV. The calspermin transcript is produced by utilization of a testis-specific promoter within an intron of the calmodulin kinase IV gene [29]. These similarities in intron/exon organization and promoter location suggest a common evolutionary origin, resulting in the coordinate regulation of expression of this family of testis-specific isoforms.
Our polyclonal antisera generated against the tCAST-specific, B-cell epitope (HTC24–40:TT) recognized human sperm calpastatin on Western blots. The antibody specificity confirms this protein is not a somatic isoform. However, Rojas et al. [30] reported a cytosolic 68-kDa protein on a Western blot of human sperm probed with a mixture of two monoclonal antibodies specific to domains I and II of calpastatin. The 68-kDa molecular weight is in agreement with that described in somatic cells [19], and presumably, the commercial antibody recognizes only sCAST. From these findings, we suggest that both sCAST and tCAST are present in human spermatozoa. The isoforms may function differentially in regulating the calpains. For example, the recombinant calpastatin forms described for rat brain [13] differed in their inhibitory efficiency on the calpains. According to those authors, domain L has no inhibitory activity by itself and may be relevant in recognition of the protease. In addition, tCAST lacks domain L, suggesting (by extrapolation to our data) an effect on modulation of calpains during spermatogenesis or sperm function. Yudin (see Note Added in Proof) has localized tCAST and calpain to the head of the primate spermatozoon, between the plasma membrane and the outer acrosomal membrane. This information, together with reports of calpain II in the acrosome and equatorial regions of pig sperm [31] and calpain activity in extracts of human sperm [30], indicate that the calpain/calpastatin system might play a critical role in mammalian sperm function and fertilization. The importance of Ca2+ to calpain activation and the absolute requirement for Ca2+ influx in initiating the acrosome reaction [32, 33] support this contention.
The transfection studies described here demonstrate clearly that the functional domains of calpastatin are responsible for different subcellular localization of these protein isoforms. Whereas domain T can target tCAST to membrane structures in the cell, domains L and I together and domain I alone from sCAST target a heterologous protein to a very different site: the nucleus. Lacking domains L and I, the testis isoform is unlikely to translocate into the nucleus. However, the localization may reflect its ultimate association with the acrosome during transformation of the spermatid to a spermatozoon.
Calpain and calpastatin were found mostly in cytoplasm, but some reports describe calpain as being in the nucleus [34] and being able to degrade nuclear matrix protein [35]. An in vitro experiment demonstrated calpain I transport to the nucleus [36], but poor translocation of calpastatin was observed. However, these investigators used the erythrocyte-type calpastatin, which lacks domain L and I and the NLS in domain I. It was assumed previously that the highly basic domain L was likely to contain putative NLS. Although there are clusters of Lys and Arg, domain L failed to localize GFP into the nucleus in the absence of domain I. A search of putative NLS within domain I revealed a KKRKVE motif, which has high similarity to the large T-antigen NLS (PKKKRKVE133) [37]. This sequence is localized to the C-terminal end of domain I, and it shares no similarity with other inhibitory domains. Therefore, in addition to its well-characterized inhibitory activity, domain I seems to harbor this important function of protein import to the nucleus. This targeting may be negatively regulated by one or more of the other domains, because the fusion protein of full-length sCAST and GFP remains mainly in the cytoplasm (data not shown). Nevertheless, we have provided the first evidence, to our knowledge, that sCAST could function in the nucleus as a consequence of NLS in domain I. Kumamoto et al. [38] detected some nuclear localization of calpastatin by electron microscopy, but most of the signal was cytoplasmic. This may result from a very low abundance of sCAST, or because protein transport occurs only under certain conditions in the cell, as observed for several other proteins [39, 40]. Perhaps only a truncated sCAST containing the NLS translocates to the nucleus and is not detectable by presently available probes. Calpain has a regulatory function in degrading transcription factors, including p53 [41], c-fos, and c-jun [42] and in cell cycle control [43]. This activity is likely to be modulated by nuclear calpastatin.
The serum, which was used in the cDNA library screen, was obtained from an infertile woman [1]. Based on the results of our in vitro assays, antibodies to calpastatin could interfere with sperm function. The presence of autoantibodies to calpastatin is not uncommon. Sato et al. [44] reported that the levels of anticalpastatin antibodies show a positive correlation in patients with inflammatory joint and muscle involvement. Takada et al. [45] reported that anticalpastatin antibodies are prevalent in sera of patients with rheumatoid arthritis (RA). Lackner et al. [46] demonstrated, however, that at least two major epitopes of calpastatin are recognized by an approximately equal number of normal sera and RA sera. Agglutination of sperm by serum in this study and the presence of antibodies to calpastatin are not necessarily related in terms of the patient's infertility.
NOTE ADDED IN PROOF
Yudin AI, Goldberg E, Robertson KR, Overstreet JW. Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa. J Androl 2000; (in press).
FOOTNOTES
First decision: 17 December 1999.
1 Supported by NIH Sub-5-U54-HD29099, by P30HD28048, and by fellowships for G.Y.W. from the Andrew W. Mellon Foundation and the World Health Organization. 
2 Correspondence: Erwin Goldberg, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 North Campus Dr., Evanston, IL 60208. FAX: 847 467 1380; erv{at}northwestern.edu
3 Current address: National Research Institute for Family Planning, Beijing, China.
4 Current address: State Teachers College, Seminar Hakibbutzim, Tel Aviv, Israel.
5 Current address: University of Tennessee Medical Center, Knoxville, TN 37920.
Accepted: February 24, 2000.
Received: November 22, 1999.
REFERENCES
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  M. J. Wolkowicz, L. Digilio, K. Klotz, J. Shetty, C. J. Flickinger, and J. C. Herr Equatorial Segment Protein (ESP) Is a Human Alloantigen Involved in Sperm-Egg Binding and Fusion J Androl, May 1, 2008; 29(3): 272 - 282. [Abstract] [Full Text] [PDF]
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 S. Li and E. Goldberg A Novel N-Terminal Domain Directs Membrane Localization of Mouse Testis-Specific Calpastatin Biol Reprod, December 1, 2000; 63(6): 1594 - 1600. [Abstract] [Full Text]
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