Pattern recognition receptor-mediated innate immune responses in seminal vesicle epithelial cell and their impacts on cellular function†.

The seminal vesicles can be infected by microorganisms, thereby resulting in vesiculitis and impairment in male fertility. Innate immune responses in seminal vesicles cells to microbial infections, which facilitate vesiculitis, have yet to be investigated. The present study aims to elucidate pattern recognition receptor-mediated innate immune responses in seminal vesicles epithelial cells. Various pattern recognition receptors, including Toll-like receptor 3, Toll-like receptor 4, cytosolic ribonucleic acid, and deoxyribonucleic acid sensors, are abundantly expressed in seminal vesicles epithelial cells. These pattern recognition receptors can recognize their respective ligands, thus activating nuclear factor kappa B and interferon regulatory factor 3. The pattern recognition receptor signaling induces expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha (Tnfa) and interleukin 6 (Il6), chemokines monocyte chemoattractant protein-1 (Mcp1) and C-X-C motif chemokine 10 (Cxcl10), and type 1 interferons Ifna and Ifnb. Moreover, pattern recognition receptor-mediated innate immune responses up-regulated the expression of microsomal prostaglandin E synthase and cyclooxygenase 2, but they down-regulated semenogelin-1 expression. These results provide novel insights into the mechanism underlying vesiculitis and its impact on the functions of the seminal vesicles.

The antigens and autoantigens of the seminal vesicle. I. Immunochemical studies on guinea pig vesicular fluid.

Guinea pig vesicular fluid was characterized both biochemically and immunologically. Biochemical analyses showed this fluid to be homogeneous by ultracentrifugal analyses, revealing a single boundary with a sedimentation coeflicient of 1.5 S. In contrast, electrophoretic separation methods revealed six components, of which three were major components, of approximately equal proportions. They were termed I, II, and III. One of these components (II) was shown to be strongly antigenic in heteroimmunization, whereas components I and III failed to show any antigenicity, even after diverse attempts. This antigen (component II) was found to be highly tissue specific and species specific. Through procedures of isoimmunization, component II was also found to be immunogenic, giving rise (in male animals) to autoantibodies, A high proportion of injected guinea pigs showed positive skin tests and many revealed tissue lesions when the seminal vesicles were examined histologically. It is therefore concluded that experimental autoimmune disease of the seminal vesicle has been induced.

[The role of seminal vesicles in male fertility].

Seminal vesical secretion is important for male fertility. It affects semen coagulation, sperm motility, stability of sperm chromatin and suppression of the immune activity in the female reproductive tract.

Murine Herc6 Plays a Critical Role in Protein ISGylation In Vivo and Has an ISGylation-Independent Function in Seminal Vesicles.

ISG15 conjugation (ISGylation) to proteins is a multistep process involving interferon (IFN)-inducible UBE1L (E1), UbcH8 (E2), and ISG15 E3 ligases (E3s). Studies performed over the past several years have shown that ISGylation plays a pivotal role in the host antiviral response against certain viruses. Recent in vitro studies revealed that human Herc5 and mouse Herc6 are major ISG15 E3 ligases, respectively. However, the global function of Herc5/6 proteins in vivo still remains unclear. Here, we report generation and initial characterization of Herc6 knockout mice. Substantial reductions of ISGylation were observed in Herc6-deficient cells after polyinosinic-polycytidylic acid double-stranded RNA injection of mice or IFN treatment of cells. On the other hand, Herc6-deficient cells and wild-type (WT) cells had similar responses to IFN stimulation, Sendai virus (Z strain) infection, and vesicular stomatitis virus infection. These results indicate that Herc6 does not play a critical role in antiviral defense of these viral infections in mice. Interestingly, male Herc6-deficient mice showed seminal vesicle hypertrophy. No such problem was detected in WT and ISG15 activating enzyme Ube1L-deficient mice. These results suggest that in addition to promoting protein ISGylation, Herc6 has a novel and protein ISGylation-independent function in the male reproductive system.

Purification of low molecular weight forms of seminal vesicle specific antigen by immunoaffinity chromatography on bound monoclonal antibody MHS-5.

A method has been developed for purification of the low molecular weight forms of seminal vesicle specific antigen (SVSA). Pooled, liquified seminal fluid was fractionated by CM cellulose chromatography followed by two cycles of monoclonal antibody affinity chromatography. Analysis of the final product shows microheterogeneity of the purified immunoreactive peptides in the range of 9-12 kDa. In one run, from 1138 mg starting material, 2.78 mg of SVSA protein was obtained, a recovery of 0.24% of the total protein in the starting material. The purified material as assessed by scanning densitometry of Coomassie stained gels is 99% pure. These findings indicate that the three-step chromatographic method is useful for purifying the low molecular weight forms of SVSA.

Inhibition of complement-mediated cytotoxicity of antisera by fluid secreted by the seminal vesicle of the house mouse.

The fluid from the seminal vesicles of the house mouse inhibited complement-mediated cytotoxicity of antisera against both sperm and lymphocytes. This inhibition was not reduced by heating or by absorption with sperm. Fractionation of the seminal vesicle fluid on Sephadex G-100 columns revealed three peaks of inhibitory activity, one of which appeared in the void volume of the columns. This inhibitory action of the seminal vesicle fluid may protect sperm from immunological attack in the female reproductive tract. It could explain the observation that immunization of female house mice with sperm does not prevent pregnancy. The relationship between this activity in the seminal vesicles of house mice, which is directed against the cytotoxicity of antisera, and the inhibition of cell-mediated responses to sperm reported for human and bull semen have not been investigated.

Localization of human seminal plasma No. 7 antigen (Ferrisplan) in accessory glands of male genital tract and spermatozoa.

The establishment of a hybridoma (1C4) producing sperm immobilizing monoclonal antibody to human seminal plasma No. 7 antigen (HSP No. 7 Ag.) and the isolation of the pure antigen by immunoaffinity chromatography bound monoclonal antibodies have been reported previously. In the present investigation, HSP No. 7 Ag. has been termed 'Ferrisplan' and its distribution in male genital organs, spermatozoa and body fluids has been studied. The amount of Ferrisplan in the body fluids was determined by radioimmunoassay. Large amounts were detected in seminal plasma and milk, trace amounts in saliva, and none in the serum and urine. The concentration of Ferrisplan was highest in the seminal plasma of azoospermic patients and gradually decreased from oligospermia to normospermia. Using an immunofluorescent method with anti-Ferrisplan monoclonal antibody, strong staining was observed on the epithelial layers of human seminal vesicles, no staining on testes and bright staining on the post-nuclear cap and mid-piece segment of spermatozoa. These results indicate that Ferrisplan is excreted mainly from the seminal vesicle and adheres to the post-nuclear cap and mid-piece of the spermatozoa as a sperm-coating antigen.

Secretory immune system of the male reproductive tract: effects of dihydrotestosterone and estradiol on IgA and secretory component levels.

Immunoglobulin A (IgA) is present at mucosal sites of the body which are exposed to the external environment. In this study we evaluated the levels of IgA and its transport protein secretory component (SC) in organs of the male reproductive tract of both intact and castrate-hormone-treated rats. Our goals were to determine whether these proteins are present in the male reproductive tract and whether sex hormones can influence the amounts of IgA and SC in selected organs. We found that in intact animals, IgA was present in the prostate, epididymis, vas deferens and testis and that SC levels in the prostate were 22-fold greater than in these same organs. Dihydrotestosterone (DHT) and/or estradiol, when administered to castrate animals, dramatically increased the levels of prostatic SC. In contrast, the levels of IgA were only minimally affected. DHT administration also resulted in a significant increase in SC found in the seminal vesicles. These studies demonstrate that IgA and SC are present in the male reproductive tract of the rat. Further, they show that androgens and estrogens act at selected sites in the male reproductive tract to play an important role in maintaining SC levels and thereby suggest that these hormones influence the movement of IgA from tissues into secretions.

The glycosylphosphatidylinositol-anchored lymphocyte antigen CDw52 is associated with the epididymal maturation of human spermatozoa.

The CAMPATH-1 (CDw52) antigen is a small glycosylphosphatidylinositol (GPI) anchored glycoprotein with a mature peptide comprising only 12 amino acids. It is abundantly expressed on human lymphocytes and is an unusually good target for complement-mediated cell lysis. The immunosuppressive and lymphocyte-depleting effects of CAMPATH-1 antibodies are being tested in a variety of diseases. Here we show that the antigen is also expressed at a high level in the male reproductive system, being found in the epididymis, seminal vesicle, seminal plasma and on the surface of mature (but not testicular) spermatozoa. Its possible transfer from epithelial cells in the epididymis to maturing sperm may represent a novel method of acquisition of cell surface antigens. In the presence of human complement, CAMPATH-1 antibodies inhibited the motility of washed sperm. However, seminal plasma blocks antibody binding and can protect sperm from this cytotoxic effect.

Seminal vesicles: a source of trophoblast lymphocyte cross-reactive antigen.

Maternal recognition of allotypic trophoblast lymphocyte cross-reactive (TLX) antigens is proposed to be involved in immunologic acceptance of the allogeneic fetus. The presence of TLX antigens in seminal plasma suggests that sensitization can occur before fertilization and implantation. In this study, the origin of TLX antigens within the male reproductive tract was investigated. Analysis of split ejaculates and immunohistological examinations of male accessory gland tissues showed the luminal epithelium of seminal vesicles as the source of seminal plasma TLX antigens. This finding suggests that seminal vesicles may play a role in the immunology of human reproduction.

Antigens of immunoglobulin G-Fc receptor III in human male reproductive tract accessory glands.

We have documented the presence of soluble antigens of immunoglobulin (Ig)G-Fc receptor type III (FcRIII) in human seminal plasma that retain an affinity for the Fc fragment of IgG. The origin of these FcRIII antigens within the male reproductive tract was not known. By using polyclonal and monoclonal antibodies directed against different epitopes on FcRIII molecules, we demonstrated FcRIII reactivity in human prostate and seminal vesicle epithelial cells as well as in their glandular secretions. The FcRIII monoclonal antibody reactivity was removed by absorption with either seminal plasma or polymorphonuclear leukocytes that express FcRIII. Absorption of FcRIII monoclonal antibody with polymorphonuclear leukocytes also removed the reactivity with seminal plasma and vice versa. These data show for the first time that male accessory glands are a source of soluble FcRIII antigens.

The use of a seminal vesicle specific protein (MHS-5 antigen) for diagnosis of agenesis of vas deferens and seminal vesicles in azoospermic men.

Azoospermia is the cause of infertility in 8% of infertile male patients. Ten percent of those patients suffer from agenesis of the seminal vesicle (SV) and vas deferens (VD) agenesis. Currently, the diagnosis of SV and VD agenesis is based on low semen volume, low pH, and low fructose content of the seminal fluid of azoospermic men who have normal serum gonadotropins. In this study, an SV-specific sperm-coating antigen, the MHS-5 antigen, was used as a marker for the presence of SVs. The SV-specific protein (SVSP), MHS-5, was present in the control group but was not found in any of the seven samples from azoospermic men with proven agenesis of SV and VD. Another semen component, the prostate-specific antigen (PSA), whose presence in the semen is not influenced by the SV and VD agenesis, was found in both the study and the control groups. Its presence ruled out the possibility of azoospermia due to ejaculatory duct obstruction. The absence of MHS-5 antigen in seminal fluid can be used as a tool for a reliable diagnosis of agenesis of SV and VD in azoospermic men.

Immunologic reactions after experimental cryosurgery to the reproductive system of the male rabbit. I. The humoral response.

Single cryoinjuries to the accessory reproductive organs and to the gonads of normal adult male rabbits elicit antibody responses in 35.8 and 11.9 per cent of the cases, respectively. The antibodies display agglutinating and precipitating properties against extracts of the organ that underwent the freezing injury, but do not react in the presence of extracts of other organs of the rabbit. Repeated cryoinjuries raise both the percentage of reacting animals (up to 100 per cent after four successive cryostimulations, in some cases) and the titer of the humoral responses, which, in addition, endure longer in blood than do responses to single stimuli. These humoral phenomena are the expression of immunologic reactions mounted by the host animals against "self" components, regarded, however, as "nonself" by their surveillance apparatus.

Function of seminal vesicles and their role on male fertility.

The present review has been designed to update the recent developments on the function of seminal vesicles and their role on male fertility. It is indicated that the true corrected fructose level is a simple method for the assessment of the seminal vesicular function. Measurement of seminal fructose used universally as a marker of the seminal vesicle function is not an appropriate approach due to its inverse relationship with the sperm count. The true corrected fructose defined as [log. motile sperm concentration] multiplied by [seminal fructose concentration] has been shown to be a better marker of the seminal vesicle function. Seminal vesicular secretion is important for semen coagulation, sperm motility, and stability of sperm chromatin and suppression of the immune activity in the female reproductive tract. In conclusion, the function of seminal vesicle is important for fertility. Parameters as sperm motility, sperm chromatin stability, and immuno-protection may be changed in case of its hypofunction.

Immunolocalization of the boar seminal immunosuppressive fraction infused via uterus on the lymphocytes populating mouse genital tract tissues.

Intrauterine deposition of the immunosuppressive fraction from boar seminal vesicle fluid (ISF) led to a suppression of antibody response to soluble and corpuscular antigens in mice. By means of an immunofluorescent method using specific monoclonal antibody, ISF was detected on the membranes of white blood cells and splenocytes of mice subjected to intrauterine treatment from the third day to the thirteenth day after its deposition. ISF was also detected on the lymphocytes populating the mucosal tissues of vagina, cervix, oviduct and uterus from day 1 to 13 after its intrauterine administration. The antibody to soluble and corpuscular antigens was inhibited in the mice treated with ISF, but after the cessation of the ISF application, a normal immune response was restored within 40 days. Sandwich immunosorbent assay revealed that intrauterine infusion of ISF decreased significantly the concentration of IgG and IgM in the sera of immunized mice both after the primary and the secondary immunizations. These findings indicate that the intrauterine infusion of semen may influence the immune defense reactions and may be an important factor in the development of viral and bacterial infections of the female reproductive tract.

H-2 associated differences in the weight of some androgen influenced organs in (C57BL/10ScSnPh X AKR/J) F2 individuals.

We have followed the possible influence of H-2 system on the differences in the relative weights of testes, vesicular gland and thymus in animals of a segregating (B10 X AKR) F2 generation. H-2b/H-2b male mice had significantly higher values of relative vesicular gland weight and significantly lower values of relative testes weight than H-2k/H-2k males. No differences in the relative thymus weight were found between these two groups of animals. The differences are caused by a complex effect of the H.2 associated genetic factor(s) because they influence simultaneously the body weight and the organ weights, the two indicators being mutually dependent to some extent.

Immunoglobulin containing cells in normal and inflamed accessory sex glands of bulls.

The peroxidase-antiperoxidase (PAP) technique was used to identify cytoplasmic immunoglobulins in the accessory sex glands of 15 normal bulls and 13 bulls with inflammation of the ASG. Immunoglobulin containing cells (ICC) of the types IgA, IgM, total IgG, IgG1 and IgG2 were measured and their percentages expressed. In accessory sex glands from normal bulls, IgA containing cells were the most frequent in prostate and bulbourethral glands (86.7% and 86.1%, respectively of all ICC present) whereas in the ampulla, IgG containing cells comprised 78.6% of the ICC. IgG1 and IgG2 containing cells were present in all the accessory sex glands in approximately equal numbers. Frequencies of IgM containing cells in the ampulla, prostate and bulbourethral glands were 6.3%, 4.0% and 3.7%, respectively. Although all isotypes of ICC were present in the seminal vesicle, the very low number precluded accurate quantification. In inflamed ampulla, seminal vesicle, bulbourethral gland and colliculus seminalis, IgG containing cells were the most frequent ICC with values of 66.2%, 83.0%, 69.0% and 53.5%, respectively; IgA containing cells were the second in prevalence with values of 21.5%, 10.3% 19.3% and 40.5%, respectively. The contribution of ICC to the locally protective immunoglobulins in accessory sex gland secretions is discussed.

[Effect of pyospermia on fertility. Studies by selective leukocyte staining, elastase determination and routine sperm parameters]. / A pyospermia hatása a fertilitásra. Vizsgálatok szelektív fehérvérsejt festéssel, elasztáz meghatározással és hagyományos ondóparaméterekkel.

The effect of cellular immune response on male fertility was investigated. 92 patients--attending the out patient practice of our Andrological department and 15 patients undergoing vasectomy for sterilisation purpose--were included in this study. For measuring the cellular immune response granulocyte concentration and PMN-elastase level were investigated in ejaculate, former according to WHO manual, and PMN-elastase concentration by ELISA technic. Elastase is a very sensitive non organ specific marker of inflammation. Pathological granulocyte concentration was present in 10.8%, slightly elevated level in 4.3% of cases, totally in 15.1%. Pathological elastase level was found in 17.4%, slightly elevated in 16.3%, totally in 33.7% of cases. High level of correlation was present between the two parameters of inflammation r = 0.695. The incidence of the two parameters in fertile and subfertile patients groups was not significant. Conventional spermaparameters (cell concentration/ml, motility %, intensive motility %, morphology %) were significantly different in the leucospermic and non leucospermic patient groups, except cell concentration. (P = 0.06, P = 0.04, P = 0.03, P = 0.001). The incidence of high elastase level was very similar P = 0.07, P = 0.005, P = 0.03, P = 0.005. As conclusion of our investigation we conclude that cellular response could have a negative influence on male fertility as "side-effect" of the cytotoxic influences of immune defense.

Seminal, clinical and colour-Doppler ultrasound correlations of prostatitis-like symptoms in males of infertile couples.

'Prostatitis-like symptoms' (PLS) are a cluster of bothersome conditions defined as 'perineal and/or ejaculatory pain or discomfort and National Institutes of Health-Chronic Prostatitis Symptom Index (NIH-CPSI) pain subdomain score ≥4' (Nickel's criteria). PLS may originate from the prostate or from other portions of the male genital tract. Although PLS could be associated with 'prostatitis', they should not be confused. The NIH-CPSI is considered the gold-standard for assessing PLS severity. Although previous studies investigated the impact of prostatitis, vesiculitis or epididymitis on semen parameters, correlations between their related symptoms and seminal or scrotal/transrectal colour-Doppler ultrasound (CDU) characteristics have not been carefully determined. And no previous study evaluated the CDU features of PLS in infertile men. This study was aimed at investigating possible associations among NIH-CPSI (total and subdomain) scores and PLS, with seminal, clinical and scrotal/transrectal CDU parameters in a cohort of males of infertile couples. PLS of 400 men (35.8 ± 7.2 years) with a suspected male factor were assessed by the NIH-CPSI. All patients underwent, during the same day, semen analysis, seminal plasma interleukin 8 (sIL-8, a marker of male genital tract inflammation), biochemical evaluation, urine/seminal cultures, scrotal/transrectal CDU. PLS was detected in 39 (9.8%) subjects. After adjusting for age, waist and total testosterone (TT), no association among NIH-CPSI (total or subdomain) scores or PLS and sperm parameters was observed. However, we found a positive association with current positive urine and/or seminal cultures, sIL-8 levels and CDU features suggestive of inflammation of the epididymis, seminal vesicles, prostate, but not of the testis. The aforementioned significant associations of PLS were further confirmed by comparing PLS patients with age-, waist- and TT-matched PLS-free patients (1 : 3 ratio). In conclusion, NIH-CPSI scores and PLS evaluated in males of infertile couples, are not related to sperm parameters, but mainly to clinical and CDU signs of infection/inflammation.

Innate immunity in the male genital tract: Chlamydia trachomatis induces keratinocyte-derived chemokine production in prostate, seminal vesicle and epididymis/vas deferens primary cultures.

Chlamydia trachomatis is an intracellular pathogen that infects mucosal epithelial cells, causing persistent infections. Although chronic inflammation is a hallmark of chlamydial disease, the proinflammatory mechanisms involved are poorly understood. Little is known about how innate immunity in the male genital tract (MGT) responds to C. trachomatis. Toll-like receptors (TLRs) are a family of receptors of the innate immunity that recognize different pathogen-associated molecular patterns (PAMPs) present in bacteria, viruses, yeasts and parasites. The study of TLR expression in the MGT has been poorly investigated. The aim of this work was to investigate the keratinocyte-derived chemokine (KC) response of MGT primary cultures from C57BL/6 mice to C. trachomatis and different PAMPs. KC production by prostate, seminal vesicle and epididymis/vas deferens cell cultures was determined by ELISA in culture supernatants. TLR2, 3, 4 and 9 agonists induced the production of KC by all MGT primary cultures assayed. In addition, we analysed the host response against C. trachomatis and Chlamydia muridarum. Chlamydial LPS (cLPS) as well as C. trachomatis and C. muridarum infection induced KC secretion by all MGT cell cultures analysed. Differences in KC levels were observed between cultures, suggesting specific sensitivity against pathogens among MGT tissues. Chemokine secretion was observed after stimulation of seminal vesicle cells with TLR agonists, cLPS and C. trachomatis. To our knowledge, this is the first report showing KC production by seminal vesicle cells after stimulation with TLR ligands, C. trachomatis or C. muridarum antigens. These results indicate that different receptors of the innate immunity are present in the MGT. Understanding specific immune responses, both innate and adaptive, against chlamydial infections, mounted in each tissue of the MGT, will be crucial to design new therapeutic approaches where innate and/or adaptive immunity would be targeted.