Michael J. Wolkowicz

SAMP32, a Testis-Specific, Isoantigenic Sperm Acrosomal Membrane-Associated Protein

Abstract To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114. Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences. Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57. SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256. Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific. SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus. The SAMP32 locus consists of seven exons on chromosome 6q15-16.2. Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel. This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence. Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome. By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis. Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic. Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.

SLLP1, A Unique, Intra-acrosomal, Non-bacteriolytic, c Lysozyme-Like Protein of Human Spermatozoa

Abstract We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by β-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P ≤ 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, ∼1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.

Equatorial Segment Protein Defines a Discrete Acrosomal Subcompartment Persisting Throughout Acrosomal Biogenesis

Abstract The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.

Cloning and Characterization of a Novel Sperm-Associated Isoantigen (E-3) with Defensin- and Lectin-Like Motifs Expressed in Rat Epididymis

Abstract In the present study we report the identification of a novel epididymis-specific secretory glycoprotein, E-3, which is a sperm-associated isoantigen containing defensin- and lectin-like motifs. E-3 was detected in rat epididymal fluid and in sperm extracts by two-dimensional (2-D) Western blotting using rat hyperimmune sera raised against rat sperm. The immunoreactive spot of approximately 28 kDa with an isoelectric point (pI) of 3.5 was cored from silver-stained gels. Microsequencing by tandem mass spectrometry and database searches revealed several peptides to be novel sequences. Degenerate deoxyinosine-containing primers corresponding to the novel peptides were used in rapid amplification of cDNA ends and polymerase chain reaction to clone E-3 from a rat epididymal cDNA library. A 449-base pair nucleotide sequence was subsequently obtained consisting of a complete open reading frame (ORF) of 111 amino acids, which showed similarity to the defensin and lectin families. The first 21 amino acids constituted a putative signal peptide, suggesting that E-3 is a secretory protein. Mature E-3 protein corresponding to amino acids 22–111 was expressed in E. coli, and chickens were immunized with recombinant E-3 (rE-3). The resulting anti-rE-3 antisera recognized the recombinant immunogen as well as a “native” protein of 28 kDa, pI 2.5–3.5 in both epididymal fluid and in sperm extracts on 2-D Western blots. Northern hybridization indicated that E-3 mRNA was present in the epididymis but not in testis or other tissues, and that E-3 mRNA was predominantly expressed in the corpus and cauda of the epididymis, but not in the initial segment or caput. Similarly, Western blots detected the E-3 protein only in the epididymal fluid and sperm from the corpus and caudal regions. Finally, indirect immunofluorescence localized E-3 on the entire tail, and with less intensity on the head of the sperm. These observations indicate that E-3 is a secreted epididymal protein that becomes associated with the sperm as it transits through the corpus and cauda. The presence of a defensin-like motif suggests that E-3 may play a role in protecting the sperm from microbial infections in the epididymis and in the female reproductive tract.

Tektin B1 Demonstrates Flagellar Localization in Human Sperm

Abstract The human flagellar protein tektin B1 (h-tekB1) in human sperm was cloned, and its sequence and subcellular location were determined. Human sperm proteins were separated by 2-dimensional electrophoresis, and a resolved protein spot of 54 kDa with an isoelectric point (pI) of 5.3 was removed from the gel, trypsinized, and microsequenced by tandem mass spectrometry. The resulting peptides did not match any protein in the (then current) protein databases. Degenerate oligonucleotides based on the microsequences were used with a polymerase chain reaction to amplify a partial cDNA clone from human testis poly(A)+ mRNA, and subsequently a full-length 1.5-kilobase (kb) clone (GenBank AF054910) was obtained from a testis cDNA library. The open reading frame encoded a 430-amino acid protein with 47% homology to the sea urchin tektin B1. Hybridization of labeled h-tekB1 cDNA to a multiple-tissue Northern blot demonstrated a transcript of 1.7 kb in human testis, and a multiple tissue dot-blot demonstrated high levels of expression in testis, trachea, and lung, intermediate levels in fetal brain and appendix, and low levels in ovary, pituitary, and fetal kidney. Rat polyclonal serum generated against a recombinant h-tekB1 demonstrated 3 h-tekB1 isoforms of pI 5.25, 5.5, and 5.35 at 53.5 kDa on a 2-dimensional Western blot of human sperm proteins. Immunofluorescent studies localized h-tekB1 to the principal piece of human sperm, but the endpiece was unstained.

Inhibition of tyrosine phosphorylation of sperm flagellar proteins, outer dense fiber protein-2 and tektin-2, is associated with impaired motility during capacitation of hamster spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor, tyrphostin‐A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin‐A47‐treated spermatozoa exhibited circular motility, which was associated with a distinct hypo‐tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000–60,000. In this study, we provide evidence on the localization of capacitation‐associated tyrosine‐phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo‐tyrosine phosphorylated major proteins of tyrphostin‐A47‐treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein‐2 and the 51 kDa protein as tektin‐2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo‐tyrosine‐phosphorylation status of outer dense fiber protein‐2 and tektin‐2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR‐tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa. Mol. Reprod. Dev. 77: 182–193, 2010.

Splicing in murine CABYR and its genomic structure.

Human calcium-binding tyrosine-phosphorylation regulated protein (CABYR) is a polymorphic, testis-specific, calcium binding protein that undergoes tyrosine phosphorylation during in vitro capacitation. A protein kinase A (PKA) regulatory subunit type II alpha (RII-alpha) homologous domain in the N-terminus, phosphorylation dependent Ca(++) binding isoforms, and localization to the principal piece of the human sperm tail suggest that CABYR may be involved in sperm motility. In this paper, four mouse orthologous cDNAs and the genomic DNA of CABYR were cloned, nucleotide and protein sequences of mouse and humans were compared, and the genomic organization of the mCABYR gene was analyzed. Human and mouse CABYR conserve potential functional motifs including a domain homologous to the dimerization interface of cyclic adenosine monophosphate dependent PKA RII-alpha, 14 PXXP motifs, and regions of homology with extensins and src homology-3-binding protein 1. mCABYR is arranged into six exons spanning about 14 kb of DNA. Mouse CABYR showed several similarities with human CABYR: (1) the protein was localized to the principal piece of mouse epididymal spermatozoa; (2) mouse CABYR has two coding regions (CR-A and CR-B), with 66 and 82% identity, respectively to human; and (3) mCABYR showed the presence of two testis-specific transcripts of approximately 1.4 and approximately 2.4 kb. Three murine splice variants were identified, two of which spliced into CR-B. Exon 4, present in all human and mouse variants and comprising 85% of CR-A appears suitable for targeted deletion. The overall 81% nucleotide identity between mouse and human CABYR, the common genomic organization, presence of similar testis-specific transcripts, localization in the principal piece of tail and occurrence of homologous splice variants indicate an authentic murine orthologue of CABYR has been identified.

Dynamic Changes in Equatorial Segment Protein 1 (SPESP1) Glycosylation During Mouse Spermiogenesis

ABSTRACT ESP1/SPESP1 is a testis-specific, postmeiotic gene expressed in round spermatids that encodes equatorial segment protein 1, an intra-acrosomal protein found in the acrosomal matrix and on the luminal surface of the inner and outer acrosomal membranes within the equatorial segment domain of mature spermatozoa. A comparison of testicular protein extracts with caput, corpus, and caudal epididymal sperm proteins revealed striking differences in the apparent masses of SPESP1 isoforms. The predominant isoforms of SPESP1 in the testis were 77 and 67 kDa, with 47-kDa forms present to a minor degree. In contrast, SPESP1 isoforms of 47 and 43 kDa were found in caput, corpus, and caudal sperm, indicating that SPESP1 undergoes noticeable mass changes during spermiogenesis and/or subsequent transport to the epididymis. On two-dimensional (2D) SDS-PAGE, testicular SPESP1 isoforms resolved as a train of pI values from 4.9 to 5.2. Immunoprecipitated 77-kDa SPESP1 from testis reacted with the glycoprofile stain after one-dimensional and 2D gel electrophoresis, indicating that the 77-kDa testicular isoform was highly glycosylated. One charge variant of the 67-kDa isoform was also glycoprofile positive after 2D gel resolution. The 47- and 43-kDa isoforms of SPESP1 from epididymal sperm did not stain with glycoprofile, suggesting an absence of, or few, glycoprofile-sensitive glycoconjugates in epididymal SPESP1. Treatment of testicular extracts with a variety of glycosidases resulted in mass shifts in immunoreactive SPESP1, indicating that testicular SPESP1 was glycosylated and that terminal sialic acid, N- and O-glycans were present. A mixture of deglycosidase enzymes (including PNGase-F, neuraminidase, beta1–4 galactosidase, endo-alpha-N-acetylgalactosaminidase, and beta N-acetyl-glucosaminidase) completely eliminated the 77- and 67-kDa SPESP1 bands and resulted in the appearance of 75-, 60-, 55-, 50-, 47-, and 43-kDa forms, confirming that both the 77- and 67-kDa testicular forms of SPESP1 contain complex carbohydrate residues. Treatment of caudal epididymal sperm with PNGase-F enzymes showed a faint deglycosylated band at 30 kDa, but neuraminidase did not result in any molecular shift, indicating that epididymal sperm SPESP1 did not contain sialic acid/N-acetylglucosamine residues. These findings are consistent with the hypothesis that SPSPESP1 undergoes significant glycosylation in the testis and that the majority of these glycoconjugates are removed by the time sperm reach the caput epididymis. Studies of the fate of SPESP1 after the acrosome reaction localized SPESP1 to the equatorial segment region in both noncapacitated and capacitated, acrosome-reacted sperm. During capacitation, SPESP1 underwent proteolysis, resulting in a 27-kDa fragment. Zona-free oocytes incubated with recSPESP1 protein showed complementary binding sites on the microvillar oolemmal domain. Both recSPESP1 and anti-recSPESP1 antibody inhibited in vitro fertilization.