Major proteins of boar seminal plasma as a tool for biotechnological preservation of spermatozoa

Boar seminal plasma is a complex mixture of secretions from the testes, epididymides, and the male accessory reproductive organs which bathe the spermatozoa at ejaculation. The seminal plasma contains factors, mostly proteins, which influence the spermatozoa, the female genital tract, and the ovum. In boars, most of the proteins belong to the spermadhesin family and bind to the sperm surface. Spermadhesins are multifunctional proteins with a wide range of ligand-binding abilities to heparin, phospholipids, protease inhibitors and carbohydrates; the family can be roughly divided into heparin-binding (AQN-1, AQN-3, AWN) and non-heparin-binding spermadhesins (PSP-I/PSP-II heterodimer). These proteins have various effects promoting or inhibiting sperm functions including motility, oviduct binding, zona binding/penetration, and ultimately fertilization. The complexity of the environmental signals that influence these actions have implications for the uses of these proteins in vivo and in vitro, and may lead to uses in improving sperm storage.     

Immunohistochemical localization of spermadhesin AWN in the porcine male genital tract

Boar spermadhesin (AWN) is a 14-kDa multifunctional protein, attached to the surface of the spermatozoa and involved in sperm capacitation and zona pellucida binding. The cellular origin of AWN was previously unknown. Moreover, the region of the male genital tract in which AWN becomes attached to the surface of spermatozoa was also uncertain. By using monospecific polyclonal antibodies against AWN, the immunohistochemical distribution pattern of AWN epitopes has been investigated in tissue sections of the porcine male genital tract. Our study has revealed that AWN is synthesized in the rete testis and in the epithelium of the seminal vesicles. The latter are also the major contributors of seminal plasma AWN. In addition, immunoblotting analysis has shown that AWN is present on epididymal spermatozoa. Our results indicate that the cellular origin of spermadhesins is species-specific. The attachment of AWN to epididymal spermatozoa is probably important in developing the capacity for fertilization.     

The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa

AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface-associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate-recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein-chemical and mass spectrometric methods. AWN exists as two isoforms, AWN-1 and AWN-2, which differ in that AWN-2 is N-terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest-neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN-1 and AQN-3, two other boar spermadhesins.     

Buck (Capra hircus) genes encode new members of the spermadhesin family

Spermadhesins are the major proteins of boar seminal plasma and form a group of polypeptides probably involved in reproduction. In previous work, a member of the spermadhesin family from buck seminal plasma, called BSFP, was characterized by mass spectrometry and N-terminal sequencing. The present study aimed to clone and characterize the BSFP gene and investigate its expression along the genital tract using real-time polymerase chain reaction (PCR). The cDNAs of the seminal vesicle, testis, epididymis, bulbourethral gland, and ductus deferens were prepared from a buck. Following 3'- and 5'-end amplifications using seminal vesicle cDNA, we cloned and sequenced four highly similar (97-98%) nucleotide sequences encoding spermadhesins, which were named Bodhesin-1(Bdh-1), Bdh-2, Bdh-3, and Bdh-4. All deduced amino acid sequences contained the CUB domain signature and were 49-52% similar to boar AWN. Among the four Bdh amino acid sequences, Bdh-2 was the most similar to the BSFP N-terminal fragment. By using real-time PCR, it was verified specific amplifications for all Bdh in the seminal vesicle, testis, epididymis, and bulbourethral gland, with the exception of Bdh-2 in epididymis. The amplicons had a melting temperature and size of approximately 78 degrees C and 130 bp, respectively. Bdh expression was higher in the seminal vesicle when compared to the other tissues. The present work confirms that goat is the fifth mammalian species, after pig, cattle, horse, and sheep, in which spermadhesin molecules are found. To the best of our knowledge, this is the first report on buck spermadhesin genes using molecular cloning and expression profile.     

Analysis of the stability of the spermadhesin PSP-I/PSP-II heterodimer. Effects of Zn2+ and acidic pH

Spermadhesins are a family of 12-16 kDa proteins with a single CUB domain. PSP-I and PSP-II, the most abundant boar spermadhesins, are present in seminal plasma as a noncovalent heterodimer. Dimerization markedly affects the binding ability of the subunits. Notably, heparin and mannose 6-phosphate binding abilities of PSP-II are abolished, indicating that the corresponding binding sites may be located at (or near) the dimer interface. Pursuing the hypothesis that cryptic binding sites in PSP-I/PSP-II may be exposed in specific physiological environments, we examined the influence of Zn2+ and acidic pH on the heterodimer stability. According to near-UV CD spectra, the core native fold is preserved in the presence of physiological concentrations of Zn2+, a cation unusually abundant in boar seminal plasma. However, the thermostability of the heterodimer decreases significantly, as observed by CD and differential scanning calorimetry. The effect is Zn2+-specific and is reversed by EDTA. Destabilization is also observed at acidic pH. Gel filtration analysis using radioiodinated PSP-I/PSP-II reveals that dissociation of the heterodimer at low (nanomolar) protein concentrations is promoted by both Zn2+ and acidic pH. Although the integrity of the heterodimer in seminal plasma seems to be guaranteed by its high concentration, dissociation may be facilitated in the female genital tract because of dilution of the protein in the intraluminal fluids of the cervix and the uterus, and the acidic fluid of the uterotubal junction. Such a mechanism may be relevant in the regulation of uterine immune reactions.     

Gap junctional communication in the male reproductive system

Male fertility is a highly controlled process that allows proliferation, meiosis and differentiation of male germ cells in the testis, final maturation in the epididymis and also requires functional male accessory glands: seminal vesicles, prostate and corpus cavernosum. In addition to classical endocrine and paracrine controls, mainly by gonadotropins LH and FSH and steroids, there is now strong evidence that all these processes are dependent upon the presence of homocellular or heterocellular junctions, including gap junctions and their specific connexins (Cxs), between the different cell types that structure the male reproductive tract. The present review is focused on the identification of Cxs, their distribution in the testis and in different structures of the male genital tract (epididymis, seminal vesicle, prostate, corpus cavernosum), their crucial role in the control of spermatogenesis and their implication in the function of the male accessory glands, including functional smooth muscle tone. Their potential dysfunctions in some testis (spermatogenic arrest, seminoma) and prostate (benign hyperplasia, adenocarcinoma) diseases and in the physiopathology of the human erectile function are also discussed.     

Isolation of non-heparin-binding and heparin-binding proteins of boar prostate

Proteins of boar prostate secretion were separated by affinity chromatography on heparin-polyacrylamide to non-heparin-binding (H) and heparin-binding (H+) protein fractions. H- and H+ fractions were then subjected to RP HPLC. Elution profiles of H-and H+ fractions of prostate secretion were compared with those of seminal plasma and the amounts of corresponding proteins were compared. Besides, the isolated proteins were characterized by SDS-PAGE. In the H- fraction of prostate secretion, PSP I and PSP II spermadhesins and in the H+ fraction AQN 2 and AWN 1 spermadhesins were found in substantially lower amounts than in seminal plasma. On the contrary, beta-microseminoprotein was identified in abundant amounts both in H- and H+ fractions of boar prostate secretion. AQN 2 and AWN 1 spermadhesins were proved by their antibodies. Some seminal plasma proteins originating mainly in seminal vesicles could also be secreted by the prostatic gland. beta-Microseminoprotein was found to be produced mainly by the prostate.     

Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1

The mannose-binding capability of recombinant wild-type boar spermadhesin AQN-1 and of its site-directed mutants in the highly-conserved region around of the single glycosylation site (asparagine 50) of some spermadhesins, where the carbohydrate binding site has been proposed to be located, was checked using a solid-phase assay and a biotinylated mannose ligand. Substitution of glycine 54 by amino acids bearing an unipolar side chain did not cause significant decrease in the mannose-binding activity. However, amino acids with uncharged polar side chains or having a charged polar side chain abolished the binding of biotinylated mannose to the corresponding AQN-1 mutants. The results suggest that the higher surface accessibility of amino acids possessing polar side chains compared to those bearing nonpolar groups may sterically interfere with monosaccharide binding. The location of the mannose-binding site in AQN-1 appears to be topologically conserved in other heparin-binding boar spermadhesins, i.e., AQN-3 and AWN, but departs from the location of the mannose-6-phosphate-recognition site of PSP-II. This indicates that different spermadhesin molecules have evolved non-equivalent carbohydrate-binding capabilities, which may underlie their distinct patterns of biological activities.     

Zinc ions induce the unfolding and self-association of boar spermadhesin PSP-I, a protein with a single CUB domain architecture, and promote its binding to heparin

Spermadhesins are a family of seminal plasma proteins composed of a single CUB domain, which appear to be involved in various aspects of the fertilization process in pigs. PSP-I and PSP-II, the most abundant porcine spermadhesins, occur in seminal plasma as noncovalent heterodimers devoid of heparin-binding capability. Of note is the stability of this dimer, which is significantly affected by physiologically relevant conditions such as Zn2+ ions. Here, we show that PSP-I and PSP-II when separated appear to conserve the overall fold of the CUB domain observed in the crystal structure of the PSP-I/PSP-II heterodimer, as concluded from gel filtration, analytical ultracentrifugation, differential scanning calorimetry, and circular dichroism analyses. However, Zn2+ concentrations in the range of those found in boar seminal plasma induce the unfolding and self-association of PSP-I, apparently as a consequence of the exposure of hydrophobic core residues, whereas they have no effect on PSP-II. Remarkably, Zn2+-denatured and self-associated (but not structured monomeric) PSP-I is retained on a heparin column, resembling the behavior of free PSP-I and homologous spermadhesins of the heparin-binding fraction of boar seminal plasma, which also exhibit different aggregation states. Thus, the modulation of the structural organization and heparin-binding ability of PSP-I by Zn2+ might be a physiological phenomenon in seminal plasma.     

Detection of viral genomes of caprine arthritis-encephalitis virus (CAEV) in semen and in genital tract tissues of male goat

The aim of this study was to determine the infectious status of semen and genital tract tissues from male goat naturally infected with the caprine lentivirus. Firstly, polymerase chain reaction (PCR) was used to detect the presence of CAEV proviral-DNA in the circulating mononuclear cells, semen (spermatozoa and non-spermatic cells), and genital tract tissues (testis, epididymis, vas deferens, and vesicular gland) of nine bucks. RT-PCR was used to detect the presence of CAEV viral RNA in seminal plasma. Secondly, in situ hybridization was performed on PCR-positive samples from the head, body, and tail of the epididymis. CAEV proviral-DNA was identified by PCR in the blood cells of 7/9 bucks and in non-spermatic cells of the seminal plasma of 3/9 bucks. No CAEV proviral-DNA was identified in the spermatozoa fraction. The presence of CAEV proviral-DNA in non-spermatic cells and the presence of CAEV in the seminal plasma was significantly higher (p<0.01) in bucks with PCR-positive blood. Two of the three bucks with positive seminal plasma cells presented with at least one PCR-positive genital tract tissue. Proviral-DNA was found in the head (3/9), body (3/9), and tail (2/9) of the epididymis. In situ hybridization confirmed the presence of viral mRNA in at least one of each of these tissues, in the periphery of the epididymal epithelium. This study clearly demonstrates the presence of viral mRNA and proviral-DNA in naturally infected male goat semen and in various tissues of the male genital tract.     

Neuropeptide Y in the human male genital tract

Neuropeptide Y (NPY) was found in high concentrations in the male genital tract. NPY levels were highest in the seminal vesicles, prostate, corpus cavernosum and vas deferens, where large numbers of immunoreactive nerve fibres were detected. Considerable quantities were also found in the epididymis and spongiosum. Lower concentrations were found in the glans penis, testis and foreskin. The presence of a large number of nerves containing NPY suggest that this active neuropeptide may play a role in control of genital function.     

Distribution of Bandeiraea simplicifolia lectin binding sites in the genital organs of female and male mice

Fluorescein isothiocyanate labelled type I lectin from Bandeiraea simplicifolia (BSA-I) known for its specific binding to alpha-D-galactopyranosyl and 2-acetamido-2-deoxy-D-galactose groups, has been used to map the distribution of the lectin specific binding sites in the genital organs of female and male mice. In non-pregnant female mice, strong lectin reactivity was restricted to the epithelium of the distal oviduct, the cervix and vagina. In pregnant mice strong BSA-I reactivity was also noted in the epithelium of uterine glands from the time of implantation on day 5 onward. In the testis BSA-I bound selectively to sperm but did not react with other cells in the seminiferous tubules. In the proximal caput epididymis BSA-I reacted with the epithelial cells, the underlying basement membranes and the intraluminal sperm. The intraluminal contents of the seminal vesicles reacted strongly with the lectin. Our data thus show a widespread but selective distribution of BSA-I lectin binding sites in the male and female genital organs and altered lectin binding in the uterus during pregnancy.     

Isolation and partial characterisation of a protein from buck seminal plasma (Capra hircus), homologous to spermadhesins

Spermadhesins are a family of secretory proteins expressed in the male genital tract of pig, horse and bull. Their function and structure have been widely studied, especially those isolated from boar. However, there are no data concerning spermadhesins isolated from buck. Buck seminal plasma was collected and subjected to ion exchange chromatography on DEAE-Sephacel column followed by chromatography in a C18 column coupled to a HPLC system. The purification of the protein was determined by SDS-PAGE and MALDI-TOF analysis exhibiting a molecular mass of 12.5 KDa and showed to be structurally homologous to spermadhesins from boar and stallion.     

Histological study of the cloacal region and associated structures in the hedgehog tenrec Echinops telfairi

The present investigation is based on several careful dissections and on extensive series of histological sections. It has led us to the conclusion that adult male and female Echinops telfairi are in the possession of a cloaca which represents a primitive feature among mammals. This cloaca is a small, bowl-shaped pouch at the ventro-posterior end of the body. Intestinal, genital and urinary tract open into this cloaca. The opening of the intestinal tract into the cloaca is regulated by a sphincter muscle. In the female the genital and the urinary tract open into the urogenital sinus, a subcompartment of the cloaca. The cloaca of the lesser hedgehog tenrec is lined by a multilayered, non-keratinized squamous epithelium without skin glands. In a small transitory zone between the cloaca and the outer skin the epithelium changes into the keratinized, multilayered squamous epithelium of the epidermis with eccrine and holocrine glands as well as hairs. In addition, there is a distinct circular cloacal sphincter muscle, built up by cross-striated skeletal muscle tissue.

In the terminal parts of intestinal, urinary and genital tracts of male animals the following glandular structures were observed: prostate gland, Cowper's glands and strongly pigmented seminal vesicles; in female animals: the urethral and the Bartholin glands. Both males and females, in addition, possess (a) a cloacal gland, the excretory ducts of which open into the cloaca and (b) a pericloacal gland which is located in the adipose tissue on both sides of the cloaca; it presumably also opens into the cloaca.     

Monoclonal antibodies, immunofluorometric assay, and detection of human semenogelin in male reproductive tract: no association with in vitro fertilizing capacity of sperm

Semenogelin plays an important role in sperm clotting and is degraded into smaller fragments by prostate-specific antigen (PSA) during clot liquefaction. Semenogelin and its fragments inhibit sperm motility in vitro. We studied the expression of semenogelin I mRNA and its localization in various tissues of the male genital tract. We also studied semenogelin concentrations with respect to sperm parameters and the outcome of in vitro fertilization. Semenogelin protein was detected by immunohistochemical staining and semenogelin I mRNA was detected by Northern blot analysis in the seminal vesicles and ampullary part of the vas deferens, whereas specimens from the prostate, epididymis, testis, and the female genital tract were negative. Using monoclonal antibodies against semenogelin, an immunofluorometric assay was developed to measure semenogelin levels in seminal plasma and to evaluate possible correlations with sperm parameters and fertilization in vitro. No correlation was found between the semenogelin concentration and the volume of the ejaculate, sperm concentration, sperm motility, or in vitro fertilization rate. Semenogelin levels were positively correlated with the total protein concentration in seminal plasma, and there was an inverse correlation between the concentration of semenogelin and that of PSA. The levels of semenogelin appear to bear no relationship to the in vitro fertilization capacity of the spermatozoa.     

The complete primary structure of the boar spermadhesin AQN-1, a carbohydrate-binding protein involved in fertilization.

Gamete recognition and adhesion are essential steps in the complex process of fertilization. In mammals and in other species, increasing evidence indicates that carbohydrate-binding proteins on the sperm surface play a pivotal role as counter-receptors for certain oligosaccharide moieties attached to the oocyte zona pellucida glycoproteins. Although different sperm-associated zona-pellucida-binding proteins have been identified in a number of species, few of them have been isolated and structurally characterized. In this paper we report the primary structural characterization of AQN-1, a 12-kDa boar-sperm-associated carbohydrate-binding and zona-pellucida-binding protein. The molecular mass of AQN-1 was determined by time-of-flight plasma-desorption mass spectrometry. Determination of its amino acid sequence and location of disulphide bridges were accomplished by a combination of proteochemical and mass spectrometric methods. The primary structure of AQN-1 failed to show any significant similarity to the protein structures deposited with the Martinsried Institute for Protein Sequences data bank, indicating that it may belong to a novel protein family involved in fertilization. AQN-1 shares extensive structural, as well as functional, similarity with two other boar sperm zona-pellucida-binding proteins, AQN-3 and AWN, which we have recently characterized. To name this protein family, we have coined the term spermadhesin. Our data may be relevant for identification of spermadhesins in other species, and thus may contribute to a better understanding of the species-specific sperm-egg recognition mechanism.     

The role of stallion seminal proteins in fertilisation

Seminal plasma proteins are secretory proteins originating mainly from the epididymis and the accessory sex glands. They are involved in the remodelling of the sperm surface which occurs during sperm transit through the male genital tract and continues later at ejaculation. During this process, collectively called post-testicular sperm maturation, the spermatozoa acquire the ability to fertilise an egg. Seminal plasma proteins have been shown to contribute to early and central steps of the fertilisation sequence, e.g. the establishment of the oviductal sperm reservoir, modulation of capacitation and gamete interaction. The major equine seminal plasma proteins belong to three protein classes, which contain widely occurring protein modules. Fn-2 type proteins are characterised by two or four tandemly arranged Fn-2 modules and have been implicated in the modulation of sperm capacitation. Multiple members of the cysteine-rich secretory proteins (CRISP) have been identified in the male genital tract of a number of species. CRISP proteins have been shown to be involved in various functions related to sperm-oocyte fusion, innate host defense function and ion channel blockage. Spermadhesins occur only in ungulate species. Their carbohydrate- and zona pellucida-binding properties would suggest a role of these proteins in gamete recognition. The major proteins of equine seminal plasma have been isolated and characterised regarding their expression along the male genital tract, protein structure and their functions.     

Immunolocalization of angiotensin 1 converting enzyme in the human male genital tract by the avidin-biotin-complex method

Summary Immunoreactivity for Angiotensin 1 Converting Enzyme was investigated in a series of 12 fixed and paraffinembedded normal human genital tract specimens. The Avidin-Biotin-Complex immunoperoxidase method was used with overnight (12 h) incubation with a polyclonal antihuman kidney Angiotensin 1 Converting Enzyme antiserum. All tissues, including testis, different parts of epididymis, ductus deferens, prostate and seminal vesicles, demonstrated a staining pattern. Immunoreactivity was observed on the luminal surface of these epithelia especially on nonmotile stereocilia. An intracellular positivity was only observed in spermatids on the acrosomal cap. Besides, an immunologic identity of Angiotensin 1 Converting Enzyme located on the different epithelia of the human male genital tract, on the endothelial cells of vessels and on the proximal tubule brush border of the kidney was observed.     

Immunolocalization of angiotensin 1 converting enzyme in the human male genital tract by the avidin-biotin-complex method

Immunoreactivity for Angiotensin 1 Converting Enzyme was investigated in a series of 12 fixed and paraffin-embedded normal human genital tract specimens. The Avidin-Biotin-Complex immunoperoxidase method was used with overnight (12 h) incubation with a polyclonal antihuman kidney Angiotensin 1 Converting Enzyme antiserum. All tissues, including testis, different parts of epididymis, ductus deferens, prostate and seminal vesicles, demonstrated a staining pattern. Immunoreactivity was observed on the luminal surface of these epithelia especially on non-motile stereocilia. An intracellular positivity was only observed in spermatids on the acrosomal cap. Besides, an immunologic identity of Angiotensin 1 Converting Enzyme located on the different epithelia of the human male genital tract, on the endothelial cells of vessels and on the proximal tubule brush border of the kidney was observed.