Esther E. Widgren

Characterization of the rabbit sperm membrane autoantigen, RSA, as a lectin-like zona binding protein☆

Adhesion between spermatozoa and the eggs extracellular coat, the zona pellucida, involves the sperms zona binding proteins (ZBP) and their interaction with the carbohydrate residues of the zona. To investigate this interaction in more detail, a purified nonenzymatic ZBP, the rabbit sperm membrane autoantigen, RSA, was used. RSA-zona binding was demonstrated on nitrocellulose blots and by using the Denny-Jaffe crosslinking reagent which identified an 87,000 molecular weight zona component as the ligand for RSA. The RSA-zona binding was of high affinity with a dissociation constant of 5.6 X 10(-13) M. Furthermore, the binding of capacitated spermatozoa to intact zona was inhibited in the presence of RSA. Characterization of the RSA-zona interaction with a variety of simple and complex carbohydrates indicated that the sulfated, complex carbohydrates fucoidin, dextran sulfate, chondroitin sulfate B, and heparin strongly inhibited RSA-zona binding while chondroitin sulfates A and C, cholesterol-3-sulfate, and monosaccharides such as galactose inhibited RSA-zona binding only weakly. It is concluded that RSA functions as a sperm lectin-like molecule to bind the spermatozoon to the zona pellucida.

Characterization of the Histone H1-binding Protein, NASP, as a Cell Cycle-regulated Somatic Protein

Nuclear autoantigenic sperm protein (NASP), initially described as a highly autoimmunogenic testis and sperm-specific protein, is a histone-binding protein that is a homologue of the N1/N2 gene expressed in oocytes of Xenopus laevis. Here, we report a somatic form of NASP (sNASP) present in all mitotic cells examined, including mouse embryonic cells and several mouse and human tissue culture cell lines. Affinity chromatography and histone isolation demonstrate that NASP from myeloma cells is complexed only with H1, linker histones. Somatic NASP is a shorter version of testicular NASP (tNASP) with two deletions in the coding region arising from alternative splicing and differs from tNASP in its 5′ untranslated regions. We examined the relationship between NASP mRNA expression and the cell cycle and report that in cultures of synchronized mouse 3T3 cells and HeLa cells sNASP mRNA levels increase during S-phase and decline in G2, concomitant with histone mRNA levels. NASP protein levels remain stable in these cells but become undetectable in confluent cultures of nondividing CV-1 cells and in nonmitotic cells in various body tissues. Expression of sNASP mRNA is regulated during the cell cycle and, consistent with a role as a histone transport protein, NASP mRNA expression parallels histone mRNA expression.

Association of Eppin with Semenogelin on Human Spermatozoa

Abstract Eppin (SPINLW1; GeneID, 57119) is a single-copy gene encoding a cysteine-rich protein found only in the testis and epididymis, which contains both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences. This study demonstrates that, in seminal plasma and on human spermatozoa following ejaculation, Eppin is bound to semenogelin I (Sg). Six different experimental approaches: 1) immunoprecipitation from spermatozoa and seminal plasma with anti-Eppin, 2) colocalization in semen and spermatozoa, 3) incubation of recombinant Eppin (rEppin) and rSg and immunoprecipitation with either anti-Eppin or anti-Sg, 4) far-Western blotting of Eppin and Sg, 5) Saturation binding of 125I-Sg to Eppin, which is competed by unlabeled Sg, and 6) direct binding of 125I-Sg to Eppin on a blot, all demonstrate that Eppin and Sg bind to each other. To study the specificity of binding, recombinant fragments of Eppin and Sg were made and demonstrate that the Eppin75–133 C-terminal fragment binds the Sg164–283 fragment containing the only cysteine in human Sg I (Cys-239). Reduction and carboxymethylation of Cys239 blocks binding of 125I-rEppin, indicating that a disulfide bond may be necessary for Eppin binding. The physiological significance of the Eppin-semenogelin complex bound on the surface of ejaculate spermatozoa lies in its ability to provide antimicrobial activity for spermatozoa, which has been reported for both Eppin and semenogelin-derived peptides, and in its ability to provide for the survival and preparation of spermatozoa for fertility in the female reproductive tract.

Characterization of an Eppin Protein Complex from Human Semen and Spermatozoa

Abstract Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells. In this study, we have isolated native eppin from ejaculate supernatants (seminal plasma) and washed ejaculate spermatozoa using column chromatography and two-dimensional SDS-PAGE, and identified by mass spectrometry and Western blots an eppin protein complex (EPC) containing lactotransferrin (LTF; also known as lactoferrin), clusterin (CLU), and semenogelin (SEMG1). To confirm the association of eppin with LTF, CLU, and SEMG1, antibodies to CLU and LTF were used to immunoprecipitate CLU and LTF from human sperm lysates. In both cases identical results were obtained, namely, the immunoprecipitate of the EPC. Additionally, we localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, implying that the EPC is present on spermatozoa from the time they leave the seminiferous tubule. On ejaculate spermatozoa eppin, LTF, and CLU colocalize on the tail. The identification of the EPC components suggests that LTF, CLU, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC, implicating the complex as a central player in a network of protein-protein interactions on the human sperm surface. The EPC may provide a surface network with microbicidal properties that protects spermatozoa as well as regulates the sperms transition to a motile, capacitated sperm.

Association of sperm protein 17 with A-kinase anchoring protein 3 in flagella

BackgroundSperm protein 17 (Sp17) is a three-domain protein that contains: 1) a highly conserved N-terminal domain that is 45% identical to the human type II alpha regulatory subunit (RII alpha) of protein kinase A (PKA); 2) a central sulphated carbohydrate-binding domain; and 3) a C-terminal Ca++/calmodulin (CaM) binding domain. Although Sp17 was originally discovered and characterized in spermatozoa, its mRNA has now been found in a variety of normal mouse and human tissues. However, Sp17 protein is found predominantly in spermatozoa, cilia and human neoplastic cell lines. This study demonstrates that Sp17 from spermatozoa binds A-kinase anchoring protein 3 (AKAP3), confirming the functionality of the N-terminal domain.MethodsIn this study in vitro precipitation and immunolocalization demonstrate that Sp17 binds to AKAP3 (AKAP110) in spermatozoa.ResultsSp17 is present in the head and tail of spermatozoa, in the tail it is in the fibrous sheath, which contains AKAP3 and AKAP4. Recombinant AKAP3 and AKAP4 RII binding domains were synthesized as glutathione S-transferase (GST) fusion proteins immobilized on glutathione-agarose resin and added to CHAPS extracts of human spermatozoa. Western blots of bound and eluted proteins probed with anti-Sp17 revealed that AKAP3 bound and precipitated a significant level of Sp17 while AKAP4 did not. AKAP4 binds AKAP3 and expression of AKAP3 is reduced in AKAP4 knockout sperm, therefore we tested AKAP4 knockout spermatozoa for Sp17 and found that there was a reduction in the amount of Sp17 expressed when compared to wild type spermatozoa. Co-localization of AKAP3 and Sp17 by immunofluorescence was demonstrated along the length of the principal piece of the flagella.ConclusionsAs predicted by its N-terminal domain that is 45% identical to the human RIIα of PKA, Sp17 from spermatozoa binds the RII binding domain of AKAP3 along the length of the flagella.

Inhibition of Human Sperm Motility by Contraceptive Anti-Eppin Antibodies from Infertile Male Monkeys: Effect on Cyclic Adenosine Monophosphate

Abstract Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.

Sequence and localization of human NASP: conservation of a Xenopus histone-binding protein.

In this study the sequence and localization of human testicular NASP (nuclear autoantigenic sperm protein) are reported. NASP cDNA contains 2561 nt encoding a protein of 787 amino acids. The open reading frame contains 2446 nt followed by an ochre stop codon (TAA) and 104 nucleotides of untranslated sequence containing a poly(A) addition signal 10 bases upstream of the poly(A) tail. Northern blot analysis of human testis poly(A) mRNA indicates a message of approximately 3.2 kb. Multiple sequence alignment (MSA) analysis of the encoded human NASP amino acid sequence with the sequence for the Xenopus histone-binding protein N1/N2 and the rabbit NASP amino acid sequence demonstrates that the human sequence and the Xenopus sequence have extensive amino acid homology upstream of the rabbit initiation codon. Significantly, there is an 85% identity between the human and the rabbit NASP sequences when the alignment starts at the N-terminal of the rabbit sequence and at amino acid 101 of the human sequence. The nuclear translocation signal found in N1/N2 and rabbit NASP is completely conserved in human NASP. The first histone-binding domain of Xenopus is 70% identical and 90% similar to the human NASP domain. The second histone-binding domain of Xenopus is 48% identical and 71% similar to the human NASP domain. MSA analysis of the three sequences generated an unrooted ancestral tree with two branches, indicating that fewer amino acid changes have occurred between the Xenopus and the human sequences than between the Xenopus and the rabbit sequences. In the human testis, NASP is localized predominantly in primary spermatocytes and round spermatids. Spermatogonia, Sertoli cells, Leydig cells, peritubular cells, and other somatic cells do not stain. Human spermatozoa contain NASP in the acrosomal region. Following the acrosome reaction, some NASP remains in the equatorial and postacrosomal regions. We propose that mammalian testes and sperm contain a histone-binding protein which may play a role in regulating the early events of spermatogenesis.

Epididymal protein targets: a brief history of the development of epididymal protease inhibitor as a contraceptive.

The Laboratories for Reproductive Biology at the University of North Carolina at Chapel Hill began collaboration with Human Genome Sciences (Rockville, Maryland) to sequence a human epididymal library and identify epididymal-specific genes. Among the first clones obtained from Human Genome Sciences was a clone for EPPIN (official symbol, SPINLW1). Our laboratory has described EPPIN (epididymal protease inhibitor) as a novel gene on human chromosome 20q12-13.2 that encodes a cysteine-rich protein containing both Kunitz-type and WAP-type 4-disulfide core consensus sequences that characterize it as a protease inhibitor. EPPIN expresses 3 mRNA splice variants that encode 2 protein isoforms found in the testis and epididymis. Of the 2 isoforms, 1 is secreted and 1 lacks a secretory signal piece. EPPIN is predominantly a dimer, although multiples often exist, and in its native form, EPPIN is found on the sperm surface complexed with lactotransferrin and clusterin. During ejaculation, semenogelin from the seminal vesicles is bound to the EPPIN protein complex, initiating a series of events that define EPPINs function: modulating prostate-specific antigen (PSA) activity, providing antimicrobial protection, and binding semenogelin, thereby inhibiting sperm motility. As PSA hydrolyzes semenogelin in the ejaculate coagulum, spermatozoa gain progressive motility. Using immunization as a tool to study antigen function, we demonstrated that EPPIN is essential for fertility because immunization of male monkeys with recombinant EPPIN results in complete, but reversible, contraception. To exploit our understanding of EPPINs function, we have developed a high-throughput screen to look for compounds that inhibit EPPIN-semenogelin interaction and mimic anti-EPPIN, inhibiting sperm motility. These compounds are now being developed into a nonhormonal male contraceptive.

Analysis of recombinant mouse zona pellucida protein 2 (ZP2) constructs for immunocontraception

In this study we have examined the potential of recombinant mouse zona pellucida glycoprotein 2 (ZP2) as a target for immunocontraception. Immunogenicity studies and fertility trials were performed in outbred Swiss-Webster mice using four ZP2 constructs: Val(35)-Gly(200) (ZP2(V35-G200)), Val(35)-Leu(331) (ZP2(V35-L331)), Pro(325)-Ala(637) (ZP2(P325-A637)), and Val(35)-Ala(637) (ZP2(V35-A637)). A significant antibody response occurred to three of the four immunogens, however antibodies capable of recognizing native ZP occurred only after immunization with ZP2(V35-A637) and ZP2(P325-A637). Only immunization with ZP2(V35-A637) correlated with a reduction in fertility. Examination of the physiological basis for infertility revealed that: (1) passive transfer of ZP2 antiserum induced infertility in non-immune mice; (2) ovaries of infertile mice appeared histologically normal; (3) infertile mice produced normal numbers of eggs and (4) ZP of ovulated eggs from infertile mice demonstrated a significant reduction in the number of sperm bound compared to eggs from adjuvant controls. Infertility can be caused entirely by ZP2 antibodies without the incidence of significant ovarian pathology. This study also demonstrated that immunization with the bioactive (sperm binding) region of ZP2, recombinant ZP2(V35-G200), did not result in a significant immune response that recognized native ZP or inhibited fertility. Consequently we designed a ZP2-sperm antigen construct, replacing the C-terminal region of ZP2 with Sp17. This construct proved to be immunogenic and reduce fertility while directing the immune response to the Val(35)-Gly(200) region of ZP2.

A mammalian sperm lectin related to rat hepatocyte lectin-2/3

In rat liver the asialoglycoprotein receptor is composed of three polypeptides, RHL-1, RHL-2 and RHL-3 [6]. In rat testis and spermatozoa a galactosyl receptor (RTG-r) which is immunologically related to RHL-2/3 has been described [7]. We now report that in addition to its presence in the rat, an antigenic species of 54 kDa related to RHL-2/3 is present on rabbit, human, pig and mouse spermatozoa. Purified rabbit testis galactosyl receptor (RbTG-r) consists of two major proteins of 54 and 49 kDa, while purified rabbit liver galactose lectin consists of two major proteins of 43 and 40 kDa. In an ELISA the purified rabbit testis galactosyl receptor was shown to bind biotinylated heat solubilized rabbit zonae, while the purified liver galactose lectin did not. We conclude that one of the mammalian sperms zona binding proteins is a galactose lectin of 54 kDa related to rat liver RHL-2/3.