Spermadhesins are a novel family of secretory proteins expressed in the male genital tract of pig, horse and bull. They are major products of the seminal plasma and have been found to be peripherally associated to the sperm surface. The structure and function of spermadhesins have been thoroughly investigated in the pig, which exhibits the greatest diversity of members: AWN, AQN-1, AQN-2, PSP-I and PSP-II and its glycosylated isoforms. They are multifunctional proteins showing a range of ligand-binding abilities, c.g. carbohydrates, sulfated glycosamino-glycans, phospholipids and protease inhibitors, suggesting that they may be involved in different steps of fertilization. Isolated porcine spermadhesins bind the zona pellucida glycoproteins in a cation-dependent manner with a Kd in a low micromolar range, and AWN, AQN-1 and AQN-3 display similar binding affinity for glyoproteins containing Galβ(1-3)-GalNAc and Galβ(1-4)-GlcNAc sequences in O-linked and N-linked oligosaccharides, respectively. During sperm passage through the epididymis AQN-3 and AWN have been shown to bind tightly to the sperm surface by interaction with the phospholipids of the membrane bilayer. At ejaculation the spermadhesins form a protective coat around the sensitive acrosomal region of the sperm head, thus possibly preventing premature acrosome reaction. During in vitro capacitation most of these aggregated sperm adhesins are lost, with the exception of phospholipid-bound spermadhesins. AWN and AQN-3 may now serve as a primary receptor for the oocyte zona pellucida, thus contributing to initial binding and recognition between sperm and egg. The amino acid sequence of spermadhesins does not show any discernible similarity with known carbohydrate recognition domains (CRD). However, they belong to the superfamily of proteins with a CUB domain with a predicted all-β structure. The crystal structure of the heterodimeric complex of the spermadhesins PSP-I/PSP-II has been solved, showing that the overall structure of both spermadhesins consists of a β-sandwich with five (parallell and antiparallel) β-strands. It is the first three-dimensional structure of a zona pellucida-binding protein and reveals the architecture of the CUB domain. The spermadhesins represent a novel class of lectins that may be involved in sequential steps of fertilization, at least in the pig.

Spermadhesins: A new protein family. Facts, hypotheses and perspectives

After mating, mammalian spermatozoa are transported to the lower oviductal isthmus. Spermatozoa are sequestered at the isthmus by attaching and interacting with oviductal epithelial cells, hence forming a sperm reservoir. In several mammalian species, specific carbohydrates mediate sperm‐ oviductal epithelial cell binding. A quantitative in vitro free cell bioassay was developed to investigate the involvement of carbohydrate recognition in pig sperm‐ oviductal epithelial cell interactions. This assay was validated. The sensitivity of the assay was such that it was possible to discriminate between different sperm concentrations and sperm‐oviductal epithelial cell co-incubation periods, spermatozoa with damaged plasma membranes and epithelial cells of non-reproductive origin. Optimal conditions were used to incubate spermatozoa and oviductal epithelial cells in the presence of six hexose sugars at concentrations of 0, 2, 10 and 50 mmol l‐1. A significant (P < 0.05) reduction in the binding of spermatozoa to the oviductal epithelium was detected with 2, 10 and 50 mmol maltose l ‐1 , 50 mmol lactose l ‐1 and 50 mmol mannose l‐1. These findings support the

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Carbohydrate mediation of boar sperm binding to oviductal epithelial cells in vitro.

The role of extrahepatic retinol binding protein in the mobilization of retinoid stores

Zona-binding inhibitory factor-1 (ZIF-1), a glycoprotein in human follicular fluid, reduces the binding of spermatozoa to the zona pellucida. ZIF-1 has a number of properties similar to those of glycodelin-A from human follicular fluid. The objective of this study was to compare the biochemical characteristics of these two glycoproteins. N-terminal sequencing and protease-digested peptide mapping showed that ZIF-1 and glycodelin-A have the same protein core. However, these glycoproteins differ in their oligosaccharide chains, as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin-binding ability, and isoelectric focusing. ZIF-1 inhibited spermatozoa-zona pellucida binding slightly more than did glycodelin-A and significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. Indirect immunofluorescence staining revealed specific binding of glycodelin-A and ZIF-1 to the acrosome region of human spermatozoa, where ZIF-1 produced a stronger signal than did glycodelin-A at the same protein concentration. These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin that potently inhibits human sperm-egg interaction. Future study on the function role of ZIF-1 would provide a better understanding of the regulation of fertilization in humans.

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Zona-Binding Inhibitory Factor-1 from Human Follicular Fluid Is an Isoform of Glycodelin

We have previously reported that Sak57 (for Spermatogenic cell/Sperm-associated keratin of molecular mass 57 kDa) is an acidic keratin found in rat spermatocytes, spermatids, and sperm. Sak57 displays conserved amino acid sequences found in the 1A and 2A regions of the alpha-helical rod domain of keratins in human, rat, and mouse. We now report indirect immunofluorescence, confocal laser scanning microscopy and immunogold electron microscopy data showing that Sak57 is associated with the microtubular mantie of the manchette, a transient microtubular structure largely regarded as formed by tubulin and microtubule-associated proteins. The immunocytochemical localization of Sak57 was detected with a polyclonal antiserum to a multiple antigenic peptide (MAP) containing an amino acid sequence known to be present in the 2A region of the alpha-helical rod domain. During spermiogenic steps 8-12, Sak57 immunoreactive sites were restricted to microtubular mantie of the manchette which encircles the spermatid nucleus during shaping and chromatin condensation. At later stages (spermiogenic steps 12-14), Sak57 immunoreactive sites in the spermatid head region disappeared gradually as specific immunoreactivity appeared along the already assembled axoneme of the developing spermatid tail. Immunogold electron microscopy confirmed the presence of Sak57 immunoreactivity among microtubules of the manchette and on outer dense fibers and the longitudinal columns linking the ribs of the fibrous sheath. Mature spermatids (spermiogenic step 19) displayed tails with an immunofluorescent banding pattern contrasting with the lack of Sak57 immunoreactivity in the head region. Results from this study suggest that, during early spermiogenesis, a microtubular-Sak57 scaffolding is associated with the spermatid nucleus during shaping and chromatin condensation. During late spermiogenesis, the dispersion of the manchette coincides with the progressive visualization of Sak57 in the paraaxonemal outer dense fibers and longitudinal columns of the fibrous sheath in the developing spermatid tail.

Sak57, an acidic keratin initially present in the spermatid manchette before becoming a component of paraaxonemal structures of the developing tail

We have previously reported the purification of rats testis galactosyl receptor, an equivalent to the Ca(2+)-dependent (C-type) minor variant of rat hepatic lectin-2/3 (RHL-2/3). We now report the purification of galactosyl receptor from rat sperm and its immunolocalization in the intact rat testis and sperm by polyclonal antibodies prepared using multiple antigen peptides (MAP) as immunogens. Two MAP antigens (designated 27-mer and 28-mer), corresponding to amino acid sequences of the carbohydrate-recognition domain (galactose) and adjacent Ca(2+)-binding sites of RHL-2/3, were used for immunization. Anti-RHL-2/3, anti-p27, and anti-p28 sera crossreacted with rat hepatocyte RHL-2/3 and its rat testis and sperm equivalent, galactosyl receptor, purified by chromatofocusing followed by galactose-Hydropore-EP affinity chromatography. Neither anti-p27 nor anti-p28 sera cross-reacted with the major hepatocyte variant, RHL-1. A RHL-1-equivalent was not detected in rat testis and sperm. Immunofluorescence studies demonstrated that anti-p27 and anti-p28 sera recognize galactosyl receptor sites at the Sertoli cell-spermatogenic cell interface and on the dorsal surface of the sperm head, overlying the acrosome. The characteristic crescent-shaped immunoreactive pattern in sperm was lost after induction of the acrosome reaction. Further studies should determine whether antisera to MAP antigens 27-mer and 28-mer, corresponding to specific protein motifs, can serve as immunological probes for examining cell-cell interaction events during spermatogenesis and at fertilization.

Rat sperm galactosyl receptor: purification and identification by polyclonal antibodies raised against multiple antigen peptides

Phospholamban (PLB) is a membrane protein implicated in the regulation of calcium ATP-ase activity in the sarcoplasmic reticulum (SR) of heart muscle cells [1]. It plays a key role in Ca2+ transport across the SR membrane and in cardiac contraction and relaxation processes [1]. PLB’s role in this regulatory activity can be explored through structural studies and through changes in structure dependent on phosphorylation activity. In order to provide a means for these studies to proceed, we have undertaken the synthesis and analysis of the C-terminal transmembrane portion of PLB. Automated chemical synthesis of PLB has previously been reported using the t-Boc synthesis strategy [2]. By employing an Fmoc based synthesis strategy and utilizing deprotection monitoring, we have been able to observe those regions of the sequence where the membrane domain of PLB exhibits difficulty.

Synthesis, Purification, and Analysis of the Transmembrane Domain of Phospholamban

Via UV monitoring on the 43 3A, the early onset of Fmoc-deprotection difficulty arises 7 to 15 residues after SPPS begins at the C-terminus of a peptide sequence [1]. Secondary structure takes shape and inhibits access of the growing chain-end to both deprotection and coupling reagents. The difficult deprotections of interest to us arise long after the SPPS process has begun, i.e. after 30 or 50 or more residues have been coupled, and they are likely signaling a different process within the peptide-resin environment, namely, tertiary structure [2].

Phillip W. Banda

Papers

Rat sperm galactosyl receptor: purification and identification by polyclonal antibodies raised against multiple antigen peptides

Synthesis, Purification, and Analysis of the Transmembrane Domain of Phospholamban

Late Deprotection Difficulties During SPPS Depend on the Preceding Sequence