Michael G. O'Rand

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.

Appearance of cell surface auto- and isoantigens during spermatogenesis in the rabbit

Abstract The appearance of spermatogenic cell surface auto- and isoantigens can be precisely determined by utilizing techniques that separate spermatogenic cells. Using cytotoxic (complement dependent) auto- and iso- rabbit antirabbit whole semen sera, specific spermatogenic auto- and isoantigens were first detected following the maturation of spermatogonia into primary pachytene spermatocytes. The antisera employed were cytotoxic (complement dependent) for pachytene diplotene and primary spermatocytes and spermatids but not for type A, intermediate, or type B spermatogonia. Furthermore, Sertoli cells, endothelial cells, Leydig cells, and erythrocytes were not lysed by the antisera. These observations support the concept of a blood-testis barrier. Only after migration of spermatogonia to the luminal side of the barrier can autoantigenic molecules be synthesized and/or inserted into the plasma membrane of spermatogenic cells. Thus, the appearance of surface autoantigens offers a model system to study the synthesis of specific molecules which are inserted into the plasma membrane at a precise time during development.

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.

Antimicrobial Activity of Human EPPIN, an Androgen-Regulated, Sperm-Bound Protein with a Whey Acidic Protein Motif

Abstract The role of epididymal sperm-binding proteins in reproductive tract immunity is now well recognized in addition to their role in sperm maturation. Spermatozoa acquire forward motility and fertilizing ability during their passage through the epididymis, where they acquire a wide variety of proteins belonging to different classes. Previously, we demonstrated that EPPIN (epididymal protease inhibitor), an androgen-regulated, sperm-binding protein containing protease-inhibitory motifs, is expressed specifically in the testis and epididymis. In the present study, we investigated the antibacterial activity of EPPIN against Escherichia coli and the mechanism of antimicrobial action. EPPIN exhibited dose- and time-dependent antibacterial activity that was relatively insensitive to salt. However, EPPIN lost its antibacterial activity completely on reduction and alkylation of its cysteines, indicating the importance of disulfide bonds for its activity. EPPIN permeabilized the outer and inner membranes of E. coli, which is consistent with its ability to induce striking morphological alterations of E. coli membranes as shown by scanning electron microscopy. EPPIN did not cause disruption of eukaryotic membranes in the rat erythrocyte hemolytic assay. The present results indicate that EPPIN has a role in the innate immune system of human epididymis.

Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit

Using a monoclonal antibody as a highly specific probe and a seminal particle-free fraction of rabbit ejaculated spermatozoa, actin has been localized in the postacrosomal region of mature rabbit spermatozoa. The sperm actin has been extracted and identified on two-dimensional PAGE immunoblots as a single spot of pI = 5.45 and Mr = 43,000. Rabbit sperm actin is present in a nonfilamentous form and is not removed by removing the plasma membrane. Unlike mature spermatozoa, however, filamentous actin is present in spermatogenic cells, as determined by rhodamine phalloidin staining. Starting as diffusely distributed in spermatocytes, actin accumulates in the subacrosomal space and appears as a band in conjunction with the developing acrosome. This band lengthens throughout the spermatid stage and becomes continuous with the postacrosomal region staining in testicular spermatozoa. Actin may therefore function during spermatogenesis to both shape the acrosome to the nucleus and to anchor inner acrosomal membrane proteins.

Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae

The binding of mammalian spermatozoa to the eggs extracellular coat, the zona pellucida, is a complex process which culminates in species-specific penetration of the sperm to the egg plasma membrane. To investigate where on the spermatozoons surface the zona binding sites are located, whole rabbit zonae were labeled with FITC, heat solubilized and used to observe the surface binding patterns on live spermatozoa. Before the acrosome reaction the zona binding sites are located either over the entire head as well as the middle piece or alternatively in patches along the apical ridge of the head. After the acrosome reaction there is a 29% loss of fluorescence and the zona binding sites are present in the posterior aspect of the acrosomal region, the anterior postacrosomal region and the middle piece. These results demonstrate the presence of zona binding sites after the acrosome reaction which would account for the sperms ability to remain bound to the zona after the acrosome reaction. Further, we report for the first time that solubilized rabbit zonae pellucidae will induce the acrosome reaction in in vitro capacitated rabbit sperm whereas solubilized pig zonae pellucidae will not. Since rabbit sperm bind pig zonae, the induction and specificity of the physiological acrosome reaction must reside in the affinity of the binding rather than the binding itself.

Restriction of a sperm surface antigen's mobility during capacitation☆

Abstract Fluorescent Fab 1 [immunoglobulin G (IgG) fragment with antigen binding capacity from papain digestion of IgG] antibody fragments and globulin from antisera prepared against a single rabbit sperm surface membrane glycoprotein antigen (MGP) were used to study surface antigen mobility. Epididymal and ejaculated spermatozoa exhibited a redistribution of MGP surface antigen over the acrosomal region when labeled at 4°C and warmed to 37°C. Following in vivo capacitation, spermatozoa did not exhibit MGP surface antigen redistribution over the acrosome. The restricted mobility of this surface antigen implies a physiological change in plasma membrane fluidity, which may be a necessary preliminary to the acrosome reaction. Furthermore, it is suggested that the presence or absence of specific peripheral membrane proteins may control the positional relationship between mobile and nonmobile integral membrane components.

Modification of the sperm membrane during capacitation.

Over thirty years ago the first reports on capacitation were made 1, when sperm injected into the ovarian capsule of the rat or oviduct of the rabbit were observed not to be immediately capable of penetrating the egg. In the ensuing three decades research has focused on both the physiological and morphological aspects of this rather remarkable endogenous change of the spermatozoon. Numerous review articles have appeared3-s during the last ten years and the reader is referred to these for more extensive discussions and background material. This paper will focus on more recent research that clearly indicates that capacitation is a membrane phenomenon consisting of several distinct but related steps. Perhaps the key to an understanding of capacitation is the realization that it is imposed on the basic parameters of fertilization so that the sperm’s ability to fertilize the egg occurs at the proper time and place. A prerequisite of fertilization is the acrosome reaction 7 , 9 . lo which releases acrosomal enzymes, allowing the spermatozoon to penetrate the zona pellucida and eventually to fuse with the egg. Consequently, capacitation could control the timing of sperm-egg interaction via control of the acrosome reaction. In fact, the changes observed to be concomitant with capacitation are probably directly related to preparing for the fusion of the sperm plasma membrane and outer acrosomal membrane rather than for sperm-egg fusion. However, changes in the sperm’s equatorial and postacrosomal regions resulting from the acrosome reaction and/ or capacitation must still be seriously considered in any general description of capacitation since these regions are involved in sperm-egg fusion.

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.

Appearance of regional surface autoantigens during spermatogenesis: comparison of anti-testis and anti-sperm autoantisera.

Abstract Autoantisera against rabbit testes and rabbit ejaculated spermatozoa have been used to study the appearance of surface autoantigens during spermatogenesis. Two distinct subclasses of autoantigens have been identified: an early subclass which first appears on pachytene spermatocytes and a late subclass which first appears on differentiating spermatids. These spermatids are just beginning to demonstrate migration of the nucleus and overlying acrosomal cap to the cell periphery and changes in nuclear shape. Some autoantigens of the early subclass do not appear on spermatozoa, but those that do are predominantly found over the acrosomal region. Autoantigens of the late subclass are predominantly found over the postacrosomal and middle-piece regions of the spermatozoon. It is suggested that morphological constraints during spermiogenesis may be responsible for the regional localization of the two subclasses.