Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

Food-grade titanium dioxide (E171) by solid or liquid matrix administration induces inflammation, germ cells sloughing in seminiferous tubules and blood-testis barrier disruption in mice.

Food-grade titanium dioxide labeled as E171 has been approved for human consumption by the Food and Drug Administration (USA) and by the European Union for five decades. However, titanium dioxide has been classified as a possible carcinogen for humans by the International Agency of Research in Cancer raising concerns of its oral intake and the translocation to bloodstream, which could disturb barriers such as the blood-testis barrier. There is evidence that titanium dioxide by intragastric/intraperitoneal/intravenous administration induced alterations on testosterone levels, testicular function and architecture, but studies of the E171 effects on the testicle structure and blood-testis barrier are limited. E171 is contained not only in foods in liquid matrix but also in solid ones, which can exert different biological effects. We aimed to compare the effects of E171 consumption in a solid matrix (0.1%, 0.5% and 1% in pellets) and liquid suspension (5 mg/kg body weight) on testis structure, inflammation infiltrate and blood-testis barrier disruption of male BALB/c mice. Results showed that none of the administration routes had influence on body weight but an increase in germ cell sloughing and the infiltrate of inflammatory cells in seminiferous tubules, together with disruption of the blood-testis barrier were similar in testis of both groups even if the dose received in mice in liquid matrix was 136 or 260 times lower than the dose reached by oral intake in solid E171 pellets in 0.5% E171 and 1% E171, respectively. This study highlights the attention on matrix food containing E171 and possible adverse effects on testis when E171 is consumed in a liquid matrix.

ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation.

Peritubular myoid cells, which form the walls of seminiferous tubules in the testis, are functionally unexplored. While they transport sperm and contribute to the spermatogonial stem cell niche, specifically their emerging role in the immune surveillance of the testis and in male infertility remains to be studied. Recently, cytokine production and activation of Toll-like receptors (TLRs) were uncovered in cultured peritubular cells. We now show that human peritubular cells express purinergic receptors P2RX4 and P2RX7, which are functionally linked to TLRs, with P2RX4 being the prevalent ATP-gated ion channel. Subsequent ATP treatment of cultured peritubular cells resulted in up-regulated (pro-)inflammatory cytokine expression and secretion, while characteristic peritubular proteins, that is smooth muscle cell markers and extracellular matrix molecules, decreased. These findings indicate that extracellular ATP may act as danger molecule on peritubular cells, able to promote inflammatory responses in the testicular environment.

Rosa damascena Mill. Essential Oil Has Protective Effect Against Testicular Damage in Diabetic Rats.

This study investigates the protective effect of Rosa damascena essential oil on diabetes-induced testicular damage in rats. Thirty-six male Wistar rats were randomly divided into 6 equal groups: Group I: negative control (no treatment); Group II: positive control (diabetic by alloxan injection); Groups III-VI that rendered diabetic and received, respectively, 50, 100, 200, and 400 µg/kg/day rose oil, orally for 28 days. Rose oil did not significantly change body weight and blood glucose level as compared to positive control. Serum testosterone level of rose oil-treated rats remained statistically the same with both negative and positive control groups (Groups I and II). Rats treated with rose oil especially at 2 higher dosages (Groups V and VI) had higher sperm count and increased diameters of seminiferous tubules as compared to Group II. Rose oil even at the lowest dosage significantly increased cell count of spermatogonia, primary spermatocytes, Sertoli cells, and Leydig cells, with better outcomes for higher dosages. It appears that short-term repeated dose administration of rose oil can dose-dependently improve structural deteriorations of testes and epididymal sperm count in diabetic rats.

Egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance.

Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.

Neonatal testicular cell transplantation restores murine spermatogenesis damaged in the course of herpes simplex virus-induced orchitis.

Genital tract infection and inflammation may affect male fertility, causing germ and Sertoli cell loss. We determined if testicular cell transplantation is effective at repairing testicular injury induced by herpes simplex virus (HSV) orchitis. ROSA26 mice were used as donors and the recipients were C57BL/6 mice after HSV testicular inoculation; some of the recipients were treated with the antiviral drug acyclovir (ACV). ACV reduced the amount of HSV antigen in testes on Day 3 after transplantation and enhanced the efficacy of transplantation at Day 30. In recipient testes, donor Sertoli cells formed new seminiferous tubules; significantly more new tubules were observed in the testes of ACV-treated mice compared with mice not treated with ACV (17.8% vs 3.6%). Over half (50.4%) of new tubules in ACV-treated testes contained germ cells and round spermatids were detected in 14.2% of new tubules compared with 15.9% and 5.3% in testes not treated with ACV, respectively. At Day 150 the seminiferous epithelium was completely recovered in some donor tubules and elongated spermatids were observed inside it. Thus, our findings reveal the effectiveness of the combination of antiviral therapy with neonatal testis-cell transplantation for the restoration of spermatogenesis damaged by viral infection.

Dual roles of endogenous and exogenous galectin-1 in the control of testicular immunopathology.

Galectin-1 (Gal-1), a proto-type member of galectin family, is highly expressed in immune privileged sites, including the testis. However, in spite of considerable progress the relevance of endogenous and exogenous Gal-1 in testis pathophysiology have not yet been explored. Here we evaluated the in vivo roles of Gal-1 in experimental autoimmune orchitis (EAO), a well-established model of autoimmune testicular inflammation associated with subfertility and infertility. A significant reduction in the incidence and severity of EAO was observed in mice genetically deficient in Gal-1 (Lgals1(-/-)) versus wild-type (WT) mice. Testicular histopathology revealed the presence of multifocal testicular damage in WT mice characterized by an interstitial mononuclear cell infiltrate and different degrees of germ cell sloughing of seminiferous tubules. TUNEL assay and assessment of active caspase-3 expression, revealed the prevalence of apoptotic spermatocytes mainly localized in the adluminal compartment of seminiferous tubules in EAO mice. A significant increased number of TUNEL-positive germ cells was detected in EAO testis from WT compared with Lgals1(-/-) mice. In contrast, exogenous administration of recombinant Gal-1 to WT mice undergoing EAO attenuated the severity of the disease. Our results unveil a dual role of endogenous versus exogenous Gal-1 in the control of autoimmune testis inflammation.

Simian immunodeficiency virus infection and immune responses in the pig-tailed macaque testis.

The testis is a site of immune privilege in rodents, and there is evidence that T cell responses are also suppressed in the primate testis. Local immunosuppression is a potential mechanism for HIV persistence in tissue reservoirs that few studies have examined. The response of the pig-tailed macaque testis to SIVmac239 infection was characterized to test this possibility. Testes were surgically removed during early-chronic (10 wk) and late-chronic (24-30 wk) SIV infection in 4 animals and compared with those from 7 uninfected animals. SIV infection caused only minor disruption to the seminiferous epithelium without marked evidence of inflammation or consistent changes in total intratesticular leukocyte numbers. Infection also led to an increase in the relative proportion of testicular effector memory CD8(+) T cell numbers and a corresponding reduction in central memory CD4(+) T cells. A decrease in the relative proportion of resident-type CD163(+) macrophages and DCs was also observed. SIV-specific CD8(+) T cells were detectable in the testis, 10-11 wk after infection by staining with SIV Gag-specific or Tat-specific MHC-I tetramers. However, testicular CD8(+) T cells from the infected animals had suppressed cytokine responses to mitogen activation. These results support the possibility that local immunosuppression in the testis may be restricting the ability of T cells to respond to SIV or HIV infection. Local immunosuppression in the testis may be an underexplored mechanism allowing HIV persistence.

Cellular evidence for selfish spermatogonial selection in aged human testes.

Owing to a recent trend for delayed paternity, the genomic integrity of spermatozoa of older men has become a focus of increased interest. Older fathers are at higher risk for their children to be born with several monogenic conditions collectively termed paternal age effect (PAE) disorders, which include achondroplasia, Apert syndrome and Costello syndrome. These disorders are caused by specific mutations originating almost exclusively from the male germline, in genes encoding components of the tyrosine kinase receptor/RAS/MAPK signalling pathway. These particular mutations, occurring randomly during mitotic divisions of spermatogonial stem cells (SSCs), are predicted to confer a selective/growth advantage on the mutant SSC. This selective advantage leads to a clonal expansion of the mutant cells over time, which generates mutant spermatozoa at levels significantly above the background mutation rate. This phenomenon, termed selfish spermatogonial selection, is likely to occur in all men. In rare cases, probably because of additional mutational events, selfish spermatogonial selection may lead to spermatocytic seminoma. The studies that initially predicted the clonal nature of selfish spermatogonial selection were based on DNA analysis, rather than the visualization of mutant clones in intact testes. In a recent study that aimed to identify these clones directly, we stained serial sections of fixed testes for expression of melanoma antigen family A4 (MAGEA4), a marker of spermatogonia. A subset of seminiferous tubules with an appearance and distribution compatible with the predicted mutant clones were identified. In these tubules, termed 'immunopositive tubules', there is an increased density of spermatogonia positive for markers related to selfish selection (FGFR3) and SSC self-renewal (phosphorylated AKT). Here we detail the properties of the immunopositive tubules and how they relate to the predicted mutant clones, as well as discussing the utility of identifying the potential cellular source of PAE mutations.

Human testicular peritubular cells: more than meets the eye.

In healthy men, several layers of inconspicuously flat cells and extracellular matrix (ECM) proteins build the wall of the seminiferous tubules. The cells of this wall, peritubular cells, are not well characterized. They are smooth-muscle-like and contractile and transport immotile sperm, a function important for male fertility. However, their full functional importance, especially their potential contribution to the paracrine regulation of the male gonad, is unknown. In men with impaired spermatogenesis, the architecture of the tubular wall is frequently altered. Deposits of ECM and morphological changes of peritubular cells imply that functions of peritubular cells may be fundamentally altered. To be able to study human peritubular cells and their functions, a culture method was established. It is based on small biopsies of patients with obstructive azoospermia but normal spermatogenesis (human testicular peritubular cells, HTPCs) and non-obstructive azoospermia, impaired spermatogenesis, and testicular fibrosis (HTPCFs). Results obtained from cellular studies and parallel examinations of biopsies provide insights into the repertoire of the secretion products, contractile properties, and plasticity of human peritubular cells. They produce ECM components, including the proteoglycan decorin, which may influence paracrine signaling between testicular cells. They may contribute to the spermatogonial stem cell niche via secreted factors. They are regulated by mast cell and macrophage products, and in response produce factors that can fuel inflammatory changes. They possess a high degree of plasticity, which results in hypertrophy and loss of contractile abilities. The data collectively indicate important roles of inconspicuous testicular peritubular cells in human male fertility and infertility.

Involvement of Fas/Fas-L and Bax/Bcl-2 systems in germ cell death following immunization with syngeneic testicular germ cells in mice.

Experimental autoimmune orchitis (EAO) is characterized by T cell-dependent lymphocytic inflammation and seminiferous tubule damage, which can result in the death of germ cells. The aim of the present study is to investigate the roles of the Fas/Fas-L and Bax/Bcl-2 systems in the death of germ cells in mice with EAO that is induced by immunization with syngeneic testicular germ cells (TGC). The results using real-time reverse transcription-polymerase chain reaction and immunostaining show that many terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining germ cells were present in seminiferous tubules during the active inflammation stage, and these cells were persistently observed in the seminiferous epithelium until the postactive inflammation stage. Intratesticular mRNA expression levels of both Fas and Bax were increased during the active inflammation stage and were dramatically decreased during the post-active inflammation stage. In contrast, the intratesticular mRNA expression levels of both Fas-L and Bcl-2 did not show significant changes during the active inflammation stage but showed extreme increases during the post-active inflammation stage. Immunohistochemically, some Fas- and Bax-positive germ cells were detected during the active inflammation stage, but these were hardly found during the post-active inflammation stage. In contrast, some Fas-L- and Bcl-2-positive germ cells were found during the active inflammation stage, and many of these were also observed during the post-active inflammation stage. These results indicate that germ cell death during TGC-induced EAO is mediated by the Fas/Fas-L and Bax/Bcl-2 systems during the active inflammation stage but not during the post-active inflammation stage.

Mice spermatogonial stem cells transplantation induces macrophage migration into the seminiferous epithelium and lipid body formation: high-resolution light microscopy and ultrastructural studies.

Transplantation of spermatogonial stem cells (SSCs), the male germline stem cells, in experimental animal models has been successfully used to study mechanisms involved in SSC self-renewal and to restore fertility. However, there are still many challenges associated with understanding the recipient immune response for SSCs use in clinical therapies. Here, we have undertaken a detailed structural study of macrophages elicited by SSCs transplantation in mice using both high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). We demonstrate that SSCs transplantation elicits a rapid and potent recruitment of macrophages into the seminiferous epithelium (SE). Infiltrating macrophages were derived from differentiation of peritubular monocyte-like cells into typical activated macrophages, which actively migrate through the SE, accumulate in the tubule lumen, and direct phagocytosis of differentiating germ cells and spermatozoa. Quantitative TEM analyses revealed increased formation of lipid bodies (LBs), organelles recognized as intracellular platforms for synthesis of inflammatory mediators and key markers of macrophage activation, within both infiltrating macrophages and Sertoli cells. LBs significantly increased in number and size in parallel to the augmented macrophage migration during different times post-transplantation. Our findings suggest that LBs may be involved with immunomodulatory mechanisms regulating the seminiferous tubule niche after SSC transplantation.

Histopathology of the tubuli recti at the start of experimental autoimmune orchitis in mice.

We previously established an experimental model of autoimmune orchitis (EAO) by means of immunization with testicular germ cells (TGC) alone in mice and confirmed that the disease can be transferred to mice that had received CD4+ but not CD8+ or B lymphocytes obtained from TGC-immunized donor mice. The tubuli recti (TR) are special in that lymphocytes first accumulate around them before spreading to the peripheral seminiferous tubules in EAO. However, the minute changes in the TR remain unknown. Therefore, we investigated the histopathology of the TR before the induction of spermatogenic disturbance. The results revealed that the first infiltrating lymphocytes around the TR were not only of T-cell but also of B-cell lineage. Moreover, it was also shown that some of these infiltrating lymphocytes migrated into the TR, with resultant degeneration of the TR epithelium before damage to the seminiferous epithelium. These findings indicate that TR epithelial cells are the first targets of autoreactive T and B lymphocytes in EAO.

Effects of immunization against alpha-inhibin using two adjuvants on daily sperm production and hormone concentrations in ram lambs.

Twenty-five ram lambs were immunized against alpha-inhibin peptide emulsified in Freund's adjuvant (FRA), Emulsigen (EML) containing an oligodeoxynucleotide as an immunostimulant, or adjuvant without alpha-inhibin antigen (control). Four immunizations were administered during an 85-d period, after which testes were obtained for determination of daily sperm production (DSP) and histological evaluation. alpha-Inhibin antibody (Ab) titers were 70-fold greater in lambs treated with FRA than in EML-treated ram lambs. alpha-Inhibin immunization had no effect on testes weight or on plasma concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Mean DSP/g tended (P=0.1) to be greater in alpha-inhibin-immunized (EML=17.6x10(6); FRA=15.8x10(6)) ram lambs than in control animals (14.4x10(6)). One of the 8 control ram lambs had an elevated DSP/g, which was a statistical outlier. Without data from this lamb, DSP/g was increased (P<0.01) in alpha-inhibin-immunized ram lambs by 28% over controls. No association was found between the titer of alpha-inhibin Ab developed and DSP/g. Histologically, the percentage of testicular area occupied by seminiferous tubules differed (P=0.01) by treatment and was greatest (82%) in EML-treated ram alpha-inhibin-immunized lambs and lowest (74%) in control animals. Percentage tubular area and DSP/g were correlated (r=0.57, P=0.003). Findings show that (1) the extent of the increase in DSP/g is not dependent on the titer of alpha-inhibin Ab; (2) the increase in DSP/g is achieved through an increase in the mass of seminiferous tubules; and (3) FRA elicits a greater alpha-inhibin Ab titer than EML containing an oligodeoxynucleotide.

Evaluation of effects of methamphetamine repeated dosing on proliferation and apoptosis of rat germ cells.

Methamphetamine (MAMP) is a central nervous system stimulant that is increasingly abused especially by teenagers and young adults, a group in its reproductive age. MAMP effects on the male reproductive system are not clear. In this experimental study, we evaluated the effects of MAMP administration on proliferation and apoptosis in seminiferous tubules of rat testis. Methamphetamine hydrochloride was synthesized by iodination of norephedrine hydrochloride and reduction to methamphetamine. Mature male rats were randomly divided into four groups (n=7) and were injected intraperitoneally with MAMP (1, 5 or 10 mg/kg) or saline at the same time, once daily for 14 consecutive days. Twenty four hs after the last injection, perfused testis were fixed, sectioned and stained by TUNEL labeling or proliferating cell nuclear antigen (PCNA) immunostaining. Apoptotis and proliferation indices were calculated and ratios of proliferation/apoptosis in the seminiferous tubules were obtained. Cell proliferation and the ratio of proliferation to apoptosis decreased significantly in all experimental groups compared to the control group. Conversely, apoptosis was increased in these groups. Such differences were observed in both spermatogonia and primary spermatocytes. In the control group, more than 95% of spermatogonia were PCNA-positive. However, 10 mg/kg of MAMP caused a reduction to approximately 75% PCNA staining in spermatogonia. In some tubules of the experimental groups, more than 10 TUNEL-positive germ cells were seen, although in the control group, the tubules with 3 TUNEL-positive germ cells were rarely observed and the majority of tubules were without such cells. There were significant differences in the indices between the 1 mg/kg group and the higher dose groups, but there was no such difference between the 5 mg/kg and 10 mg/kg groups. In some tubules of the experimental groups, significant gaps in the epithelium between the spermatogonia layer and other cell layers were observed. These results show that repeated administration of MAMP, especially at higher doses, may cause a decrease in cellular proliferation, induce apoptosis and change the proliferation/apoptosis ratio in testis. This might explain the MAMP effect on the spermatogenesis process. It is suggested that studies on the consequence of MAMP consumption on male fertility is warranted.

The presence of intra-tubular lymphocytes in normal testis of the mouse.

Spermatoza contain various autoimmunogenic materials, which are recognized as foreign by the self immune system. Therefore, the blood-tesits-barrier (BTB) formed by Sertoli cells, basal lamina and myoid cells protects autoimmunogeneic spermatozoa from attack by the self immune system. However, the BTB at the tubuli recti (TR) and the rete testis (RT) is known to be incomplete against humoral substances. We investigated here whether the BTB is physiologically penetrated by lymphocytes in mice. We performed light and electron microscopical observation of the seminiferous tubules (ST), the TR and the RT in normal C3H/IHe mice. Although no lymphocytes were observed inside the ST, a very few lymphocytes could be found beyond the basal lamina of the TR and the RT. These lymphocytes were close to testicular spermatozoa in the TR lumen. These findings provide a possibility that lymphocytes may gain access to autoantigens of spermatozoa inside the TR and RT under physiological conditions in mice.

Changes in the testis seminiferous tubules and interstitium in prepubertal bull calves immunised against inhibin at the time of gonadotropin administration.

The aim of the present study was to evaluate the effects of gonadotropin administration at initiation of inhibin passive immunisation in Jersey bull calves (age 27 +/- 5 days) on testicular morphology and development. Primary treatments consisted of control (keyhole limpet haemocyanin, KLH; n = 9) or immunisation against inhibin (INH; n = 9). Subsets of calves were randomly assigned within primary treatments (TRT) to receive saline ( n = 3 per TRT), follicle-stimulating hormone (FSH; n = 3 per TRT) or gonadotrophin-releasing hormone (GnRH, n = 3 per TRT). The right testis was removed (age 118 +/- 5 days) to determine volumes of testicular components and cell numbers per testis using stereology. Data were analysed using the MIXED procedure of the SAS program. Antibody titres against inhibin were increased in INH bulls compared with KLH bulls (P < 0.05). In addition, a significant immunisation x hormone treatment interaction was noted for the number of germ cells. Administration of FSH at the time of initial immunisation against inhibin significantly increased the number of germ cells (92.2 +/- 9 x 10(6) cells) compared with INH+saline bulls (54.9 +/- 10 x 10(6) cells), with INH+GnRH bulls having an intermediate number of cells (64.5 +/- 9 x 10(6) cells; P < 0.05). These results suggest that gonadotropin administration at the time of inhibin immunisation increases the number of germ cells in the testis.

[Testicular perfusion injury. Cytokines and cell adhesion molecules in humans]. / Testikuläre Ischämie. Verlauf von Zytokinen und Zelladhäsionsmolekülen beim Menschen.

BACKGROUND: Ischemia and reperfusion (I/R) lead to cellular damage. A disturbance of testicular perfusion occurs during the therapy of cryptorchidism and in cases of testicular torsion. This results in the activation of mediator cells with an increasing synthesis of mediators of infection like TNF-alpha and the expression of cell adhesion molecules like ICAM (intercellular adhesion molecule) and VCAM (vascular cell adhesion molecule) at the cellular surface. METHODS: The expression of the cytokines IL-10 and TNF-alpha and the adhesion molecules ICAM and VCAM after defined testicular I/R injury in nine male transsexuals was evaluated with rt-PCR. Furthermore we examined lactate and the diameter of the testicular tubulus under ischemic conditions. RESULTS: During ischemia ICAM, IL-10, and VCAM do not show significant changes on the side of testicular ischemia and the contralateral side; the same was seen for the tubulus diameter. TNF-alpha and the testicular lactate values showed a significant change of the expression pattern. DISCUSSION: The statistical changes of TNF-alpha and testicular lactate are the expression of leukocyte migration, infectious reaction, and immune response. To what extent the TNF-alpha expression represents a severe immunological reaction remains undefined. This human study shows primary results for the immunological understanding of and cellular response to testicular ischemia.

Ultrastructural localization of glycodelin oligosaccharides Le-x and Le-y in human seminal vesicles by immunogold staining.

Histo-blood group antigens Le-x and Le-y are oligosaccharidic terminals that characterize many glycoproteins in the human tissues. In seminal plasma, they are expressed as part of the so-called glycodelin S, which is suggested to regulate sperm capacitation/decapacitation. It has recently been demonstrated that the core protein of glycodelin S is secreted by seminal vesicles. Here we show that epithelial cells of human seminal vesicles also release the Le-x and Le-y antigens. The presence of these substances in secretory material was revealed by means of an immunogold staining method in normal surgical samples. The results suggest that glycodelin S is secreted by seminal vesicles in its finished glycosylated form. Moreover, antigen reactivity was also revealed associated with plasma membranes.

Sertoli cells initiate testicular innate immune responses through TLR activation.

TLRs play a crucial role in early host defense against invading pathogens. In the seminiferous epithelium, Sertoli cells are the somatic nurse cells that mechanically segregate germ cell autoantigens by means of the blood-tubular barrier and create a microenvironment that protects germ cells from both interstitial and ascending invading pathogens. The objective of this study was to examine TLR expression and their functional responses to specific agonists in mouse Sertoli cells. We measured the expression of TLR2, TLR4, TLR5, and TLR6 mRNAs and confirmed by FACS analysis the presence of proteins TLR2 and TLR5 on which we focused our study. Stimulation of Sertoli cells with macrophage-activating lipopeptide-2, agonist of TLR2/TLR6, and with flagellin, agonist of TLR5, induces augmented secretion of the chemokine MCP-1. To assess the functional significance of MCP-1 production following TLR stimulation, conditioned medium from either macrophage-activating lipopeptide-2 or flagellin-treated Sertoli cells was tested for in vitro chemotaxis assay, and a significant increase of macrophage migration was observed in comparison with unstimulated conditioned medium. Moreover, we studied the role of NF-kappaB and of MAPKs in regulating TLR-mediated MCP-1 secretion by using inhibitors specific for each transduction pathway and we demonstrated a pivotal role of the IkappaB/NF-kappaB and JNK systems. In addition, TLR2/TLR6 and TLR5 stimulation induces increased ICAM-1 expression in Sertoli cells. Collectively, this study demonstrates the novel ability of Sertoli cells to potentially respond to a wide variety of bacteria through TLR stimulation.