Spermadhesin AQN1 is a candidate receptor molecule involved in the formation of the oviductal sperm reservoir in the pig

Sperm are stored in the isthmic region of the oviduct under conditions that maintain viability and suppress early capacitation steps until ovulation occurs. The initial contact between sperm and oviductal epithelium is mediated by carbohydrate-protein interactions. In the pig, the carbohydrate recognition system has been shown to involve oligomannosyl structures. The spermadhesins AWN and AQN1 are the dominant porcine carbohydrate-binding sperm proteins. The objective of this study was to demonstrate that AQN1 contributes to sperm binding to the oviductal epithelium. AQN1 showed a broad carbohydrate-binding pattern as it recognizes both alpha- and beta-linked galactose as well as Manalpha1-3(Manalpha1-6)Man structures, whereas AWN bound only the galactose species. Binding of ejaculated sperm to oviductal epithelium was inhibited by addition of AQN1 but not by AWN. Mannose-binding sites were localized over the rostral region of the sperm head. Flow cytometry showed that, under capacitating conditions, the population of live sperm was shifted within 30 min toward an increase in the proportion of cells with low mannose- and high galactose-binding. The loss of mannose-binding sites was accompanied by the loss of AQN1 in sperm extracts and the significant reduction in the sperm-oviduct binding. The oviductal epithelium was shown by GNA-lectin histochemistry and by SDS-PAGE and lectin blotting of the apical membrane fraction to express mannose components that could be recognized by AQN1. These results demonstrate that the sperm lectin AQN1 fulfils the criteria for an oviduct receptor in the pig and may play a role in the formation of the oviductal sperm reservoir.     

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.     

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.     

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.     

Separation, characterization and identification of boar seminal plasma proteins

Methods used for the isolation, separation and characterization of boar seminal plasma proteins are discussed, as well as techniques applied to study their binding properties. Attention is paid to interactions of these proteins with different types of saccharides and glycoconjugates, with membrane phospholipids, and to interactions between proteins. Boar seminal plasma contains different types of proteins: spermadhesins of the AQN and AWN families; DQH and PSP proteins belong to the most abundant. Some of these proteins are bound to the sperm surface during ejaculation and thus protein-coating layers of sperm are formed. Sperms coated with proteins participate in different types of interactions occurring in the course of the reproduction process, e.g. formation of the oviductal sperm reservoir, sperm capacitation, oocyte recognition and sperm binding to the oocyte.     

The effect of oestradiolum benzoicum and progesterone on AChE activity in the nerves of the female reproductive system of immature pigs

The present paper describes the effect of exogenous oestradiol and progesterone on the AChE activity in nerves of the ovary, oviduct, uterus, and vagina, and on morphological features of the AChE-positive nerves of these organs in sexually immature pigs. Studies were carried out on 30 sexually immature pigs, 37 to 43 kg in weight, at the age of 115 to 125 d, of the BWP (Big White Polish) race. The animals were divided into 4 groups. Group I (n = 6) constituted the control. Animals in group II (n = 8) received oestradiolum benzoicum. Those in group III (n = 8) were given progesterone, and in group IV (n = 8) oestradiol and progesterone. Oestradiol injections resulted in a 1.2-fold increase of AChE activity in the ovary, 1.3-fold in the oviduct, 1.7-fold in the uterine horn, and a 1.2-fold decrease of this activity in the cervix, while no differences were noted in the vagina. Progesterone injections did not affect AChE activity in the ovary, while a 1.1-fold increase was observed in the oviduct, 1.2-fold in the uterine horn and 1.1-fold in the cervix compared to the control. A 1.1-fold decrease of this activity was observed in the vagina. Joint injections of oestradiol and progesterone resulted in about a 1.4-fold increase of AChE activity in the ovary, 1.3-fold in the oviduct, 1.4-fold in the uterine horn, 1.6-fold in the vagina, while no changes were observed in the cervix. The results suggest the effect of the administered hormones upon AChE activity in the organs under study, mainly in the oviduct, uterine horn, and vagina. They also point to the possibility of a synergic effect of oestradiol and progesterone as regards to an increase of the activity.     

Comparative distribution of neuropeptide tyrosine-,vasoactive intestinal polypeptide-, substance P-immunoreactive,acetylcholinesterase-positive and noradrenergic nerves in the reproductive tract of the female rat

Summary Location, distribution and density of nerve fibers immunoreactive to neuropeptide tyrosine, vasoactive intestinal polypeptide and substance P were studied in the reproductive tract of the female rat and compared with acetylcholinesterase-positive (cholinergic) and noradrenergic nerves. Plexuses of all types of fibers were present in the vagina, uterine cervix, uterine horn and oviduct. In the tubular reproductive organs all of these types of nerve fibers appeared to innervate vascular and non-vascular smooth muscle and nearly all types of fibers formed plexuses subjacent to the epithelium lining the organs. Individual fibers of all classes appeared to innervate fascicles of smooth muscle in the mesometrium of the uterine horn. A few acetylcholinesterase-positive and substance P-immunoreactive fibers were present in the ovary but no vasoactive intestinal polypeptide-immunoreactive nerves were observed. Noradrenergic and neuropeptide tyrosine-immunoreactive nerves were numerous in the ovary where they were seen in the interstitial gland tissue and associated with follicles and blood vessels. It is suggested that these nerves may influence hemodynamic events and non-vascular smooth muscle in such functions as transport of sperm and ova and parturition. Substance P-immunoreactive nerve fibers are likely to be sensory fibers that could have roles in neurohormonal reflexes.     

Sperm binding glycoprotein (SBG) produces calcium and bicarbonate dependent alteration of acrosome morphology and protein tyrosine phosphorylation on boar sperm

The oviduct is a dynamic organ which modulates gamete physiology. Two subpopulations of sperm have been described in the oviduct of sows, a majority with normal appearance in the deep furrows and a minority, centrally located, and showing damaged membranes. Sperm-oviduct interaction provides the formation of a sperm storage and allows the selection of sperm with certain qualities. Pig (Sus scrofa) oviductal sperm binding glycoprotein (SBG) binds to sperm and exposes Gal beta1-3GalNAc. This disaccharide may be recognized by boar spermadhesin AQN1, which seems to be involved in sperm interaction with the oviduct. SBG is present at the apical surface of the epithelial cells that surround the lumen of the oviduct rather than at the bottom of the crypts. These characteristics imply it could be involved in sperm interaction with this organ. In this study, we evaluate the effect of SBG over boar sperm. We show that the presence of SBG produces alterations of the acrosome morphology of sperm only when they are incubated in capacitating conditions. SBG binds to the periacrosomal region of sperm undergoing capacitation. Its presence induces an increase on the tyrosine-phosphorylation of a polypeptide of apparent molecular mass 97 kDa, as occurs with a 95 kDa protein in other mammalian sperm upon acrosomic reaction. Altogether, these results suggest that SBG might be involved in sperm selection by alteration of the acrosome of sperm that have already begun the capacitation process when they arrive to the oviduct.     

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.     

Sperm-uterine interactions: a review.

The uterus of domestic animals, including the horse, has a dual role in the interaction of the uterus and sperm. On one hand, uterine contractions carry sperm toward the oviduct, and on the other hand the uterus eliminates excessive sperm. The selection of sperm for the small numbers of "good" cells that gain access to the oviduct and for the majority of sperm that will be destroyed takes place in the uterus. The sperm-uterine interaction works both ways; sperm and seminal plasma also have several effects on the uterus. Sperm and seminal plasma probably provoke uterine contractions. Sperm induce leukocytosis in the equine uterus by activating complement. Seminal plasma has immuno-suppressive effects in the uterus. The sperm-uterine interaction can be modified by the type of the inseminate: concentration, numbers, motility/viability of sperm, volume, and absence or presence of seminal plasma. In mares, relaxation of the cervix, myometrial contractions, lymphatic drainage, and stallion contact affect elimination of sperm.     

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.     

Gamma-aminobutyric acid in the genital tract of the rat during the oestrous cycle

The highest values of gamma-aminobutyric acid (GABA) in the genital tract of the rat at different stages of the oestrous cycle were found in the oviduct (3.5-7 micrograms/mg protein) and the lowest in the ovary (50-100 ng/mg protein). The values for uterus and vagina ranged between 80 and 150 ng/mg protein. GABA (10-30 ng/microliter) was also found in fluid in the ovarian bursa. At 11:00 h, on the day of oestrus, GABA content increased in the ovaries but values in the oviducts were maximal at 11:00 h on the day of pro-oestrus. Variations in GABA content of the vagina were also found. Uterine cervix or uterine horn showed no changes during the oestrous cycle. The GABA content was not uniform throughout the oviduct: the highest values were found in the portion next to the ovary. At 10 days after removal of the right oviduct, GABA values in the ovary and ovarian bursa fluid decreased on the operated side. At 1 month after surgery, the values in ovary were normal but the values in ovarian bursa fluid were still low, suggesting that the source of ovarian GABA was not the oviduct. The variations observed in the present paper suggest an involvement of GABA in reproductive physiology.     

Proteinase inhibitors in aggregated forms of boar seminal plasma proteins

Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I–V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5–25 kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (M_r>100 kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also β-microseminoprotein (β-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5–25 kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with M_r 8 kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown.     

Sperm penetration in vitro of pig follicular oocytes matured in culture.

Boar ejaculated and epididymal spermatozoa were preincubated in modified KRB or the isolated oviduct and uterine horn of an oestrous sow for 4.5-5 h at 37 degrees C before introduction into medium containing ovarian oocytes previously cultured for 24 h. At examination 17-20 h after insemination 60.6% of the total oocytes had reached at least the 2nd metaphase. The proportions of oocytes penetrated (i.e. enlarged sperm head or male pronucleus and corresponding sperm tail) were 0, 10.0 and 16.7% with ejaculated spermatozoa, and 3.3, 19.6 and 26.4% with epididymal spermatozoa preincubated in modified KRB, oviduct and uterus, respectively. Although the proportion of oocytes with morphologically normal male and female pronuclei was low (10/36 = 27.8%), the results suggest that boar spermatozoa can be capacitated in the isolated genital tract of an oestrous sow and that capacitation of epididymal is better than that of ejaculated spermatozoa.