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Proceedings of the Society for Experimental Biology and Medicine 224:123-132 (2000)
© 2000 Society for Experimental Biology and Medicine

Review Article

Antisperm Antibodies Associated with Infertility: Properties and Encoding Genes of Target Antigens

Samuel S. Koide*,1, Linfang Wang{dagger} and Masaharu Kamada{ddagger}


* Center for Biomedical Research, Population Council, New York, New York 10021;
{dagger} National Laboratory of Medical Molecular Biology, Beijing 10005, China; and
{ddagger} Department of Obstetrics and Gynecology, University of Tokushima, School of Medicine, Tokushima 770, Japan


   Abstract
Top
 Abstract
Introduction
 YWK-II
 BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
 Concluding Remarks
 References
 
Infertility among couples of reproductive age is a perplexing condition when the cause is indeterminate. These cases are classified as unexplained infertility. In a subset of subjects, antisperm antibodies with sperm agglutinating and/or immobilizing activities have been detected in the blood or fluids of the reproductive tract. These cases are designated as immunologic infertility although a cause and effect relationship of the antibodies to infertility has not been established. In this review, seven target sperm antigens to antibodies associated with infertility and their encoding genes are described. The antisperm antibodies (ASAs) examined were obtained from infertile women or were monoclonal antibodies (mAb) raised against human sperm proteins. All the ASAs studied possessed potent sperm agglutinating and/or immobilizing activities. The target antigens were isolated from human and other mammalian sperm, and the encoding genes identified. The seven antigens are YWK-II, BE-20, rSMP-B, BS-63 (nucleoporin-related), BS-17 (calpastatin), HED-2 (zyxin), and 75- kDa. Each antigen is a distinct and separate entity and is produced by different cells of the reproductive tract, (e.g., germ cells, epididymal epithelial cells, and Sertoli cells). No single predominant target component has been found to interact with the ASAs. It is proposed that immunologic infertility is the consequence of the combined actions of multiple ASAs in immobilizing and/or agglutinating spermatozoa, blocking spermegg interaction, preventing implantation, and/or arresting embryo development.


   Introduction
Top
 Abstract
 Introduction
YWK-II
 BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
 Concluding Remarks
 References
 
Infertility occurs in one out of five couples of reproductive age. In 10%–20% of these cases, no definitive cause of the infertility has been uncovered; therefore, they are classified as unexplained infertility (1, 2). A subset of infertile subjects has been found to possess antisperm antibodies (ASAs) in blood, semen, ovarian follicular fluid, and/or vaginal and cervical fluids (3, 4). The ASAs associated with infertility induce agglutination and/or immobilization of sperm. Infertile subjects possessing ASAs are classified as having immunologic infertility (5-20). Although the ASAs can agglutinate and/or immobilize human sperm, a cause-effect relationship to infertility has not been established (21-33). The major obstacle is the complexity of the ASA. It is composed of numerous antibodies interacting with multiple sperm components, so that no single predominant target antigen has been identified (5, 34). To resolve this issue of whether a specific ASA with sperm agglutinating and/or immobilizing activities can cause infertility, several target sperm antigens of ASAs were isolated and characterized, and their encoding genes determined. The ASAs were tested for antifertility potential, and/or the target antigens were assessed as antifertility immunogens (35-40). In the present review, the properties of seven human sperm components, identified as target antigens to ASAs involved with infertility, and their encoding genes are described.


   YWK-II
Top
 Abstract
 Introduction
 YWK-II
BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
 Concluding Remarks
 References
 
Characteristics of YWK-II Component.
Monoclonal antibodies (mAbs) to human sperm proteins were raised, and those with sperm agglutinating activity were selected (41). One of the selected mAbs, designated as YWK-II, interacted with two human sperm components, namely 60- and 72-kDa proteins, determined by Western blot (42, 43). The YWK-II antigen was immunolocalized to the equatorial sector of the human sperm head. In rat spermatozoa the YWK-II mAb also interacted with two target antigens, namely 88- and 115-kDa proteins (44, 45). These antigens were immunolocated on the convex surface of the rat sperm head. A noteworthy observation was that the amount or ratio of the isoantigens found in rat spermatozoa varied with their location in the different segments of the epididymis. This finding suggests that a differential production of the isoforms of YWK-II antigen occurs, perhaps as sperm mature and acquire motility and fertilizing potential.

Encoding Gene.
The YWK-II mAb was used to screen a rat {lambda}gt11 expression library; the YWK-II mAb yielded a positive clone from which a cDNA insert, composed of 1837 base pairs with an open reading frame of 573 base pairs coding a polypeptide consisting of 191 amino acid residues, was isolated (46, 47). The cDNA and the deduced polypeptide were assigned the accession number M31322. Comparing the amino acid sequence of the deduced YWK-II polypeptide with other proteins deposited in GenBank, the C-terminus of YWK-II and the transmembrane-cytoplasmic domain of BA4-amyloid precursor protein (APP), found in brain plaques of Alzheimer's disease patients, have a high degree of similarity (35, 48-52). A notable structural difference between the two polypeptides is the occurrence of the tripeptide, Arg-Lys-Arg, in YWK-II and the triplet, Lys-Lys-Lys, in APP. The N-terminals of the two polypeptides, however, are distinctly different. Thus the YWK-II polypeptide is related to the APP family of proteins, based on the high sequence similarity in their transmembrane-cytoplasmic domains.

ßA4-Amyloid Precursor Protein (APP).
APP is composed of 751 amino acid residues and structurally resembles cell-surface receptors. It consists of an extracellular Kunitz protease inhibitor domain, a signal peptide, and a transmembrane-cytoplasmic sequence (51, 53, 54). A full-length cDNA related to the APP gene was cloned from a human placenta expression library and designated as the amyloid precursor homolog (APPH) (49).

The transmembrane-cytoplasmic domains of YWK-II and APPH have high similarity (46, 49). Moreover, the rat amyloid precursor-like protein2 (APLP2), APPH, and murine CDE1-binding protein are apparently species-specific forms of the same entity (55-57). APP is present in the testis and may be involved in sperm development (58). The APLP gene has been mapped to the long arm of human chromosome 19 (59), and the APLP2 gene has been assigned to 11q23-q25 (60, 61).

APP has been implicated as a critical factor required in the organogenesis of developing embryos (62). The APP gene is expressed in the liver and nervous system, including the mesencephalic nucleus of the trigeminal and spinal ganglions, ependymal cells, choroid plexus, and cerebellum of rat embryos (63).

Signal Transduction Pathway.
APP may act as a neuronal receptor coupled to an intracellular signal transduction system by interacting with the GTP-binding protein, G(0) (64). The YWK-II component may play a similar role in sperm. To show that the YWK-II protein interacts with G proteins, a truncated YWK-II cDNA (220-base pair) fragment designated as RSD-220 was prepared and inserted into the expression vector, pQE-31. The construct was transfected into E. coli M15 strain and expressed. The recombinant protein with an estimated relative molecular mass (Mr) of 41 kDa was isolated and purified. Identification of the specific interacting G proteins is being performed. These G proteins are pertussis-toxin-sensitive, undergo ADP-ribosylation, and are involved in cAMP-triggered targeting of water channel aquaporin 2 (AQ2) found in intracellular vesicles and in the apical membranes of kidney epithelial cells (65).

Heterotrimeric G proteins play an important role in the signal transduction system of cells that regulate growth. The G-{alpha} interacting protein (GAIP) is considered to be a regulator of G protein signaling pathways and interacts with G{alpha}i3 protein (66). Also, multiple trimeric G proteins are present in the trans-Golgi network and regulate the formation of constitutive secretory vesicles (67). G{alpha}i3 and G{alpha}i2 proteins interact with nucleobindin, a mammalian calcium binding protein located in the Golgi (68, 69). On the other hand, G{alpha}i2 is involved in receptor-mediated activity. Human adipocyte plasma membrane possesses a multireceptor-linked NADPH-dependent H2O2-generating system activated by insulin and mediated by G{alpha}i2 (70). Thus the potential binding of G-{alpha} proteins by the YWK-II component may influence cyclic nucleotide signal pathways and protein phosphorylation in spermatozoa, thereby influencing their motility (71, 72) and promoting capacitation (73, 74).

Fertility Regulation.
To show that the production of anti-YWK-II antibodies can influence fertility, three oligopeptides corresponding to the extracellular domain of the deduced YWK-II polypeptide were synthesized as multiple antigen peptides (MAP) (75). A significant number of female rats, immunized with these synthetic oligopeptides, or MAP, became infertile. The most effective antifertility immunogen was YAL-198. The antifertility activity of anti-YWK-II antibodies was confirmed when a significant number of female rats, immunized with a conjugate of YAL-198 peptide to tetanus toxoid, became infertile (76). These results suggests that anti-YWK-II antibodies may be a cause of infertility.

Antifertility Action of Antisperm Antibodies.
Although anti-YWK-II antibody possesses potent sperm agglutinating activity, its antifertility effects may be due to additional actions. For example, passive immunization of female mice with anti-YWK-II mAb caused an arrest in the development of fertilized ova (77). Also anti-YAL-198 mAbs added in vitro to cultivated fertilized mouse zygotes blocked their development at the two-cell stage (Kamada M et al., unpublished data). Thus anti-YWK-II antibody has multiple actions; it agglutinates human sperm, interferes with sperm-egg interaction, and arrests the growth and development of zygotes or embryos. By these multiple actions, anti-YWK-II antibodies can interfere with fertility in the female.

Apoptosis and Cell Differentiation.
Limonene, a monoterpene administered in the diet to rats bearing mammary carcinoma will induce apoptosis and differentiation of the cancer cells (78). During the limoneme-induced regression of the tumor, a marked expression of the YWK-II gene occurs. This finding suggests that the YWK-II component may participate in cell apoptosis and differentiation. On the other hand, it is hypothesized that anti-YWK-II antibodies would block apoptosis during spermatogenesis, arrest germ cell differentiation, and interfere with organogenesis of developing embryos.


   BE-20: Epididymal-Specific Protein
Top
 Abstract
 Introduction
 YWK-II
 BE-20: Epididymal-Specific...
Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
 Concluding Remarks
 References
 
Mammalian spermatozoa released from the testis acquire motility and the capacity to fertilize eggs during their passage through the epididymis by undergoing maturation (79-81). Sperm maturation is an androgen-dependent process that involves surface and structural remodeling and chromatin condensation (82-86). The maturation-promoting activity of the epididymis has been attributed to its secretory components, including murine glutathione peroxidase (87), CD52/HE5 (81, 88, 89), CDW 52 (90), and extracellular protease inhibitor (91, 92). However, the specific factor responsible for promoting maturation has not been identified.

BE-20 Component.
In our studies to identify the maturation promoting factor, a 20-kDa protein was isolated from the caudal epididymal fluid of rabbits by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by HPLC on a monoQ HR5/5 anion exchange column, and designated as BE-20 (93). Polyclonal antibodies to purified BE-20 antigen were raised, and the IgG fraction was separated by chromatography on a Protein A-Sepharose column. The antibodies agglutinated and immobilized human spermatozoa and blocked their capacity to penetrate in vitro zona-free hamster eggs (93). The BE-20 protein was immunolocalized in the epithelial cells of the corpus epididymis and proximal segment of the caudal epididymis of rabbits.

Encoding Gene.
The amino acid sequence of the N-terminus of BE-20 protein, consisting of eight amino acid residues, was determined. A 23-mer oligonucleotide coding the octapeptide was synthesized and used as the sense primer with rabbit epididymal mRNAs as a template in an RT-PCR system (94). A truncated cDNA coding the BE-20 was isolated and extended by the method of rapid amplification of cDNA ends (RACE) (95), yielding the full-length BE-20 cDNA (96). The cDNA consisted of 585 base pairs, containing an open reading frame of 369 base pairs, encoding a 13-kDa polypeptide and assigned the accession number U26725. The nucleotide sequence has high similarity to the proteinase inhibitors of human epididymis, designated as HE4 (76.8%), HE5 (52%), and HE6 (48%) (91, 92, 96). The N-terminus of the deduced polypeptide is enriched in leucine (i.e., 11 out of 23 amino acids). Moreover, the deduced polypeptide contains 16 cysteine residues and is related to members of the four-disulfide core family of extracellular proteinase inhibitors (91, 92).

Epididymis-Specific Expression.
The mRNAs prepared from 17 different human tissues were analyzed by Northern blot using the 0.5-kb BE-20 cDNA fragment as probe (96). Positive reaction was elicited only with the epididymis preparation. The specific cells of various tissues expressing the BE-20 gene were identified by in situ hybridization using a Dig-labeled BE-20 cDNA as probe. Positive reaction occurred with the epithelial cells of the cauda epididymis and proximal segment of ductus deferens of the male reproductive tract (93).

Postulated Functions.
BE-20 protein is produced by the epididymis and coats spermatozoa during their passage through the lumen of this organ while acquiring motility and fertilizing potential (79, 93). These associations raise the possibility that the BE-20 component may be the maturation-promoting factor. Moreover, it is structurally related to members of the four-disulfide core family of extracellular proteinase inhibitors (85, 88, 91, 92, 97). Hence, it may act as a proteinase inhibitor, maintaining the integrity of acrosomes and preventing their spontaneous or premature lysis. It is postulated that anti-BE-20 antibodies may interfere with sperm maturation by interacting with the proteinase inhibitor, thereby releasing the protease; this would induce premature lysis of the acrosomes and nullify the fertilizing capacity of sperm.


   Sperm Tail Component (rSMP-B)
Top
 Abstract
 Introduction
 YWK-II
 BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
 Concluding Remarks
 References
 
A unique cell structure of spermatozoa is the tail. This structure is formed in germ cells at the spermatid stage of spermiogenesis (98). Presumably, a specific factor(s) is required that will induce synthesis of specialized components involved in the formation of the tail. This postulated factor has not been identified.

Identification and Immunolocalization.
To identify the specialized component(s), rabbit sperm tails were prepared by nitrogen cavitation. A 20.1-kDa protein was isolated from the sperm tails and designated as rSMP-B (35, 99). Polyclonal antibodies to rSMP-B were raised and used to immunolocalize the antigen on spermatozoa and somatic cells. With rabbit sperm, positive staining occurred over the mid-piece and tail. In testis sections, only spermatids stained positively. In sections of the epididymis, sperm within the lumen were stained, whereas the epididymal cells remained unstained. The immunostaining results suggest that rSMP-B is produced by germ cells during the spermatid stage of spermiogenesis.

Anti-rSMP-B mAb was used to identify the corresponding target component in human sperm by Western blot. A 72-kDa protein was identified as the antigen (100). It should be pointed out that the Mrs of rabbit and human target antigens were markedly different, 20.1 and 72 kDa, respectively.

Antifertility Studies.
The antifertility activity of anti-rSMP-B mAb was demonstrated by its capacity to block in vitro the penetration and fertilization of zona-free hamster eggs by human sperm (100). The 20.1-kDa protein was assessed as an antifertility immunogen in the following study.

Male rabbits were immunized with the 20.1-kDa protein isolated from rabbit sperm tails. The seminiferous tubules of the testis of immunized animals showed marked thinning of the germ cell layer accompanied by a complete absence of spermatozoa (99). Moreover, the lumen of the epididymis was devoid of sperm. The experimental results demonstrated that immunization with rSMP-B caused blockage of spermatogenesis, resulting in azoospermia.

To validate that anti-rSMP-B antibodies can cause infertility, two peptides designated as rSMP-229 and rSMP-230, corresponding to two different sections of the extracellular domain of the deduced rSMP-B polypeptide, were synthesized as MAPs (75). Twelve male and 12 female rats were immunized with the MAPs. Infertility was induced in only 25% of the female rats immunized with rSMP-229, whereas the male rats remained fertile. On the other hand, a significant number (10 out of 12) of female rats immunized with rSMP-230 became infertile. Moreover, none of the male rats immunized with rSMP-230 MAP mated with normal cycling female rats. Blood LH and testosterone concentrations in the immunized male rats were markedly reduced. The treated animals regained their fertility 45 days postimmunization, accompanied by a return of the low blood LH and testosterone values to normal levels. The present results show that immunization with rSMP-230 MAP, to induce production of anti-rSMP-B antibodies, reduces fertility in male and female rats and that the antifertility effect is reversible.

To show clinical relevance of these animal studies, ASAs present in the blood of infertile women were assessed for their capacity to interact with rSMP-230. A noteworthy finding was that a significant number of sera (10 out of 29), obtained from infertile women containing antibodies with sperm immobilizing activity, interacted with rSMP-230, a segment of rSMP-B protein (101). Polyclonal antibodies to rSMP-230 also immobilize human sperm in vitro and block the binding of human sperm to zona membranes. These recent findings suggest that one of the target antigens of antisperm antibodies with sperm immobilizing activity is rSMP-B. Thus anti-rSMP-B antibodies may be a cause of infertility in women (102). Anti-rSMP-B antibodies may interfere with fertility by immobilizing sperm and by blocking sperm-egg interaction.

rSMP-B cDNA (RSD-1).
Using polyclonal antibodies to the 20.1-kDa protein, a positive clone was isolated from a rat testis {lambda}gtll cDNA expression library. The cDNA encoding rSMP-B was isolated and designated as RSD-1 (103, 104). The RSD-1 cDNA is composed of 2069 base pairs with an open reading frame consisting of 438 base pairs, encoding a polypeptide containing 146 amino acid residues, with a calculated Mr of 15,507.

Expression of rSMP-B Gene.
Using the single-stranded 35S-labeled complementary RNA of rSMP-B cDNA as probe, the presence of rSMP-B mRNA in rat and rabbit testis sections was determined by in situ hybridization (105). Among the testicular cells, only spermatids reacted positively showing that the rSMP-B gene was expressed in germ cells during spermiogenesis. The results concur with the immunolocalization observation that the rSMP-B component is produced in spermatids of rabbits testis (99). The rSMP-B gene was also expressed in Chinese hamster ovarian (CHO) cells. RSD-1 was inserted into the EcoRI site of the pSV2-EP vector and transfected into CHO cells (106). One of the transformed cell lines stained positively with anti-rSMP-B antibodies.


   Testis Nucleoporin-Related Component
Top
 Abstract
 Introduction
 YWK-II
 BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
 Concluding Remarks
 References
 
A critical metabolic process in cells is the transport of nutrients, ions, and metabolites from the cytoplasm to various organelles. The bidirectional transport of proteins and nucleoproteins between the nucleus and cytoplasm in cells is selectively controlled by nuclear pore complexes (NPCs) (107, 108). NPCs are large biostructures composed of nearly a thousand components with multiple (ranging from 8–16) copies of 50–100 different proteins (109). The NPC outer diameter is about 120 nm having an estimated Mr of 125 kDa. It is interesting to note that a component of human sperm tails has been identified as a nucleoporin-related protein (110). Moreover, nucleoporin-related mRNA has been shown to be transcribed in germ cells of rat testis (111).

BS-63 cDNA.
DIG-labeled RSD-1 (2069 base pairs) was prepared and used as a probe to screen a human testis {lambda}ZAPII cDNA library. A positive clone was isolated that yielded a cDNA consisting of 1933 base pairs, with an open reading frame composed of 1824 base pairs, encoding a polypeptide, containing 608 amino acid residues (110). The cDNA and deduced polypeptide were designated as BS-63 and assigned the accession number U64675 by GenBank. The 3' terminus contained an untranslatable segment of 104 base pairs and a poly(A) tail. The primary sequence consisted of five segments of XFXF/FG repeats, two LRE motifs, and a Ran binding site (RAN BD). The deduced BS-63 polypeptide could be delineated into four domains based on these structures. Domains I and II contained two and three XFXFX/FG repeats that are characteristics of NPC proteins (112). Domain II was the Ran binding site (Ran BD) (113). Domains I, II, and III of BS-63 had high similarity (96%) to nucleoporin Ran BP2/Nup358. Domain IV was a unique segment and had no similarity to any reported gene product. Domains I and IV contained the tripeptide motif LRE, which acts as a site for the attachment of cell adhesive proteins, including laminin family members and intermediate filament proteins (114).

Tissue Expression.
The mRNAs were prepared from 16 different human tissues and analyzed for BS-63 transcripts by Northern blot using BS-63 cDNA as probe (110). Two transcripts, namely 6.0- and 8.5-kb, were detected. The 6.0-kb transcript was found only in the testis, whereas the 8.5-kb transcript occurred in all tissues. Expression and transcription of the BS-63 gene in the testis were determined by in situ hybridization, using sense and antisense mRNA as probes. Germ cells in all stages of spermatogenesis expressed the BS-63 gene (110). These findings suggest that the 6.0-kb species is involved in the differentiation of spermatogonia to spermatozoa.

Chromosome Mapping.
Southern blot analysis of Oncor Somatic Cell Hybrid Panel of human male and female genomic DNAs was performed using BS-63 cDNA as probe. Three positive bands (3.0, 5.0, and 6.0 kb) were found. Similar positive bands were detected on chromosome 2. Mitotic chromosomes were prepared from human blood leukocytes and analyzed by fluorescent in situ hybridization (FISH) and DAPI(4, 6-diamidino-2-phenylindole) banding (110, 115). Chromosome 2 was identified by DAPI banding and was shown to emit two FISH signals at locus 2q11-q12. The present results show conclusively that the gene coding BS-63 is located on chromosome 2 and at locus 2q11-q12.

Expression of BS-63 Gene in E. coli.
A truncated BS-63 cDNA (1599 base pairs) was inserted into the expression vector, pET30a (+), and transfected into E. coli (DE3). A 66-kDa protein was detected in the lysate prepared from IPTG-induced transfected E. coli, indicating that the BS-63 cDNA can be expressed to produce the recombinant protein (110).

Relation of BS-63 and Ran BD.
The N-terminus of BS-63 has high similarity to the corresponding sequence of RanBP2/Nup358 derived from a HeLa cell lambda expression library (112), showing structural relationship of BS-63 to nucleoporins of NPC. BS-63 cDNA has 99% identity with RanBP2 over 1913 base pairs (116). Also, BS-63 and RanBP2 genes were localized to 2q11-q12, indicating that both genes reside at the same, if not identical, locus. The presence of RanBD in BS-63 will bestow to the protein an affinity to interact with the GTP binding protein, Ran/TC4, suggesting a mode of regulating GTPase activity, an energy-producing system of NPC (113, 117). In the evolutionary scale, RanBD exists as an integral domain of nucleoporins obtained from species ranging from yeast to mammals (118), indicating that it is a conserved gene. RanBP2/Nup258 is a component of NPC and involved in the control of bidirectional transport of proteins and nucleoproteins by supplying the required energy using a Ras-like GTPase activity (119). In mouse testis, two forms of Ran mRNA are transcribed (120). One isoform is Ran/TC4, and the other is a variant with 94% homology. The variant is expressed only in the testis. The activity of Ran is regulated by a Ran-GTPase–activating protein (RanGAP1) (121). Two transcripts encoding RanGAP (2.8 and 3.5 kb) were identified. The 2.8-kb transcript is expressed only in the testis. These findings, which show that Ran/TC4 variant, 2.8-kb Ran GAP1, and 6.0-kb BS-63 are expressed only in the testis, suggest a close functional relationship.


   BS-17 Component: Calpastatin
Top
 Abstract
 Introduction
 YWK-II
 BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
 Concluding Remarks
 References
 
BS-17 Component.
A serum sample from an infertile woman containing ASAs with potent sperm-agglutinating activity was used in a Western blot analysis of human sperm proteins. The target antigen was identified as a 17.5-kDa glycoprotein containing 17.6% neutral hexoses and designated as BS-17 (122). Polyclonal antibodies to BS-17 were raised and used to immunolocalize the antigen on human, rat, rabbit, mouse, and hamster spermatozoa. Intense staining occurred over the acrosomal region of all the mammalian sperm tested. With hamster, rat, and mouse sperm, additional staining occurred over the principal segment of the tail. With rabbit sperm, the principal and middle segments of the tail stained intensely. With human sperm, slight staining of the tail occurred. Thus, BS-17 component is located primarily on the surface of the acrosomal region of mammalian spermatozoa, and variable amounts are present in the tail depending on the species.

BS-17 Gene.
Polyclonal anti-BS-17 antibodies were used to isolate a positive clone from a human testis {lambda}gt11 cDNA expression library (123). The positive clone yielded a cDNA consisting of 758 base pairs with an open reading frame of 558 base pairs, encoding a polypeptide composed of 186 amino acid residues. It has 99.7% homology in 758 nucleotides, overlapping with the 3'-terminus of the gene (2493 base pairs) coding calpastatin (124, 125). The nonidentity nucleotide occurred at position 1937 (A in place of G) and an extra nucleotide (T) at position 2456 of the calpastatin gene (124, 125). The deduced BS-17 polypeptide had 99.5% identity in 186 amino acids overlapping with the carboxyl terminus of calpastatin (708 residues). The nonidentity amino acid (glutamic acid in place of glycine) in the deduced BS-17 polypeptide occurred at position 592 of calpastatin (123). Thus BS-17 appears to be calpastatin. To validate this conclusion, anti-BS-17 antibody was used to identify the target antigen in human sperm by Western blot. Two sperm components, 17.1 and 84 kDa, stained positively. The estimated Mr of the latter component is the same as that of calpastatin (126), indicating that the BS-17 component corresponds to the carboxyl terminal segment of calpastatin (123).

Antifertility Studies.
Polyclonal anti-BS-17 antibodies added in vitro to a fertilization system block the ability of human sperm to penetrate and fertilize zona-free hamster ova (122). However, the antibodies did not influence the attachment of human sperm to the surface of the ova or sperm motility. The antifertility potential of anti-BS-17 antibodies was further demonstrated by the following in vivo experiment. Mouse sperm were incubated with the anti-BS-17 antiserum, and the treated sperm were instilled into the oviduct of ovulated female mice. The animals were examined for fertilized ova and developing embryos several days later. The numbers of developing embryos were significantly reduced in the group instilled with the treated sperm compared with the control group. The results suggest that anti-BS-17 antibodies nullified the fertilizing capacity of mouse sperm to fertilize ova.

Expression of BS-17 Gene.
Single-stranded 35S-labeled RNA probe was prepared from calpastatin cDNA and used to determine the stage of germ cell differentiation when calpastatin (BS-17) gene is expressed. In situ hybridization examination of human testis sections showed that the calpastatin mRNA is transcribed only in spermatids (127). This finding concurs with the previous immunolocalization observation that the BS-17 component is produced in spermatids (122). BS-17 appears to play a role in the late stage of germ cell differentiation (i.e., during the period of spermiogenesis).

Proposed Function of Sperm Calpastatin.
Calpastatin is known to be bound to calpain, a Ca2+-dependent cysteine endopeptidase by forming an inactive complex. Calpain is activated upon dissociation of the complex (126). It is proposed that at the time of fertilization the calpain-calpastatin complex dissociates, releasing the protease that triggers the acrosome reaction. In the presence of anti-BS-17 antibodies, the complex is destabilized by interacting with calpastatin, releasing calpain, to trigger prematurely the acrosomal reaction thereby nullifying the fertilizing potential of spermatozoa.


   HED-2 (Zyxin): A Sertoli Cell Component
Top
 Abstract
 Introduction
 YWK-II
 BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
75-kDa Protein and Gene
 Concluding Remarks
 References
 
During spermatogenesis, germ cells residing in the seminiferous tubules undergo a complex series of structural transformations and biochemical modifications associated with the expression of specific genes to form spermatozoa (128-130). Subsequently, the spermatozoa are released into the lumen of the seminiferous tubules and pass on to the epididymis to undergo maturation, thereby acquiring motility and fertilizing potential (86). It has been hypothesized that Sertoli and epididymal cells produce factors that regulate this remarkable metamorphosis of germ cells to mature spermatozoa (131). The regulatory factors promoting spermatogenesis, spermiation, and sperm maturation have not been identified. To determine the active factors involved, a glycoprotein with an estimated Mr of 20 kDa was isolated from rabbit epididymal fluid, designated EP-20, and found to be produced by Sertoli and epididymal cells, suggesting a dual role of influencing spermatogenesis and maturation (132-134).

EP-20 Component.
A 20-kDa glycoprotein, designated EP-20, was isolated from rabbit caudal epididymal fluid by preparative polyacrylamide gel electrophoresis (132). Polyclonal antibodies to the EP-20 protein were raised and used to immunolocalize the antigen in cells of rabbit testis and epididymis. The principal cells of the distal segment of the corpus epididymis and proximal segment of the caudal epididymis, including the stereocilia of the principal cells, were immunostained. Also, spermatozoa present within the lumen were stained. However, the mucus-secreting cells of the epididymis did not stain. A notable finding was the intense staining of the Sertoli cells of rabbit and human testes, whereas germ cells and interstitial cells remained unstained. Moreover, nine other somatic tissues analyzed did not stain. These results show that EP-20 protein is produced by Sertoli cells of the testis and the principal cells of the epididymis (135).

Antifertility Activity.
Polyclonal anti-EP-20 antibodies immobilized and agglutinated human sperm in vitro (132, 135) and inhibited the penetration of zona-free hamster eggs by human sperm (136).

HED-2 Gene.
Polyclonal anti-EP-20 antibodies were used to screen a human testis {lambda}gtll cDNA expression library (133). A positive clone was isolated and yielded a cDNA designated as HED-2. It consisted of 1908 base pairs with an open reading frame composed of 1479 base pairs, encoding a polypeptide of 493 amino acid residues and assigned the accession number U15158 by GenBank.

Cellular Expression of HED-2 Gene.
The mRNA was prepared from 16 human tissues and analyzed by Northern blot for EP-20 transcript using radiolabeled HED-2 cDNA as probe. The probe interacted with a 2.4-kb mRNA that was present in all tissues analyzed (134). To determine which cells contained the 2.4-kb RNA, human testis sections were examined by in situ hybridization using Dig-labeled antisense HED-2 mRNA as probe. Positive reaction was elicited only with Sertoli cells. The present findings concur with the immunolocalization results that the Sertoli cells of the testis are the source of EP-20 protein.

Structure-Function Relation of HED-2 and Zyxin.
The deduced HED-2 polypeptide contains high proline content (about 24%) compared with common proteins (average content of 4%–5%) (134). In addition, the C-terminal of HED-2 contains three LIM domains and a cysteine-rich motif. These two features are also found in zyxin, a component of the adhesive matrix of cell-cell junctions (137, 138). HED-2 and zyxin have 99% identity in their amino acid sequences with differences in only three amino acids. These amino acids are located at the following positions in HED-2/zyxin: Leu(121)/Try(200); Leu(144)/His(232); and Glu(193)/Lys(272). Zyxin is composed of 572 amino acid residues and is a constituent of the adhesive matrix of cell-cell and cell-basement membrane junctions (137, 138). It is located on the cytoplasmic surface in proximity to actin filament and is closely associated with {alpha}-actinin, a cross-linked protein of actin found in nonmuscle cells (139). Zyxin is a phosphoprotein, having the characteristics of an intracellular signal transducer. It contains a proline-rich domain, a nuclear export signal, LIM motifs, and a double zinc finger domain (140-142). It interacts or is associated with important intracellular structural and metabolic components, including {alpha}-actinin, cysteine-rich proteins, Src-related proteins, and Eva/VASP family members (143). These properties bestow to zyxin the capacity to promote cell differentiation and control the spatial assembly of actin filaments.

Structurally, HED-2 protein also consists of three distinct features: i) three LIM segments; ii) high proline content (24 out of 100 amino acids) in the N-terminus; and iii) multiple serine residues (9 out of 100) in the N-terminus. The LIM domain is a specific metallic ion binding structure containing two zinc binding segments and participates in protein-protein interactions (144, 145). It is found in transcriptional regulators, proto-oncogene products, and adhesive matrix constituents (145). LIM-related components appear to regulate transcription and cell differentiation and act as gene regulators during embryonic development.

A proline-rich N-terminus will tend to alter the alpha helix structure of the polypeptide and induce conformational changes. The proline-rich motif, E/DF PPPPXD/E, is present in vinculin and in ActA protein of the intracellular pathogen, Listeria monocytogenes, and is purported to be involved in the remodeling of actin-based cytoskeleton (146, 147). In addition, several proline-rich proteins act as transcriptional factors, (e.g., CTF/NFI, APZ, Jun, etc.) and as a structural frame for ligand binding (148).

Serine residues in the N-terminus are potential sites for phosphorylation. Activation of a kinase might be the mechanism by which the HED-2 component may trigger the signal transduction system of germ cells to undergo differentiation.

Postulated Function.
Stem germ cells (spermatogonia) in seminiferous tubules of testis are situated next to the basement membrane and proliferate by mitosis. One group of the progeny cells, A-1 spermatogonia, become detached from the basement membrane and are engulfed by the Sertoli cells. While residing within the microenvironment of the Sertoli cells, germ cells will undergo meiosis and differentiate into spermatozoa. Zyxin, a constituent of the adhesive matrix of cell-cell junctions, is involved in the intercellular exchange and transport of essential nutrients and metabolites and in the binding of ligands to the plasma membranes (140). HED-2 protein may play a similar role in the nurturing of germ cells by the Sertoli cells by promoting specific gene expression, by acting as a transcriptional regulator, and by facilitating intracellular signal transduction, thus modulating the differentiation of spermatogonia to spermatozoa.


   75-kDa Protein and Gene
Top
 Abstract
 Introduction
 YWK-II
 BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
Concluding Remarks
 References
 
Serum obtained from an infertile woman, inducing head-to-head agglutination of human sperm, was used to screen a human testis {lambda}gt11 cDNA expression library (149). A positive clone yielded a 2.4-kb cDNA composed of 2427 base pairs (accession number S58544) with an open reading frame of 1584 base pairs, encoding a polypeptide containing 528 amino acid residues. Epitope-selected antibodies were prepared and used to identify the target antigen. By Western blot, positive staining occurred with a 75-kDa human sperm protein. No homology in the nucleotide sequence of the 2.4-kb cDNA, or in the amino acid sequence of the deduced polypeptide, was found with the reported sequences deposited in the Genetic Sequence Data Bank and the Data Bank of The National Biomedical Research Foundation Protein Identification Resource.


   Concluding Remarks
Top
 Abstract
 Introduction
 YWK-II
 BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
 Concluding Remarks
References
 
Seven target antigens to antisperm antibodies with sperm agglutinating or immobilizing activities have been identified and their encoding genes determined. No single predominant target component of the ASAs studied has been found. Antibodies to all target antigens are associated with clinical infertility, reduced fertility in experimental animals, or interference with in vitro fertilization. Based on these findings, immunologic infertility can be attributed to the combined effects of multiple antibodies to a variety of sperm components.


   Footnotes
 
1 To whom requests for reprints should be addressed at the Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021. E-mail: koide{at}popcbr.rockefeller.edu Back


   References
Top
 Abstract
 Introduction
 YWK-II
 BE-20: Epididymal-Specific...
 Sperm Tail Component (rSMP-B)
 Testis Nucleoporin-Related...
 BS-17 Component: Calpastatin
 HED-2 (Zyxin): A Sertoli...
 75-kDa Protein and Gene
 Concluding Remarks
 References
 

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