Seminal Vesicle Secretory Protein Research Articles

Glucagon-Like Peptide 1 Receptor Agonist Improves Renal Tubular Damage in Mice with Diabetic Kidney Disease.

PurposeThis study aims to investigate the renal protective effect of glucagon-like peptide 1 receptor agonist (GLP-1RA) on improving renal tubular damage in diabetic kidney disease (DKD) and to explore the potential mechanism of GLP-1RA on renal tubular protection.MethodsLong-acting GLP-1RA was used to treat DKD mice for 12 weeks. The label-free quantitative proteomic analysis of renal proteins was conducted to explore the differentially expressed proteins (DEPs) in the renal tissues of the control, DKD and GLP-1RA groups. The DEPs and markers of renal tubular injury were verified by qPCR in vivo and in vitro. The expression of glucagon-likepeptide-1 receptor (GLP-1R) in renal tubules was determined by immunofluorescence staining.ResultsGLP-1RA treatment significantly improved the tubular damages in kidney tissues of DKD mice and mTEC cells stimulated by high glucose (HG). Proteomics analysis revealed that 30 proteins in kidney tissue were differentially expressed among three groups. Seminal vesicle secretory protein 6 (SVS6) was the most differentially expressed protein in kidney tissues among three groups of mice. The expression changes of Svs6 mRNA in vitro and in vivo detected by qPCR were consistent with the results of proteomic analysis. Furthermore, reduction of Svs6 expression by SVS6 siRNA could attenuate HG-stimulated tubular injury in mTEC cells. Immunofluorescence staining also found that GLP-1R was widely expressed in renal tubules in vitro and in vivo.ConclusionGLP-1RA significantly improved renal tubular damage in DKD mice. SVS6 may be a potential therapeutic target for GLP-1RA in the treatment of DKD.

Elucidation of the protein composition of mouse seminal vesicle fluid.

The seminal vesicles are male accessory sex glands that contribute the major portion of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to conveying sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation, and influence the female reproductive tract to promote receptivity to pregnancy. Analysis of seminal vesicle fluid (SVF) composition by proteomics has proven challenging, due to its highly biased protein signature with a small subset of dominant proteins and the difficulty of solubilizing this viscous fluid. As such, publicly available proteomic datasets identify only 85 SVF proteins in total. To address this limitation, we report a new preparative methodology involving sequential solubilization of mouse SVF in guanidine hydrochloride, acetone precipitation, and analysis by label-free mass spectrometry. Using this strategy, we identified 126 SVF proteins, including 83 previously undetected in SVF. Members of the seminal vesicle secretory protein family were the most abundant, accounting for 79% of all peptide spectrum matches. Functional analysis identified inflammation and formation of the vaginal plug as the two most prominent biological processes. Other notable processes included modulation of sperm function and regulation of the female reproductive tract immune environment. Together, these findings provide a robust methodological framework for future SVF studies and identify novel proteins with potential to influence both male and female reproductive physiology.

Experimental autoimmune prostatitis: different antigens induction and antigen-specific therapy.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has uncertain etiology and lacks effective treatment. Autoimmunity is an important pathogeny, and experimental autoimmune prostatitis (EAP) models have long been used for studying CP/CPPS. This review presents the detailed current knowledge of EAP models based on evaluation criteria aspects to provide a tool for model selection in pathogenesis studies and therapeutic drug screening. We extensively searched the published literature on CP/CPPS and different antigen-induced EAP models focusing on the histopathology, clinical-related phenotypes, and biochemical indicators. We also cover the changes in the prostate function and other organs in EAP. Finally, we try to get some insights about antigen-based therapeutic approaches for CP/CPPS. Several inciting autoantigens were reported in EAP, including male accessory gland extracts, prostate extracts (PE), prostatic steroid-binding protein, prostatic spermine-binding protein (p25), prostatic acid phosphatase, seminal vesicle secretory protein 2, and T2 peptide. All of these models mimicked histological prostatitis, however only p25- and T2-induced models developed both pelvic pain and voiding behaviors. PE immunization is the most widely used method. Diminished fertility and mental health disorders can be found in PE model. Oral and intravenous T2 peptide have been studied for antigen-specific therapy and achieved preliminary progress in EAP models. PE-induced model is the most commonly used, while T2- or p25-model could serve as a promising CP/CPPS model. Antigen-specific therapy in CP/CPPS deserves further study.

Sexual dimorphism in brain transcriptomes of Amami spiny rats (Tokudaia osimensis): a rodent species where males lack the Y chromosome

BackgroundBrain sexual differentiation is sculpted by precise coordination of steroid hormones during development. Programming of several brain regions in males depends upon aromatase conversion of testosterone to estrogen. However, it is not clear the direct contribution that Y chromosome associated genes, especially sex-determining region Y (Sry), might exert on brain sexual differentiation in therian mammals. Two species of spiny rats: Amami spiny rat (Tokudaia osimensis) and Tokunoshima spiny rat (T. tokunoshimensis) lack a Y chromosome/Sry, and these individuals possess an XO chromosome system in both sexes. Both Tokudaia species are highly endangered. To assess the neural transcriptome profile in male and female Amami spiny rats, RNA was isolated from brain samples of adult male and female spiny rats that had died accidentally and used for RNAseq analyses.ResultsRNAseq analyses confirmed that several genes and individual transcripts were differentially expressed between males and females. In males, seminal vesicle secretory protein 5 (Svs5) and cytochrome P450 1B1 (Cyp1b1) genes were significantly elevated compared to females, whereas serine (or cysteine) peptidase inhibitor, clade A, member 3 N (Serpina3n) was upregulated in females. Many individual transcripts elevated in males included those encoding for zinc finger proteins, e.g. zinc finger protein X-linked (Zfx).ConclusionsThis method successfully identified several genes and transcripts that showed expression differences in the brain of adult male and female Amami spiny rat. The functional significance of these findings, especially differential expression of transcripts encoding zinc finger proteins, in this unusual rodent species remains to be determined.

Seminal vesicle secretory protein 7, PATE4, is not required for sperm function but for copulatory plug formation to ensure fecundity†.

Seminal vesicle secretions (SVSs), together with spermatozoa, are ejaculated into the female reproductive tract. SVS7, also known as PATE4, is one of the major SVS proteins found in the seminal vesicle, copulatory plug, and uterine fluid after copulation. Here, we generated Pate4 knockout (−/−) mice and examined the detailed function of PATE4 on male fecundity. The morphology and weight of Pate4−/− seminal vesicles were comparable to the control. Although Pate4−/− cauda epididymal spermatozoa have no overt defects during in vitro fertilization, Pate4−/− males were subfertile. We found that the copulatory plugs were smaller in the vagina of females mated with Pate4−/− males, leading to semen leakage and a decreased sperm count in the uterus. When the females mated with Pate4−/− males were immediately re-caged with Pate4+/+ males, the females had subsequent productive matings. When the cauda epididymal spermatozoa were injected into the uterus and plugged artificially [artificial insemination (AI)], Pate4−/− spermatozoa could efficiently fertilize eggs as compared to wild-type spermatozoa. We finally examined the effect of SVSs on AI, and observed no difference in fertilization rates between Pate4+/+ and Pate4−/− SVSs. In conclusion, PATE4 is a novel factor in forming the copulatory plug that inhibits sequential matings and maintains spermatozoa in the uterus to ensure male fecundity.

Testicular Dysgenesis Syndrome and Long-Lasting Epigenetic Silencing of Mouse Sperm Genes Involved in the Reproductive System after Prenatal Exposure to DEHP.

The endocrine disruptor bis(2-ethylhexyl) phthalate (DEHP) has been shown to exert adverse effects on the male animal reproductive system. However, its mode of action is unclear and a systematic analysis of its molecular targets is needed. In the present study, we investigated the effects of prenatal exposure to 300 mg/kg/day DEHP during a critical period for gonads differentiation to testes on male mice offspring reproductive parameters, including the genome-wide RNA expression and associated promoter methylation status in the sperm of the first filial generation. It was observed that adult male offspring displayed symptoms similar to the human testicular dysgenesis syndrome. A combination of sperm transcriptome and methylome data analysis allowed to detect a long-lasting DEHP-induced and robust promoter methylation-associated silencing of almost the entire cluster of the seminal vesicle secretory proteins and antigen genes, which are known to play a fundamental role in sperm physiology. It also resulted in the detection of a DEHP-induced promoter demethylation associated with an up-regulation of three genes apparently not relevant for sperm physiology and partially related to the immune system. As previously reported, DEHP induced an increase in mir-615 microRNA expression and a genome-wide decrease in microRNA promoter methylation. A functional analysis revealed DEHP-induced enrichments in down-regulated gene transcripts coding for peroxisome proliferator-activated receptors and tumor necrosis factor signaling pathways, and in up-regulated gene transcripts coding for calcium binding and numerous myosin proteins. All these enriched pathways and networks have been described to be associated in some way with the reproductive system. This study identifies a large new array of genes dysregulated by DEHP that may play a role in the complex system controlling the development of the male reproductive system.

Conformational Analysis of Rat Seminal Vesicle Secretory Protein 4, an Intrinsically Disordered Protein Having Interesting Pharmacological Properties

Rat seminal vesicle protein 4 (RSV4) is a member of the seminal vesicle protein family present in rats, 90 residues long. When secreted, it is involved in many functions related to the reproduction ranging from semen coagulation to sperm capacitation, but its fragments have also shown in vitro pharmacological properties such as anti-inflammatory and pro-coagulant activity, important in the cancer development. However, no three-dimensional model of this protein is yet available probably because of the presence of intrinsic disorder in the structure. In this article we report structural studies in solution of RSV4 by SEC (size exclusion chromatography) and CD (circular dichroism). In solution the monomer is highly flexible and poorly organized with the presence of highly fluctuating helical segments and, as also suggested by SEC, is classifiable as NU-PMG (natively unfolded pre-molten globule). The lack of a cooperative sigmoidal structural transition induced by thermal and GdmCl perturbation in monomer supports the poor structural organization expected for a NU-PMG conformational model. The structure of RSV4 monomer was modeled computationally and subjected to molecular dynamics simulations to study its conformational changes and energetic stability.

Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland

Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands.

Diethylstilbestrol (DES)-Stimulated Hormonal Toxicity is Mediated by ER α Alteration of Target Gene Methylation Patterns and Epigenetic Modifiers ( DNMT3A , MBD2 , and HDAC2 ) in the Mouse Seminal Vesicle

Background: Diethylstilbestrol (DES) is a synthetic estrogen associated with adverse effects on reproductive organs. DES-induced toxicity of the mouse seminal vesicle (SV) is mediated by estrogen receptor α (ERα), which alters expression of seminal vesicle secretory protein IV (Svs4) and lactoferrin (Ltf) genes.Objectives: We examined a role for nuclear receptor activity in association with DNA methylation and altered gene expression.Methods: We used the neonatal DES exposure mouse model to examine DNA methylation patterns via bisulfite conversion sequencing in SVs of wild-type (WT) and ERα-knockout (αERKO) mice.Results: The DNA methylation status at four specific CpGs (–160, –237, –306, and –367) in the Svs4 gene promoter changed during mouse development from methylated to unmethylated, and DES prevented this change at 10 weeks of age in WT SV. At two specific CpGs (–449 and –459) of the Ltf gene promoter, DES altered the methylation status from methylated to unmethylated. Alterations in DNA methylation of Svs4 and Ltf were not observed in αERKO SVs, suggesting that changes of methylation status at these CpGs are ERα dependent. The methylation status was associated with the level of gene expression. In addition, gene expression of three epigenetic modifiers—DNMT3A, MBD2, and HDAC2—increased in the SV of DES-exposed WT mice.Conclusion: DES-induced hormonal toxicity resulted from altered gene expression of Svs4 and Ltf associated with changes in DNA methylation that were mediated by ERα. Alterations in gene expression of DNMT3A, MBD2, and HDAC2 in DES-exposed male mice may be involved in mediating the changes in methylation status in the SV.Citation: Li Y, Hamilton KJ, Lai AY, Burns KA, Li L, Wade PA, Korach KS. 2014. Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle. Environ Health Perspect 122:262–268; http://dx.doi.org/10.1289/ehp.1307351

Estrogen Receptor-α Mediates Diethylstilbestrol-Induced Feminization of the Seminal Vesicle in Male Mice

Background: Studies have shown that perinatal exposure to the synthetic estrogen diethylstilbestrol (DES) leads to feminization of the seminal vesicle (SV) in male mice, as illustrated by tissue hyperplasia, ectopic expression of the major estrogen-inducible uterine secretory protein lactoferrin (LF), and reduced expression of SV secretory protein IV (SVS IV).Objectives: The present study was designed to evaluate the role of the estrogen receptor (ER) in this action by using ER-knockout (ERKO) mice.Methods: Wild-type (WT), ERα-null (αERKO), and ERβ-null (βERKO) male mice were treated with either vehicle or DES (2 μg/day) on neonatal days 1–5. These mice were divided into two groups: In the first group, intact mice were sacrificed at 10 weeks of age; in the second group, mice were castrated at 10 weeks of age, allowed to recover for 10 days, treated with dihydrotestosterone (DHT) or placebo, and sacrificed 2 weeks later. Body weights and SV weights were recorded, and mRNA expression levels of Ltf (lactoferrin), Svs4, and androgen receptor (Ar) were assessed.Results: In DES-treated intact mice, SV weights were reduced in WT and βERKO mice but not in αERKO mice. DES-treated WT and βERKO males, but not αERKO males, exhibited ectopic expression of LF in the SV. DES treatment resulted in decreased SVS IV protein and mRNA expression in WT males, but no effect was seen in αERKO mice. In addition, DES-treated βERKO mice exhibited reduced Svs4 mRNA expression but maintained control levels of SVS IV protein. In DES-treated castrated mice, DHT implants restored SV weights to normal levels in αERKO mice but not in WT mice, suggesting full androgen responsiveness in αERKO mice.Conclusions: These data suggest that DES-induced SV toxicity and feminization are primarily mediated by ERα; however, some aspects of androgen response may require the action of ERβ.

An aberrant prostate antigen–specific immune response causes prostatitis in mice and is associated with chronic prostatitis in humans

Chronic prostatitis is a common disease of unclear etiology and has no specific treatment. Mice deficient in the expression of the autoimmune regulator (Aire) gene, which are defective in thymic expression of self antigens and central tolerance, develop spontaneous prostatitis. In this study, we found that Aire-deficient mice developed spontaneous B and T cell immune responses to a prostate autoantigen, seminal vesicle secretory protein 2 (SVS2), which we believe to be novel. We show that thymic expression of this self antigen was Aire dependent. Moreover, prostatitis was induced in WT mice through immunization with SVS2, demonstrating that immunity to SVS2 was sufficient to induce prostatitis. The clinical relevance of this antigen was highlighted by our observation that patients with chronic prostatitis possessed specific autoantibodies against the human SVS2-like seminal vesicle protein semenogelin. These results provide direct evidence that spontaneous chronic prostatitis is an autoimmune disease and is regulated by both central and peripheral tolerance. Moreover, SVS2 and semenogelin are among the relevant autoantigens in mice and humans, respectively.

Gene expression in bovine seminal vesicles: Genexpression in der Bullenbläschendrüse

Seminal vesicle secretion contributes significantly to the proteins of bovine seminal plasma. The following proteins from bull seminal vesicle were isolated and characterized: major protein (PDC 109), the basic proteins BUSI II, RNAse BS1, protein P6 and seminal antimicrobial protein (SAP). Using antibodies against the proteins BUSI II, RNAse BS1, SAP and major protein, the seminal vesicle epithelium was identified as the source of the respective antigens. The biosynthesis of bovine seminal vesicle secretory proteins was studied by cell free translation of poly (A)-RNA from seminal vesicles and the respective mRNAs were characterized by cDNA cloning. Recombinant clones (103) of a cDNA library of bull seminal vesicle poly (A) + RNA were screened by colony hybridisation using radioactively labelled synthetic probes. The respective clone containing the longest cDNA insert was sequenced. In case of major protein the Mr of the 134 amino acid residue precursor polypeptide was 15,480 as deduced from direct mRNA sequencing. The precursor sequence of 25 amino acid residues has a hydrophobic character and very likely constitutes a signal peptide, directing the protein towards the secretory pathway. The deduced amino acid sequence contained no consensus sequence indicative of N-glycosylation.

Annexins Are Candidate Oviductal Receptors for Bovine Sperm Surface Proteins and Thus May Serve to Hold Bovine Sperm in the Oviductal Reservoir1

The sperm of eutherian mammals are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. The reservoir serves to maintain the fertility of sperm during storage and to reduce the incidence of polyspermic fertilization. Bovine sperm bind to the epithelium via seminal vesicle secretory proteins in the bovine seminal plasma protein (BSP) family, namely, PDC109 (BSPA1/A2), BSPA3, and BSP30K, which coat the sperm head. Our objective was to identify the receptors for bull sperm on the oviductal epithelium. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were subjected to affinity purification using purified BSPs bound to corresponding antibodies conjugated to Protein A agarose beads. Oviductal protein bands of approximately 34 and 36 kDa were eluted by EGTA from the beads and identified by tandem mass spectrometry as annexins (ANXAs) 1, 2, 4, and 5. Subsequently, antibodies to each of the ANXAs were found to inhibit sperm binding to explants of oviductal epithelium. Anti-ANXA antibodies labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of ANXAs in apical plasma membranes. Because fucose had been determined to be a critical component of the oviductal receptor, the ANXAs were immunoprecipitated from solubilized apical plasma membranes and were probed with Lotus tetragonolobus lectin to verify the presence of fucose. Thus, these ANXAs are strong candidates for the sperm receptors on bovine oviductal epithelium.

THE OVIDUCTAL SPERM RESERVOIR: OVIDUCTAL ANNEXINS AS RECEPTORS FOR BULL SPERM BSP PROTEINS

Mammalian sperm are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. In the bovine, we determined that seminal vesicle secretory proteins in the BSP family, namely PDC-109 (BSP A1/A2), BSP-A3, and BSP-30-kDa, coat the sperm head and enable sperm to bind to the epithelium. Our next objective was to identify the receptors for sperm on the oviductal epithelium. In order to capture oviductal ligands, a co-immunoprecipitation system was constructed using purified BSPs immobilized with corresponding antibodies covalently bound to Protein A agarose beads. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were passed through the beads packed into columns. Unbound proteins were washed from the beads and then bound proteins were eluted using EGTA, because sperm binding to oviductal epithelium is calcium-dependent. Protein bands of 34 and 38 kDa were detected in silver-stained SDS PAGE of the eluates from each of the three columns. Protein constituents of the bands were identified by tandem mass spectrometry as annexins (ANXA) 1, 2, 4, and 5. Subsequently, commercial antibodies to each of the ANXAs were found to inhibit binding of washed ejaculated sperm to explants of oviductal epithelium. Antibodies to ANXA 1, 2, 4, and 5 reduced binding by 76, 67, 70, and 84 %, respectively, compared to nonspecific IgGs (P<0.05). Anti-annexin antibodies also labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of annexins in apical plasma membranes, and 2-D blots demonstrated multiple forms of the annexins, suggesting post-translational modifications. Because fucose had been determined to be a critical component of the oviductal receptor for sperm, the ANXAs were immunoprecipitated from solubilized apical membranes and probed with fucose lectins to verify the presence of fucose. ANXA 1, 2, 4, and 5 exhibited staining with Anguilla anguilla lectin that was blocked by fucose. Annexins comprise a large multigene family of calcium-regulated phospholipid- and membrane-binding proteins whose diverse functions include cell adhesion, ion channel modulation, signal transduction, and modulation of membrane architecture. Thus, annexins are strong candidates for the sperm receptors on bovine oviductal epithelium. NRICGP Grant no. 2004-35203-14952 from USDA CREES. (platform)

Suppression effect of seminal vesicle autoantigen on platelet‐activating factor‐induced mouse sperm capacitation

Mammalian sperm gain the ability to fertilize an egg successfully by the capacitation process. An unregulated capacitation process causes sperm to undergo a spontaneous acrosome reaction (AR) and resulting in loss of their fertilization activity. Thus, functional sperm activation is tightly regulated by a capacitation and suppression (decapacitation) mechanism. Factors, such as platelet-activating factor (PAF) present in both sperm and the female genital tract, are able to stimulate sperm capacitation. Seminal plasma is thought to have the ability to suppress sperm capacitation; however, the regulatory mechanisms of seminal plasma protein on sperm capacitation are not well understood. Recently, we demonstrated that seminal vesicle autoantigen (SVA), a major seminal vesicle secretory protein, is able to suppress mouse sperm capacitation. To further study the suppression spectra of SVA on sperm capacitation, we investigated the effect of SVA on PAF-induced mouse sperm capacitation-related signals. Here, we demonstrate that SVA decreases the [Ca(2+)](i) to suppress the PAF's effects on [Ca(2+)](i), the cAMP level, protein tyrosine phosphorylation, and capacitation. The inhibition of PAF-induced protein tyrosine phosphorylation and capacitation by SVA can be reversed by cAMP agonists. Characterization of the interactions of SVA with PAF by TLC overlay and tryptophan fluorescence spectrum analyses indicates that SVA is capable of binding PAF with an apparent dissociation constant K(d) > 50 microM. Together with these results, we demonstrate that SVA deceases [Ca(2+)](i) and cross-talks with PAF-induced intracellular signals to regulate mouse sperm capacitation.

Cytokeratin‐20 and seminal vesicle secretory protein VI as possible marker proteins in urinary bladder preneoplastic lesions induced by N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine

A simple and non-invasive methods for the diagnosis of transitional cell carcinoma of the bladder are needed for the prevention of invasive tumor. A proteomic technology has recently been developed to facilitate protein profiling of biological mixtures. We tried to detect the marker proteins by proteomic approach during the initiation stages on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder carcinogenesis in rats. Ten rats in group A were given 0.05% BBN in drinking water for 12 weeks. Other 10 rats in group B with 10 rats were designated as a control group and were not given BBN. Whole urinary bladders of all rats were excised at 12 weeks from the beginning of the experiment. Conventional proteomics was performed with high resolution 2-dimension gel electrophoresis followed by computational image analysis and protein identification using mass spectrometry. A comparison of urinary bladder hyperplasia tissue with control tissue showed that five proteins; actin gamma2 propeptide, cytokeratin-20 (CK-20), proapolipoprotein, alpha2 actin (alpha-cardiac actin) and heat shock 27 kDa protein-1 were over-expressed in hyperplastic tissues. Three proteins; transcription factor myocardin, seminal vesicle secretory protein VI (SVS-VI) precursor and hypothetical protein RMT-7 were under-expressed in hyperplastic tissues. In our animal mode, BBN-induced urinary bladder mucosal hyperplasia resulted in an increase in the expression of five proteins and a decrease in the expression of three proteins. Of these, CK-20 and SVS-VI seem to be of particular interest. However other method such as Western blotting seems to be needed for confirmation of these proteins and more information on human bladder tissue is needed for clinical application.

Signals of seminal vesicle autoantigen suppresses bovine serum albumin-induced capacitation in mouse sperm

Capacitation is the prerequisite process for sperm to gain the ability for successful fertilization. Unregulated capacitation will cause sperm to undergo a spontaneous acrosome reaction and then fail to fertilize an egg. Seminal plasma is thought to have the ability to suppress sperm capacitation. However, the mechanisms by which seminal proteins suppress capacitation have not been well understood. Recently, we demonstrated that a major seminal vesicle secretory protein, seminal vesicle autoantigen (SVA), is able to suppress bovine serum albumin (BSA)-induced mouse sperm capacitation. To further identify the mechanism of SVA action, we determine the molecular events associated with SVA suppression of BSA’s activity. In this communication, we demonstrate that SVA suppresses the BSA-induced increase of intracellular calcium concentration ([Ca 2+] i), intracellular pH (pH i), the cAMP level, PKA activity, protein tyrosine phosphorylation, and capacitation in mouse sperm. Besides, we also found that the suppression ability of SVA against BSA-induced protein tyrosine phosphorylation and capacitation could be reversed by dbcAMP (a cAMP agonist).

Androgen-dependent expression, gene structure, and molecular evolution of guinea pig caltrin II, a WAP-motif protein.

We determined the cDNA and gene structures of guinea pig caltrin II, a unique member of the calcium transporter inhibitors containing a whey acidic protein (WAP) motif, and we established that it is a secretory protein with a potential 21-amino acid signal peptide in its N-terminus. Northern blot analysis and in situ hybridization histochemistry indicated that the expression of caltrin II is restricted to luminal epithelial cells in the seminal vesicles. Its message levels markedly decreased either after castration (and were restored by simultaneous administration of testosterone) or after treatment of the animals with estradiol, suggesting that the expression of caltrin II is androgen-dependent. Recombinant caltrin II had an elastase-inhibitor activity. Comparison of sequence between the caltrin II and related genes and their molecular evolutionary analyses revealed that caltrin II and seminal vesicle secretory proteins (SVPs) appear to be evolved from a common ancestor gene that is made by the fusion of semenogelin and trappin genes. Caltrin II and SVPs lost the transglutaminase substrate domain and the WAP motif, respectively, within a single exon, resulting in the exertion of different functions.

Isolation and characterization of gelatin-binding bison seminal vesicle secretory proteins.

Bovine seminal plasma (BSP) contains a family of major proteins designated BSP-A1/A2, BSP-A3, and BSP-30kDa (collectively called BSP proteins) that bind to sperm at ejaculation and potentiate sperm capacitation. Homologous proteins have been identified in stallion, boar, goat, and ram seminal plasma. We report here the isolation and characterization of homologous proteins from bison seminal vesicle secretions. Seminal vesicle secretory proteins were precipitated by adding cold ethanol and recovered by centrifugation. The precipitates were resuspended in ammonium bicarbonate, dialyzed, and lyophilized. Lyophilized proteins were dissolved in 0.05 M phosphate buffer (PB) and loaded onto a gelatin-agarose column. The unadsorbed proteins and adsorbed proteins were eluted with PB and 5 M urea in PB, respectively. The gelatin-adsorbed fraction was analyzed by SDS-PAGE and revealed the presence of four major proteins designated BiSV-16kDa, BiSV-17kDa, BiSV-18kDa, and BiSV-28kDa (BiSV: bison seminal vesicle proteins). Heparin-Sepharose chromatography allowed the separation of BiSV-16kDa, which did not bind heparin from other BiSV proteins, which bound heparin. Immunoblotting revealed that BiSV-16kDa cross-reacted with BSP-A3 antibodies, BiSV-17kDa and BiSV-18kDa cross-reacted with BSP-A1/-A2 antibodies, and BiSV-28kDa cross-reacted with BSP-30kDa antibodies. Radioimmunoassays indicated that approximately 25% of bison seminal vesicle total proteins are related to BSP proteins. The amino-terminal sequencing indicated that BiSV proteins share almost 100% sequence identity with BSP proteins. In addition, BiSV proteins bind to low-density lipoproteins isolated from hen's egg yolk. These results confirm that BSP protein homologs are present in mammalian seminal plasma and they may share the same biological role.

Localization of the Transglutaminase Cross-linking Site in SVS III, a Novel Glycoprotein Secreted from Mouse Seminal Vesicle

The nucleotide sequence of MpSv-1, a novel androgen-regulated gene exclusively expressed in mouse seminal vesicle, was analyzed to establish a 5'-flanking region of 2123 bp, three exons of 95, 765, and 330 bp, and two introns of 222 and 811 bp. The transcription unit is organized with the first exon encoding a signal peptide, and the second a secreted protein, whereas the third encompasses a 3'-non-translated nucleotide that shares common features of rapid evolving substrates of transglutaminase gene family. The protein sequence deduced from this gene contains 265 amino acid residues in which the central part, residues 116-145, is a region composed of five short tandem repeats, consisting of four amino acid residues, QXK(S/T), where X is an aliphatic amino acid residue. Among the mouse seminal vesicle secretory proteins that could be resolved by SDS-PAGE into seven major components, SVS I-VII, the antiserum against residues 77-109 of the MpSv-1-translated protein only reacted with SVS III. Matrix-assisted laser desorption/ionization-time of flight mass spectral analysis from a trypsin digest of SVS III supported this protein as derived from MpSv-1. SVS III was immunolocalized to the epithelium of both the primary and secondary folds of the seminal vesicle and the copulatory plug. All of mouse SVS I-III were proven to be substrates of transglutaminase and could be cross-linked readily after the enzyme reaction. The transglutaminase cross-linking site of SVS III was identified to be the tandem repeats of QXK(S/T) in the central part of this protein molecule.