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Regucalcin is a multi-functional protein having roles in calcium homeostasis as well as in anti-apoptotic, anti-prolific and anti-oxidative functions. Recently, it has been reported from the male reproductive tract, but its role in male reproduction needs further investigation; for which the native regucalcin of reproductive origin will be more appropriate. The gel exclusion chromatography followed by diethyl aminoethane cellulose chromatography and two-dimentional cellulose acetate membrane electrophoresis used for its purification are time consuming and less specific. Here, the regucalcin gene from buffalo testis has been cloned, expressed and purified in recombinant form, and subsequently used for raising hyper-immune serum. The Western blot of seminal vesicular fluid probed with anti-regucalcin polyclonal and monoclonal antibodies showed the presence of 28 and 34 kDa bands specific to regucalcin. Further, an affinity matrix has been prepared using anti-regucalcin polyclonal antibodies. An immuno-affinity chromatography method has been standardized to isolate regucalcin from seminal vesicular fluid. The initial complexity of the protein mixture in the seminal vesicular fluid has been reduced by a heat coagulation step. The purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band at 68 kDa that has been further confirmed as regucalcin by Liquid chromatography-mass spectrometry/mass spectrometry. The RGN purified from seminal vesicular fluid will be more appropriate for studying its possible role in male reproduction, especially sperm cell capacitation, hyperactivation, acrosome reaction and cryopreservation. The study can be applied in purifying regucalcin from different tissues or species with minor modifications in the methodology.

In honeybees, reproductive females usually mate early in their life with more than 10 males in free flight, often within 10 minutes, and then store male gametes for up to five years. Because of the extreme polyandry and mating in free flight special adaptations in males are most likely. We present here the results of an investigation of the protein content of four types of male reproductive glands from the Western honeybee (Apis mellifera) drone, namely seminal vesicles (secretion in ejaculate), as well as bulbus, cornua and mucus glands (secretions for the mating plug). Using high resolution and accuracy mass spectrometry and a combination of database searching and de novo sequencing techniques it was possible to identify 50 different proteins in total, inside all mentioned glands, except in the mucus gland. Most of the proteins are unique for a specific gland type, only one of them (H9KEY1/ATP synthase subunit O) was found in three glands, and 7 proteins were found in two types of glands. The identified proteins represent a wide variety of biological functions and can be assigned to several physiological classes, such as protection, energy generation, maintaining optimal conditions, associated mainly with vesicula seminalis; signaling, cuticle proteins, icarpin and apolipoproteins located mainly in the bulbus and cornua glands; and some other classes. Most of the discovered proteins were not found earlier during investigation of semen, seminal fluid and tissue of reproductive glands of the bee drone. Moreover, we provide here the origin of each protein. Thus, the presented data might shed light on the role of each reproductive gland.

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Male reproductive fitness is strongly affected by seminal fluid. In addition to interacting with the female environment, seminal fluid mediates important physiological characteristics of sperm, including capacitation and motility. In mammals, the male reproductive tract shows a striking degree of compartmentalization, with at least six distinct tissue types contributing material that is combined with sperm in an ejaculate. Although studies of whole ejaculates have been undertaken in some species, we lack a comprehensive picture of the specific proteins produced by different accessory tissues. Here, we perform proteomic investigations of six regions of the male reproductive tract in mice -- seminal vesicles, anterior prostate, dorsolateral prostate, ventral prostate, bulbourethral gland, and bulbourethral diverticulum. We identify 766 proteins that could be mapped to 506 unique genes and compare them with a high-quality human seminal fluid data set. We find that Gene Ontology functions of seminal proteins are largely conserved between mice and humans. By placing these data in an evolutionary framework, we show that seminal vesicle proteins have experienced a significantly higher rate of nonsynonymous substitution compared with the genome, which could be the result of adaptive evolution. In contrast, proteins from the other five tissues showed significantly lower nonsynonymous substitution, revealing a previously unappreciated level of evolutionary constraint acting on the majority of male reproductive proteins.

Dietary vitamin E supplementation plays a key role in animal reproduction by protecting germ cells from oxidative damage. Recently, alpha-tocopherol homologues (namely, beta-, gamma- and delta-tocopherol) have been the object of increasing research because of their peculiar nonantioxidant properties. We found that these tocol-derived compounds are not homogeneously distributed among semen components. Alpha-T was the major vitamin E homologue found in all semen fractions. Half of the total gamma (+beta)-T was found in germ cells, while more than 50% of total delta-T was preferentially accumulated in seminal plasma. The concentration of various tocol-derived compounds depended on their relative amounts in diet and the competition for saturable enzymes implicated in their metabolism. A higher concentration of delta-T in seminal plasma may be related to its more polar nature. However, the biological function of this compound in semen remains to be cleared. To our knowledge, this is the first study aimed at identifying alpha-tocopherol homologues in rabbit semen fractions.

The Ly-6 protein family refers to a group of glycophosphatidyl inositol-anchored membrane proteins with ten conserved cysteines. They are thought to be involved in cellular adhesion and signaling. Recently, a subfamily of secreted Ly-6 proteins has been identified. In the present study, we report a secreted Ly-6 protein, secreted seminal vesicle Ly-6 protein 1 (SSLP-1) purified from mouse seminal vesicles using a series of steps including ion-exchange chromatography on a diethylaminoethyl (DEAE)-Sephacel column, gel filtration on a Sephadex G-75 column, and ion-exchange HPLC on a sulfopropyl column. Further analysis demonstrated it to be a novel, previously unnamed, 17 kDa glycoprotein. N-glycosidase F treatment revealed a core protein with a molecular mass of 8720 Da. By Basic Local Alignment Search Tool Protein analysis, we found that SSLP-1 had ten conserved cysteine residues identical with other secreted Ly-6 proteins. The gene Gm191, which is located on chromosome 9, encodes SSLP-1. By Northern blotting with 21 different mouse tissues, we found that Sslp-1 mRNA was predominantly expressed in the seminal vesicle. Immunohistochemistry revealed SSLP-1 protein in the luminal fluid and mucosal epithelium of the seminal vesicles. The amount of Sslp-1 mRNA and SSLP-1 protein in the seminal vesicle was regulated by testosterone and correlated with the stage of animal maturation. The tissue-specific expression pattern suggests that SSLP-1 may play a physiological role in male mouse reproduction.

It is known that abnormal androgen dynamics in the tissues is a cause of androgen-dependent disorders. Investigation of tissue androgen levels could provide a clue to the elucidation of disorders. However, it is difficult to measure a trace amount of androgen in the tissues. We established a highly sensitive simultaneous quantification method of testosterone and dihydrotestosterone (DHT), which play the most important roles in the body among androgenic steroids in trace amounts, and investigated time course changes in testosterone and DHT levels in male accessory sex organs, serum, and seminal fluid after castration in rat models. In addition, changes in the testosterone/DHT ratio of male accessory sex organs and seminal fluid were observed. The simultaneous testosterone and DHT measurement method established by us was validated. Intra-assay variation and interassay precision and accuracy were all within +/-20%, and the quantification limits of testosterone and DHT were both 15.6 pg/g. With the use of this method, the testosterone and DHT levels in the prostate, seminal vesicles, and serum immediately after castration were similar to those previously reported. The testosterone and DHT levels were 350 pg/g and 605 pg/g, respectively; which showed dominance of DHT in seminal fluid, although it was not as marked as that in the male accessory sex organs. Androgens decreased with time after castration in the accessory sex organs, serum, and seminal fluid. In the prostate and seminal vesicles, testosterone and DHT decreased to about 50% and about 2% of the normal levels, respectively, 72 hours after castration. The serum levels were under the quantification limits 6 hours after castration and thereafter. In seminal fluid, the testosterone and DHT levels decreased to 49% and 35% of normal levels, respectively, 72 hours after castration. The testosterone/DHT ratio in the male accessory sex organs was lower in the prostate (0.06) than in the seminal vesicles (0.13) immediately after castration. In the seminal fluid, changes in the ratio were small compared with those in the accessory sex organs and serum. These results showed that our method was capable of measuring testosterone and DHT in very small amounts of samples such as prostate biopsy specimens, and it might provide a clue to the elucidation of the pathology of androgen-dependent disorders.

The objective of this study was to investigate the correlation between lead and cadmium with seminal vesicle and prostatic markers. Semen samples categorized into fertile and infertile were evaluated for the presence of lead and cadmium and biochemical markers in the seminal plasma. Associations between lead and fructose, acid phosphatase and gamma-glutamyl transpeptidase (gamma-GT) were observed. However, no such relationships were noticed for cadmium. It is concluded that lead may be one of the pollutants indirectly affecting semen quality by altering the functions of accessory sex glands.

High resolution 600 MHz 1H NMR spectroscopy was used to investigate the changes in biochemical composition of whole human seminal fluid (SF) and an artificial mixture of prostatic (PF) and seminal vesicle fluid (SVF). A variety of time-related biochemical changes were monitored simultaneously and non-invasively in SF, including enzymatic hydrolysis of phosphorylcholine to choline and polypeptides to amino acids. The fastest NMR-observable reactions in SF were the conversion of phosphorylcholine to choline (t1/2 approximately equal to 9 min) and uridine-5'-monophosphate (UMP) to uridine (t1/2 < 2 min). UMP has not previously been detected in SF because of its rapid hydrolysis. Artificial mixtures of separately obtained prostatic and SVF showed very similar biochemical changes to those observed in whole SF. Addition of EDTA to SF incubated for 2 min post ejaculation strongly inhibited peptide hydrolysis. Zn2+, present in whole SF was shown to be non EDTA-chelatable 2 min after ejaculation, whereas after 7 min, a singlet signal from the ethylenic protons of the Zn-EDTA2- complex was clearly observed which remained constant after 7 min. This indicates that soon after ejaculation (< 5 min) Zn2+ is immobilised in a macromolecular complex which is rapidly broken down by proteolytic enzymes, the released Zn2+ then being free to react with EDTA. Mg- and Ca-EDTA2- complexes were observed at 2 min and remained constant (at 1.4 and 2.1 mM, respectively) throughout the entire time course of the experiment. These studies cast new light on the time-related biochemical changes occurring in the post-ejaculatory SF which may have an important role in reproductive function.

Problem: The effect of seminal immunosuppressive component (ISF) on the primary and secondary antibody response, induced by soluble and/or corpuscular antigens, was evaluated in the sera obtained at different intervals before and after immunizations. The duration of the immune suppression induced by ISF treatment within the primary and secondary immunizations was also determined., Method of Study: The ability of the seminal immunosuppressive component to suppress the primary and secondary antibody response was evaluated by enzyme-linked immunoadsorbent assay (ELISA) in the sera of mice treated in vivo with the immunosuppressor before and after immunization with antigens. Likewise, the duration of the immune suppression induced by the seminal immunosuppressor administered before the primary and secondary immunizations was tested by ELISA with antisera to keyhole limpets hemocyanin., Results: Intravenous and rectal deposition of ISF led to a suppression of the primary and secondary antibody response to soluble and corpuscular antigens. The most effective suppression of the immune response was achieved in mice treated with immunosuppressor 3 days before the immunization with antigens. Also the secondary antibody response to the challenging antigen was significantly suppressed by ISF. The production of immunoglobulin G (IgG), IgM, and IgA to keyhole limpets hemocyanin was depressed for a relatively long period in mice treated with the immunosuppressor. The results indicated that the preexposure is needed for maximal immunosuppression of the primary antibody production. The treatment with ISF led to a prolonged immunosuppression but not to permanent tolerance to the challenging antigen., Conclusions: The in vivo deposition of semen may compromise some aspects of the immune system and may be an important factor in the development of viral and bacterial infections. The suppression of humoral immune response suggests potential uses of seminal immunosuppressor for the animal model study in the therapy of antibody-mediated diseases.

Purpose: Vascular permeability factor (VPF) is a potent inducer of microvascular hyperpermeability and stimulates endothelial cell growth and angiogenesis. This study examines expression of VPF in the male genital tract., Materials and Methods: Vascular permeability factor in seminal plasma was quantified by immunoassay. Vascular permeability factor mRNA and protein expression in tissue were studied by situ hybridization and immunohistochemistry., Results: All seminal plasmas studied contained high levels of VPF. Prostatic and seminal vesicle epithelium labeled strongly for VPF mRNA and protein., Conclusions: The strong expression of VPF in prostate and seminal vesicle and the high concentration of VPF in semen suggest an important role for this cytokine in male fertility.

The aspartic protease progastricsin (EC 3.4.23.3) is found in all parts of the mammalian stomach and has also been found extragastrically. In humans and monkeys, seminal fluid usually contains high concentrations of progastricsin. Using immunohistochemistry and in situ hybridization, we determined in this investigation the origin of seminal progastricsin to be the epithelia of both the prostatic gland and the seminal vesicles. In addition, Northern (RNA) blotting showed the presence of a 1.8-kb transcript in both tissues. Seminal progastricsin clones from two human prostatic gland cDNA libraries were isolated and sequenced. The combined sequence manifested only six nucleotide differences from the published genomic and gastric cDNA sequence. One conservative base substitution was present in both libraries. N-Terminal amino acid sequencing of all 43 residues of the seminal proenzyme and the first 34 residues of the mature enzyme yielded sequences identical to those deduced from cDNAs derived from both gastric and prostatic origin. The results obtained indicate that gastric and seminal progastricsin are products of the same gene and that the observed molecular differences between the zymogen from the two sources are probably due to posttranslational modifications.

Sperm were detectable by microscopic examination in human cervicovaginal lavage (CVL) specimens < or = 8 h after intercourse, whereas an enzyme-linked immunosorbent assay using the monoclonal antibody MHS-5, specific for a seminal vesicle antigen present in semen detected semen at a concentration of 1:2,500,000 (0.00004%) in CVL specimens and was positive < or = 24 h following unprotected intercourse. We recommend the routine use of semen detection assays to reduce false-positive results attributable to semen contamination in assays of pathogens, antibodies, or other factors in cervicovaginal secretions.

A cDNA expression library in lambda gt11 prepared from cDNA derived of seminal vesicle tissue was screened by means of monospecific rabbit anti-aSFP IgG. The sequence of clone pTF21, containing an insert of 668 bp comprised an open reading frame from position 7 to 411 terminated by two stop codons. From this sequence a protein of 134 amino acid residues can be deduced. The mature aSFP was preceded by a signal peptide of 20 amino acids length. The protein sequence contains no signal for N-glycosylation. The molecular weight calculated from the amino acid sequence is 12922 Da. The start codon ATG is part of the sequence AAGATGA which fulfills the criteria of an initiation consensus sequence. The coding region was followed by 257bp of the complete 3'-untranslated region (3'UTR). A putative polyadenylation signal AATAAT, although not of the standard type, is observed at position 650. According to Northern analysis, aSFP mRNA is expressed in seminal vesicle tissue, ampulla and weakly in tissue of epididymis, but not in testis or other bovine tissue. aSFP is specified by a single copy gene. Attempts to detect homologies to known protein sequences were not successful.

A new acidic seminal fluid protein (aSFP) was purified from bovine seminal fluid, using anion exchange chromatography and FPLC on MonoQ. The purified aSFP displays a pI of 4.8 and an apparent molecular weight of 14 kDa. Homogeneity of aSFP was demonstrated by FPLC and SDS-polyacrylamide gel electrophoresis. Monospecific anti-aSFP IgGs were employed to characterize aSFP in bovine seminal plasma and seminal vesicle secretion by immuno blot analysis. Proteinchemical characterization of aSFP included amino acid analysis as well as determination of 23 amino acid residues of the N-terminal sequence of aSFP. According to this sequence, aSFP appears to represent a hitherto unknown protein. aSFP stimulated cell division and progesterone secretion of bovine granulosa cells in vitro in a potent and dose dependent manner. aSFP appears to be a potent growth factor with effects on ovarian granulosa cells.

Three hundred eight-nine healthy, infertile patients were studied to determine the effects of inflammation on genital tract organs. Clinically silent inflammation was diagnosed by measuring polymorphonuclear granulocyte (PMN) elastase in semen. Seminal vesicle, prostate, and epididymis functions were assessed by measuring fructose, citric acid, and neutral alpha-glucosidase in semen. There was a significant relationship between high PMN elastase levels and low citric acid levels in semen; fructose and neutral alpha-glucosidase were not related to PMN elastase. Semen samples with increased PMN elastase levels (greater than 250 and greater than 1,000 ng/ml) showed a high incidence of pathologic citric acid levels (67% and 73%, respectively). These biochemical data indicate that the prostate is the main target in clinically silent male genital tract inflammation.