Purification, molecular characterization and ligand binding properties of the major donkey seminal plasma protein DSP-1.

Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the protein is heterogeneously glycosylated and also contains multiple acetylations, occurring in the attached glycans. Structural and thermal stability studies on DSP-1 employing CD spectroscopy and differential scanning calorimetry showed that the protein unfolds at ~43 °C and binding to phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases its thermal stability. Intrinsic fluorescence titrations revealed that DSP-1 recognizes lyso-phosphatidylcholine with over 100-fold higher affinity than PrC. Further, interaction of DSP-1 with erythrocytes, a model cell membrane, revealed that DSP-1 binding is mediated by a specific interaction with choline phospholipids and results in membrane perturbation, suggesting that binding of this protein to sperm plasma membrane could be physiologically significant.

Expression and purification of recombinant mouse CRISP4 using a baculovirus system.

Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield. Thus, defining the functions of CRISPs have been impeded. In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4.

Identification and functional analysis of bull (Bos taurus) cauda epididymal fluid proteome.

Despite recent advances in bull epididymal fluid proteome research, significant numbers of proteins secreted to epididymal lumen remain unidentified. The objective of this study was to expand the number of identified cauda epididymal fluid proteins in bulls and to contextualize them in a broader view of their mutual interactions and involvement in biological processes and pathways, to fully elucidate the ways in which epididymal fluid proteins are involved in storage and maturation of spermatozoa in epididymis. We collected postmortem cauda epididymal fluid from 6 mature Holstein Friesian bulls. We performed the identification of proteins using 2-dimensional electrophoresis coupled with MALDI mass spectrometry. Analysis of functionality and pathway involvement of identified proteins was performed using Ingenuity Pathway Analysis software. We identified a total of 189 epididymal fluid proteins, out of which 100 were newly identified in bull epididymal fluid. We have combined our data with 2 previously performed bull epididymal fluid proteome identifications, yielding 280 proteins total, and analyzed it. The main canonical pathways involving epididymal proteins were glycolysis, gluconeogenesis, protein ubiquitination pathway, nuclear factor-erythroid 2-related factor 2-mediated oxidative stress response, and farnesoid X receptor/retinoid X receptor activation. The main biological functions potentially performed by epididymal fluid proteins included carbohydrate metabolism, cellular growth and proliferation, cell death and survival, and small molecule biochemistry. Overall, our results have pointed out multiple novel pathways in bull epididymal fluid that might take part in various aspects of maturation and protection processes of epididymal spermatozoa.

SL15: A seminal plasma-derived lectin from the sperm of llama (Lama glama).

The oviductal sperm reservoir of South American camelids is formed when sperm bind to N-acetylgalactosamine (GalNAc) on the surface of oviductal epithelium. The aim of this study was to characterize the GalNAc-binding proteins on llama sperm, and to establish their origin. Sperm-adsorbed proteins were extracted with 0.5 M KCl in Hepes-balanced salts. Sperm-adsorbed and seminal plasma proteins were then subjected to ligand blotting for their GalNAc affinity, and the labeled bands were identified by mass spectrometry. Three proteins were identified in seminal plasma versus only one in the sperm-adsorbed population; SL15, a seminal lectin, was common to both. SL15 is a homologue of Zymogen granule protein 16, homolog B-like, which belongs to the Jacalin-related lectin family. This lectin is likely presented to sperm via seminal plasma since epididymal sperm are not capable of binding GalNAc, whereas ejaculated sperm does, and its transcript was enriched predominantly in the prostate and bulbourethral glands. This is the first report of a seminal lectin in South American camelids that originates in the male reproductive tract, and is probably involved in sperm reservoir formation.

Recombinant expression of porcine spermadhesin AWN and its phospholipid interaction: Indication for a novel lipid binding property.

AWN is a porcine (Sus scrofa domestica) seminal plasma protein and has been linked to a variety of processes related to fertilization. To acquire the protein in sufficient amount and purity for functional studies, we established its recombinant expression in E. coli and a three-step purification protocol based on different chromatographies. The test for AWN-phospholipid interaction revealed phosphatidic acid and cardiolipin as potential binding partners. As phosphatidic acid is surmised to play a role in cation-induced membrane destabilization and fusion events, we propose a membrane protective function of the presented binding affinity. Further studies with recombinant AWN will allow new insights into the mechanism of sperm-spermadhesin interaction and might provide new approaches for artificial reproduction techniques.

Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins.

Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as well as hydrogen bonding network between (NAG)3 and proteins were mostly conserved. LYZL5 in buffalo and other mammalian species contained additional 10-12 amino acid sequence at c-terminal that matched with ankyrin repeat domain-containing protein 27. Phylogenetic analysis indicated LYZL2 to be most ancient among all the LYZL proteins and that the evolution of LYZL proteins occurred through several gene duplications preceding the speciation of mammals from other vertebrates as distant as reptiles and amphibians.

Purification and characterization of CRISP-3 from human seminal plasma and its real-time binding kinetics with PSP94.

Cysteine-rich secretory proteins (CRISPs) have been postulated to have a role in male reproduction and prostate pathophysiology. Of the mammalian CRISPs, CRISP-3 levels in particular have been shown to be upregulated in prostate cancer. Efforts have been made to obtain highly pure CRISP-3 for gaining structure-function information of this protein. However, well characterized and highly pure protein is not available yet. CRISPs from snake venom have been purified using prostate secretory protein of 94 amino acids (PSP94) has been reported earlier. In the present study, CRISP-3 was purified to homogeneity from human seminal plasma using human PSP94-immnobilized affinity column. The molecular mass of the purified protein was determined by SDS-PAGE followed by immunoblotting and found to be ∼26kDa and ∼28kDa. The purity was further verified using MALDI-TOF MS analysis, where two peaks at m/z 25509 and 27715 were obtained. The lower molecular weight peak corresponds to the calculated molecular mass of CRISP-3 (∼26kDa); whereas the higher molecular weight peak was confirmed to be the glycosylated form (∼28kDa) from the deglycosylation experiment. Binding of PSP94 in increasing concentrations to purified CRISP-3 immobilized chip was further validated using surface plasmon resonance. The kinetics data suggested that purified CRISP-3 binds specifically and with high affinity to PSP94. In conclusion, a homogeneous preparation of highly pure CRISP-3 protein is obtained from human seminal plasma.

A 31-kDa seminal plasma heparin-binding protein reduces cold shock stress during cryopreservation of cross-bred cattle bull semen.

In the present study, a 31-kDa protein, purified from cattle bull seminal plasma heparin-binding proteins (SP-HBP), was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. Raw semen of six cross-bred bulls was treated with 31-kDa HBP before cryopreservation to observe its effect on motility, viability, hypo-osmotic swelling test, acrosome integrity, in vitro capacitation/acrosome reaction, and oxidative stress at pre-freeze and frozen-thawed phases of cryopreservation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 31-kDa protein eluted and purified from SP-HBP (separated on acrylamide gels) resulted in a single band of 40 kDa. In matrix-assisted laser desorption/ionization-time of flight analysis, 12 peptides were identified with matching significantly (P < 0.05) to interlukin-6 of bovine with a top score of 55. Addition of 25 µg/mL of fluorescein isothiocyanate-conjugated 31-kDa protein to raw semen and incubation at 37 °C for 20 minutes before cryopreservation resulted in its binding mainly to head region. Treatment of semen with 31-kDa HBP resulted in a significant (P < 0.05) average increase of 9.2%, 6.8%, and 11.7% and 5.5%, 6.5%, and 11.0% in motile, viable, hypo-osmotic swelling-responsive spermatozoa in six bulls at pre-freeze and frozen-thawed phases of cryopreservation, respectively. Percentage of spermatozoa with intact acrosomes nonsignificantly enhanced in the semen treated with 31-kDa HBP at both phases of cryopreservation. An average nonsignificant increase of 3.1% in in vitro capacitated and acrosome-reacted spermatozoa was obtained in semen supplemented with 31-kDa HBP. Addition of 31-kDa HBP also nonsignificantly reduced Malonadialdehyde (MDA) level by 10.7 and 19.3 µM/10(9) spermatozoa in prefrozen and frozen-thawed semen, respectively. The results obtained here indicate to conclude that treatment of cross-bred cattle bull semen with 31-kDa HBP protects the spermatozoa from cold shock effect by coating the sperm surface.

Individual and seasonal variations in the quality of fractionated boar ejaculates.

Reproductive seasonality has been shown to affect the quality of boar semen. In this study, effects of seasonal variations in the characteristics of spermatozoa and seminal plasma (SP) of fractioned ejaculates from individual boars have been investigated. Fractionated ejaculates, designated as fraction 1 (F1), fraction 2 (F2), and fraction 3 (F3), were collected from five mature boars during the autumn-winter (October through March) and spring-summer periods (April through September). A total of 10 fractionated ejaculates (F1, F2, and F3) were collected from each boar within each seasonal period. Assessments of the sperm quality characteristics included computer-assisted sperm analysis motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity, normal apical ridge acrosomes, and DNA fragmentation. Besides SDS-PAGE and densitometric analyses of the SP proteins, the antiperoxidant activity was monitored. There were marked differences in the sperm quality characteristics among the boars, except for sperm MMP. Distinct seasonal differences (P < 0.05) were observed in the ejaculate volume of F3 during the autumn-winter and spring-summer periods (107.78 ± 5.45 and 87.80 ± 4.75 mL, respectively). Significantly higher (P < 0.05) sperm concentration and the total number of spermatozoa in the fraction were observed during the autumn-winter period. Seasonal effects in MMP and plasma membrane integrity were manifested in significantly higher (P < 0.05) percentages of spermatozoa with functional mitochondria and intact plasma membrane during the autumn-winter period. However, the seasonal effects were less marked in either sperm normal apical ridge acrosomes or sperm DNA fragmentation. Sodium dodecyl sulfate-PAGE and densitometric analyses revealed marked variations in the protein composition of the SP profiles among the boars, regardless of the ejaculate fraction and seasonal period. Distinct seasonal variations, observed in the SDS-PAGE profiles, were associated with an abundance of protein fractions of low-molecular and high-molecular weight components, particularly during the autumn-winter period. There were wide variations in antiperoxidant activity in the SP among the boars, being significantly higher in the autumn-winter period, irrespective of the ejaculate fraction. It can be suggested that marked deterioration of the quality of fractionated ejaculates during the spring-summer period was probably caused by impaired reproductive function in the boar.

Isolation and characterization of an ovoinhibitor, a multidomain Kazal-like inhibitor from Turkey (Meleagris gallopavo) seminal plasma.

Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse.

While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

Evolutionary rate covariation identifies new members of a protein network required for Drosophila melanogaster female post-mating responses.

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks.

Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.

The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research.

Sperm and seminal fluid proteomes of the field cricket Teleogryllus oceanicus: identification of novel proteins transferred to females at mating.

Reproductive proteins are amongst the most evolutionarily divergent proteins known, and research on genetically well-characterized species suggests that postcopulatory sexual selection might be important in their evolution; however, we lack the taxonomic breadth of information on reproductive proteins that is required to determine the general importance of sexual selection for their evolution. We used transcriptome sequencing and proteomics to characterize the sperm and seminal fluid proteins of a cricket, Teleogryllus oceanicus, that has been widely used in the study of postcopulatory sexual selection. We identified 57 proteins from the sperm of these crickets. Many of these had predicted function in glycolysis and metabolism, or were structural, and had sequence similarity to sperm proteins found across taxa ranging from flies to humans. We identified 21 seminal fluid proteins, some of which resemble those found to be involved in postmating changes to female reproduction in other species. Some 27% of sperm proteins and 48% of seminal fluid proteins were of unknown function. The characterization of seminal fluid proteins in this species will allow us to explore their adaptive significance, and to contribute comparative data that will facilitate a general appreciation of the evolution of reproductive proteins within and among animal taxa.

Aggregation analysis of Con A binding proteins of human seminal plasma: a dynamic light scattering study.

Concanavalin A (Con A) binding fraction of human seminal plasma is vital as it shows decapacitating activity and contains proteins which have critical roles in fertility related processes. Con A binding proteins were isolated by lectin affinity chromatography. These proteins form high molecular weight aggregates at near physiological pH (7.0) as inferred by gel filtration. Aggregation analysis was performed by dynamic light scattering (DLS). DLS analysis was also performed at different pH values and in presence of various additives including NaCl, EDTA, cholesterol and sugars, such as d-glucose, d-fructose and d-mannose to identify their effect on aggregation size. The results indicate that degree of aggregation was highly reduced in presence of d-fructose, EDTA and at lower and higher pH values as depicted by lowering of hydrodynamic radii. This aggregation behaviour might be decisive for fertility related events with a suggestive role towards inhibition of premature capacitation.

ß-Nerve growth factor is a major component of alpaca seminal plasma and induces ovulation in female alpacas.

Ovulation in camelids is induced by an unidentified protein in the seminal plasma of the male termed 'ovulation-inducing factor'. This protein has been reported to be a 14-kDa protein under reducing conditions, which, when purified from seminal plasma, induces ovulation in llamas. The identification of this protein and investigation of its potential to induce ovulation in camelids may aid the development of protocols for the induction of ovulation. In the present study, alpaca seminal plasma proteins were separated using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the most abundant protein of 14 kDa was identified as ß-nerve growth factor (ß-NGF) by liquid chromatography mass spectrometry. Female alpacas (n = 5 per group) were given intramuscular injections of: (1) 1 mL of 0.9% saline; (2) 4 µg buserelin, a gonadotrophin-releasing hormone agonist; (3) 2 mL alpaca seminal plasma; or (4) 1mg human ß-NGF. Ovulation was detected by transrectal ultrasonography 8 days after treatment and confirmed by plasma progesterone concentrations. Ovulation occurred in 0%, 80%, 80% and 80% of animals treated with saline, buserelin, seminal plasma and ß-NGF, respectively. Treatment type did not affect the diameter of the corpus luteum, but plasma progesterone concentrations were lower in saline-treated animals than in the other treatment groups owing to the lack of a corpus luteum. The present study is the first to identify the ovulation-inducing factor protein in alpacas. ß-NGF successfully induces ovulation in alpacas and this finding may lead to new methods for the induction of ovulation in camelids.

Expression, purification and structural analysis of recombinant rBdh-2His6, a spermadhesin from buck (Capra hircus) seminal plasma.

Spermadhesins, a family of secretory proteins from the male genital tract of ungulate species, belong to the group of animal lectins. Spermadhesins have a prominent role in different aspects of fertilisation, such as spermatozoid capacitation, acrosomal stabilisation, sperm-oviduct interaction and during sperm-oocyte fusion. Proteins (spermadhesins) in buck seminal plasma were described. In the present study, bodhesin Bdh-2 cDNA present in buck seminal plasma was subcloned with the expression plasmid pTrcHis TOPO used to transform Escherichia coli Top10 One shot cells. The recombinant clones were selected by growth in 50 µg mL⁻¹ ampicillin-containing LB broth and polymerase chain reaction amplification. Recombinant rBdh-2His6 synthesis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by immunoblotting using monoclonal anti-His antibody. Production of rBdh-2 using low temperatures was not satisfactory. Greater production of rBdh-2 occurred with 1.5mM isopropyl ßd-thiogalactoside after 2h of induction. The method used to purify rBdh-2 was affinity chromatography on a His-Trap column following ion-exchange chromatography on a DEAE-Sephacel column. The secondary structure of the rBdh-2His6 was evaluated by spectral profile circular dichroism (CD). The prevalence of secondary structures like ß-sheets, with fewer unfolded structures and α-helices, was confirmed. The structure of rBdh-2His6 remained stable up to 35°C. However, significant structural changes were observed at temperatures higher than 40 °C related to a distortion of the CD spectrum.

Identification of a new mouse sperm acrosome-associated protein.

The binding of capacitated spermatozoa to the egg's extracellular coat and induction of acrosome reaction are necessary for successful fertilization in mammals. Biogenesis of acrosome is complicated, and not all proteins involved in this process are known. In this study, we have cloned a novel mouse gene, Spaca7, that is expressed exclusively in the testes. During the postnatal development, transcripts of the gene could be detected at a very low level in 18-day-old mouse testes and at a higher level in 21-day-old mouse testes and later, which corresponds to an expansion of round spermatids. In the stably transfected PT67 cells, SPACA7 fused with EGFP was predominantly localized in the Golgi apparatus. In transgenic mouse testes, the fusion protein was found in acrosome (starting from the first stages of acrosome formation in late pachytene spermatocytes and finally in spermatozoa isolated from caput and cauda of epididymis). Confocal microscopy studies revealed an intra-acrosomal not membrane-bound localization of SPACA7/EGFP, which suggests that the protein can be released during acrosome reaction and involved in fertilization. Acrosomal localization of endogenous SPACA7 protein was also found in human spermatozoa.

S100A7 is present in human sperm and a homologous pig sperm protein interacts with sperm-binding glycoprotein (SBG).

In sows, the oviductal sperm-binding glycoprotein (SBG), which binds to the periacrosomal region of boar sperm, has been shown to be involved in sperm selection. In this work, we isolated porcine sperm proteins that interact with SBG. One of them is identified as a homologue of human S100A7 (psoriasin). Anti-human S100A7 antibodies show that this homologous protein localises to the head of sperm. The isolation of a homologue of S100A7 based on affinity to SBG and its localisation at the head of sperm leads us to suggest that S100A7's homologous protein may be involved in the negative selection of sperm by SBG in pigs. Human S100A7 shows antibacterial properties, particularly over Escherichia coli, a species that has demonstrated deleterious effects on human sperm. We searched for S100A7 in human sperm and found that it is present and localises at the acrosomal region. Thus, we report the presence of S100A7 in human sperm and of a homologous protein in pig, with similar localisations. In humans, an antimicrobial role seems likely for psoriasin; in porcine sperm the studied protein binds to SBG suggesting a function in sperm selection, but an antimicrobial function cannot be ruled out.

Isolation and identification of Concanavalin A binding glycoproteins from human seminal plasma: a step towards identification of male infertility marker proteins.

Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma.