Systemic LPS induces toll-like receptor 3 (TLR3) expression and apoptosis in testicular mouse tissue.

It is well known that sepsis and inflammation reduce male fertility. Within the testis, toll-like receptor 3 (TLR3) is constitutively expressed and recognizes double-stranded RNA (dsRNA) from viruses, degraded bacteria, damaged tissues and necrotic cells. To characterize the potential role of TLR3 in response to testicular infections, its expression and downstream signaling were investigated upon challenge with lipopolysaccharides (LPS) in two mouse strains that differ in their immuno-competence regarding T cell-regulated immunity. Thereto, Balb/c and Foxn1nu mice were randomized into six interventional groups treated with either i.v. application of saline or LPS followed by 20 min, 5 h 30 min and 18 h of observation and two sham-treated control groups. LPS administration induced a significant stress response; the amplification was manifested for TLR3 and interleukin 6 (IL6) mRNA in the impaired testis 5 h 30 min after LPS injection. TLR3 immunostaining revealed that TLR3 was primarily localized in spermatocytes. The TLR3 expression displayed different temporal dynamics between both mouse strains. However, immunofluorescence staining indicated only punctual interferon regulatory factor 3 (IRF3) expression upon LPS treatment along with minor alterations in interferon ß (IFNß) mRNA expression. Induction of acute inflammation was closely followed by a significant shift of the Bax/Bcl2 ratio to pro-apoptotic signaling accompanied by augmented TUNEL-positive cells 18 h after LPS injection with again differing patterns in both mouse strains. In conclusion, this study shows the involvement of TLR3 in response to LPS-induced testicular inflammation in immuno-competent and -incompetent mice, yet lacking transmission into its signaling pathway.

Cellular changes in the hamster testicular interstitium with ageing and after exposure to short photoperiod.

The aim of this study was to evaluate the cellular changes that occur in the hamster testicular interstitium in two very different physiological situations involving testicular involution: ageing and exposure to a short photoperiod. The animals were divided into an 'age group' with three subgroups - young, adult and old animals - and a 'regressed group' with animals subjected to a short photoperiod. The testicular interstitium was characterised by light and electron microscopy. Interstitial cells were studied histochemically with regard to their proliferation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling (TUNEL+) and testosterone synthetic activity. We identified two types of Leydig cell: Type A cells showed a normal morphology, while Type B cells appeared necrotic. With ageing, pericyte proliferation decreased but there was no variation in the index of TUNEL-positive Leydig cells. In the regressed group, pericyte proliferation was greater and TUNEL-positive cells were not observed in the interstitium. The testicular interstitium suffered few ultrastructural changes during ageing and necrotic Leydig cells were observed. In contrast, an ultrastructural involution of Leydig cells with no necrosis was observed in the regressed group. In conclusion, the testicular interstitium of Mesocricetus auratus showed different cellular changes in the two groups (age and regressed), probably due to the irreversible nature of ageing and the reversible character of changes induced by short photoperiod.

Toll-like receptor 3-initiated antiviral responses in mouse male germ cells in vitro.

The testis is an immunoprivileged site where local cell-initiated innate immunity plays a crucial role in antimicrobial responses. Toll-like receptors (TLRs) mediate innate immune responses in testicular somatic cells. Although several TLRs are expressed in some stages of male germ cells, the potential role of TLRs in triggering antimicrobial responses in the germ cells has yet to be exclusively studied. The current study demonstrates that TLR3 is constitutively expressed in spermatogonia and spermatocytes and can be activated by a synthetic double-strained RNA analog, polyinosinic-polycytidylic acid. TLR3 activation in these male germ cells up-regulates the expression of proinflammatory cytokines, such as interleukin IL1B, IL6, and tumor necrosis factor alpha, through activation of nuclear factor kappa B; it also induces production of type 1 interferons (IFNA and IFNB) through the activation of IFN regulatory factor 3. In addition, TLR3 activation increases the production of two major antiviral proteins, namely, double-stranded RNA-activated protein kinase and MX1 protein, by germ cells. Data in this article describe an antiviral response of male germ cells through the activation of TLR3 in vitro.

Biochemical, immunohistochemical and morphometrical investigation of the effect of thymoquinone on the rat testis following exposure to a 900-MHz electromagnetic field.

Long-term use of cell phones emitting electromagnetic fields (EMFs) have raised concerns regarding public health in recent year. We aimed to investigate the possible effects of 900 MHz EMF exposure (60 min/day for 28 days) on the rat testis. Another objective was to determine whether the deleterious effect of EMF radiation would be reduced by the administration of thymoquinone (TQ) (10 mg/kg/day). Twenty-four male adult Wistar albino rats were randomly selected, then assigned into four groups as followControl, EMF, TQ and EMF + TQ. Testicular samples were analyzed using histological, stereological, biochemical and immunohistochemical techniques. Total numbers of primary spermatocytes and spermatids as well as Leydig cells were significantly decreased in the EMF group compared to the Control group (p < 0.05). In the EMF + TQ group, the total number of primary spermatocytes was significantly increased compared to the EMF group (p < 0.05). Superoxide dismutase (SOD) activity was significantly increased in the EMF group compared to the Control group (p < 0.05). Also, serum testosterone levels and wet weight of testes were significantly decreased in the EMF group compared to the Control group (p < 0.05). Our findings suggested that exposure to a 900 MHz EMF had adverse effects on rat testicular tissue and that the administration of TQ partially mitigated testicular oxidative damages caused by EMF radiation.

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.

Membrane and cytoplasmic proteins are transported in the same organelle complex during nematode spermatogenesis.

During the development of pseudopodial spermatozoa of the nematode, Caenorhabditis elegans, protein synthesis stops before differentiation is completed. Colloidal gold conjugates of monoclonal antibody SP56, which binds to the surface of spermatozoa, and TR20, which recognizes the major sperm cytoplasmic protein (MSP), were used to label thin sections of testes embedded in Lowicryl K4M in order to follow polypeptides from their synthesis early in spermatogenesis to their segregation to specific compartments of the mature cell. Both antigens are synthesized in primary spermatocytes and are assembled into a unique double organelle, the fibrous body-membranous organelle (FB-MO) complex. However, the antigens are localized in different regions of this FB-MO complex. As described in detail, the assembly of proteins into the FB-MO complex allows both membrane and cytoplasmic components to be concentrated in the spermatids after meiosis. Then, the stepwise disassembly of this transient structure ensures delivery of each component to its final destination in the mature spermatozoan: MSP filaments in the fibrous body depolymerize, releasing MSP into the cytoplasm and the membranous organelles fuse with the plasma membrane, delivering SP56 antigen to the surface.

Pattern of expression of immune-relevant genes in the gonad of a teleost, the gilthead seabream (Sparus aurata L.).

Immune responses in the testis are regulated in a way that provides protection for the developing male germ cells, while permitting qualitatively normal inflammatory responses and protection against infection. In addition, germ cells are potent targets for the growth factors and cytokines which regulate the reproductive process. Our study analyzes for the first time the pattern of expression of several immune-relevant genes in the gonad of a seasonal breeding teleost fish. The immune molecules analyzed include (i) inflammatory molecules, such as interleukin-1b (il1b), il6, tumor necrosis factor-a (tnfa), cyclooxygenase-2 (cox2) and the NADPH oxidase subunit p40(phox) (ncf4 gene); (ii) the anti-inflammatory cytokine transforming growth factor-b1 (tgfb1) and its type 2 receptor tgfbr2; (iii) innate immune receptors, including toll-like receptor 9 (tlr9), tlr5, tlr22 and macrophage-colony stimulating factor receptor (mcsfr); (iv) lymphocyte receptors, such as the beta subunit of T-cell receptor (Tcrb) and the heavy chain of immunoglobulin M (ighm); (v) the anti-bacterial molecules lysozyme (lyz), hepcidin (hamp) and complement component 3 (c3); (vi) the anti-viral molecule myxovirus (influenza) resistance protein (mx); and (vii) molecules related to leukocyte infiltration, including the CC chemokine ccl4, the CXC chemokine il8 and the leukocyte adhesion molecule E-selectin (Sele). Notably, all of them show a pattern of expression that depends on the reproductive stage of the first two reproductive cycles when the fish develop and function as males. Furthermore, we demonstrate that some of these immune-relevant molecules, such as Il1b and Mcsfr, are produced by germ cells (Il1b) and ovarian and testicular somatic cells (Mcsfr). These data suggest that, as occurs in mammals, there is a critical balance between immune molecules and that these may play an essential role in the orchestration of gametogenesis and the maintenance of gonad tissue homeostasis in fish.

Expression of POTE protein in human testis detected by novel monoclonal antibodies.

The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2gammaC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2gammaC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.

Obtaining and characterization of anti-testis monoclonal antibodies: Invaluable tools toward the identification of testis antigens involved in fertilization.

BACKGROUND: Generation and utilization of the specific monoclonal antibodies against testis antigens is reported to identify the antigens that are important in reproductive field. OBJECTIVE: Current study aimed to introduce a hybridoma that producing a specific anti-testis monoclonal antibody to identify the testis antigens that can be important in the reproduction field. METHODS: To make hybridoma against testis antigens, mice were immunized with testis cell lysate. After cell fusion, resulted hybridomas were screened by indirect ELISA, then cloned by limiting dilution and finally the produced monoclonal antibody were characterized by some of the molecular laboratory techniques such as immunohistochemistry, immunocytochemistry and western blot. RESULTS: By using hybridoma technique, cell fusion was performed and ten (8A11, 8D6, 8D7, 9F6, 9G11, 10C3, 10B3, 10B2, 10C2 and 10H7) antibodies specific to the testis antigens were produced finally. Among the produced antibodies, 10C3 was found to cross-react with testis, but not detected in other tissues. mAb 10C3 recognized the sperm and testis antigens, specifically the intertestitial tissue of testis, spermatogonia, primary and secondary spermatocyte antigens, so they were most likely the target of generated mAb. Also our mAb could totally detect the mouse sperm antigens and the specific antigens of head and tail of human sperm. In western blotting analysis, mAb 10C3 could recognize the specific protein bands of sperm and testis extracts. Also in this study the testis specific genes that were target of generated mAb, were selected according to the mouse EST profile available at UniGene part of NCBI. CONCLUSIONS: So this stable anti-testis mAb has a potential for laboratory researches and also for diagnostic procedures in fertilization. Thus it could be exploited as a suitable tool for target-specific diagnosis and research in several diseases.

A rat spermatocyte surface protein is involved in adhesion of pachytene spermatocytes to Sertoli cells in vitro.

Immunochemical approaches were used in trying to identify rat spermatocyte molecules involved in the adhesion to Sertoli cells in coculture. The results show that a surface protein of 80,000 apparent molecular weight strongly inhibits spermatocyte adhesion, suggesting it to be the germ cell surface component involved in the recognition of Sertoli cells.

Proteolytic cleavage of ALF into alpha- and beta-subunits that form homologous and heterologous complexes with somatic TFIIA and TRF2 in male germ cells.

Male germ cells specifically express paralogues of components of the general transcription apparatus including ALF a paralogue of TFIIAalpha/beta. We show that endogenous ALF is proteolytically cleaved to give alpha- and beta-subunits and we map the proteolytic cleavage site by mass spectrometry. Immunoprecipitations show that ALFalpha- and beta-subunits form a series of homologous and heterologous complexes with somatic TFIIA which is coexpressed in male germ cells. In addition, we show that ALF is coexpressed in late pachytene spermatocytes and in haploid round spermatids with transcription factor TRF2, and that these proteins form stable complexes in testis extracts. Our observations highlight how cleavage of ALF and coexpression with TFIIA and TRF2 increases the combinatorial possibilities for gene regulation at different developmental stages of spermatogenesis.

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.

The time of appearance of Ia antigens during spermatogenesis in the mouse.

Ia specificities were studied on germinal cells from C3H and 129 mice by using an AT.H anti-AT.L antiserum. By absorption experiments and immunofluorescence studies on sections of testes from mice of various ages, we were able to detect Ia specificities on cells of the germinal line from primary spermatocyte to spermatozoa. Ia specificities were not detectable on gonocytes or spermatogonia.

AJ-p97: a novel antigen of the human sperm tail fibrous sheath detected by a neurofilament monoclonal antibody.

Using indirect immunofluorescence (IIF), the RT97 anti-neurofilament monoclonal antibody (MoAb) detected an intracellular antigen in the principal piece of human ejaculated sperm tails. Its localisation to the tail fibrous sheath (FS) was confirmed by immunoelectron microscopy (IEM), which showed the binding of the gold particles to the outer FS surface. During spermatogenesis the antigen was first expressed on the spermatid FS, and its expression was continued on ejaculated mature sperm. In Western blotting of sperm lysates, the RT97 reacted with a 97 kDa protein (AJ-p97) which lacked disulphide bonds. This antigen was not detected on mouse or rat sperm tail FS, suggesting a sequence divergence of the AJ-p97 during evolution. The significance of these results and the relationship of AJ-p97 to neurofilaments are discussed, together with the use of the antibody as a probe for the structural dissection of the FS and for analysing the molecular events that take place during spermiogenesis, especially those involved in sperm tail morphogenesis.

Characterization by enriched polyclonal antibodies of developmentally regulated and cell type specific mouse testis antigens.

Polyclonal antisera directed against testicular proteins were characterized by immunocytochemistry and Western blot analysis. Antibodies binding to testis-specific, developmentally regulated protein bands were eluted from their antigens and used for further characterization of the developmental profile and cell type-specific expression of two antigens, PSM33 and NNA75. While PSM33 was found to be present in spermatocytes from the late pachytene stage on, NNA75 could be localized in neonatal interstitial cells. NNA75 expression ceases by to postnatal day ten, when first meiosis starts within the seminiferous tubules, thus suggesting an interactive role of Leydig cells during the onset of meiotic divisions.

Analysis of various antigens in golden hamster testis by monoclonal antibodies.

A total of 38 hybridomas producing monoclonal antibodies (mAbs) was established by immunizing BALB/c mice with extracts of the golden hamster testis. Six mAbs stained the acrosome of developing spermatids by immunofluorescence. Two mAbs (1A11 and 4D8) reacted with spermatid components other than acrosome. The mAbs 1C9 and 4D3 recognized a 103 kilodalton (kDa) protein on immunoblots, and were reactive to spermatocytes and early spermatids, but not to late spermatids and spermatozoa. This finding suggests that the protein functions for meiosis or early spermiogenesis. Four mAbs (3G2, 2E5, 2G3, and 3F10) stained all stages of spermatogenic cells. The remaining 24 mAbs showed a positive reaction to the basement membrane of the seminiferous tubule. Two of them, 3D6 and 3E5, recognized approximately 150 kDa major proteins, indicating that the antigen is an extracellular matrix.

[The dynamics of the formation of spontaneous autoantibodies to the synaptonemal complex in male mice]. / Issledovanie dinamiki formirovaniia spontannykh autoantitel k sinaptonemnomu kompleksu u samtsov myshi.

Time-related changes in formation of spontaneous autoantibodies against the synaptonemal complex were studied by indirect immunofluorescence in male mice. Appearance of the spontaneous autoantibodies against the synaptonemal complex coincided with that of the cells containing synaptonemal complexes. The mouse synaptonemal complexes were binding spontaneous autoantibodies of the rabbit and human sera. The synaptonemal complexes of the equine spermatocytes were binding spontaneous autoantibodies of the mouse serum. There was no fluorescence of synaptonemal complexes on preparations of spread rye meiocytes treated with the mouse serum. Antigenic similarity was shown for the synaptonemal complex components in representatives of the different mammalian orders: rodents, odd-toed ungulates, and primates.

Molecular characterization and expression of dipeptidase 3, a testis-specific membrane-bound dipeptidase: complex formation with TEX101, a germ-cell-specific antigen in the mouse testis.

We previously established an anti-sperm head auto-monoclonal antibody designated Ts4. The immunoreactivity of this antibody was also observed in other reproduction-related cells, such as testicular germ cells and early embryos, suggesting that the Ts4-recognized molecules might play a role in the reproductive process. However, the molecular characteristics and functions of the antigens warrant further clarification. In this study, we primarily attempted identification of the mAb-recognized molecules within the mouse testis. An immunoprecipitation method, together with liquid chromatography-tandem mass spectrometry, revealed that the testicular immunoprecipitants with Ts4 contained dipeptidase 3 (DPEP3), a member of the membrane-bound dipeptidase family. A Western blot analysis using an anti-DPEP3 polyclonal antibody established in this study showed that this molecule was glycosylated and formed a disulfide-linked homodimer within the testis. Expression of DPEP3 protein was observed in the testicular germ cells, but not in the Sertoli or interstitial cells, or in any other major organs. Although Western blot analysis of testicular proteins separated by two-dimensional SDS-PAGE failed to demonstrate binding of Ts4 to DPEP3, we found that DPEP3 forms complexes with Ts4-immunoreactive molecules, such as TEX101, on the surfaces of spermatocytes, spermatids, and testicular spermatozoa. Based on data showing in the present study, further studies concerning DPEP3 on the testicular germ cells may help to clarify the molecular mechanisms of testicular germ-cell development.

Involvement of the Fas and Fas ligand in testicular germ cell apoptosis by zearalenone in rat.

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin, is known to cause testicular toxicity in animals. In the present study, the effects of ZEA on spermatogenesis and possible mechanisms involved in germ cell injury were examined in rats. Ten-week-old Sprague-Dawley rats were treated with 5 mg/kg i.p. of ZEA and euthanized 3, 6, 12, 24 or 48 h after treatment. Histopathologically, spermatogonia and spermatocytes were found to be affected selectively. They were TUNEL-positive and found to be primarily in spermatogenic stages I-VI tubules from 6 h after dosing, increasing gradually until 12 h and then gradually decreasing. Western blot analysis revealed an increase in Fas and Fas ligand (Fas-L) protein levels in the ZEAtreated rats. However, the estrogen receptor (ER)alpha expression was not changed during the study. Collectively, our data suggest that acute exposure of ZEA induces apoptosis in germ cells of male rats and that this toxicity of ZEA is partially mediated through modulation of Fas and Fas-L systems, though ERalpha may not play a significant role.

Expression of Aire and the early wave of apoptosis in spermatogenesis.

Expression of the autoimmune regulator (Aire) protein in mice and humans is thought to be restricted to the medullary epithelial and monocyte-dendritic cells of the thymus. There it mediates expression and presentation of a large variety of proteins, including those that are peripheral organ-specific and are not expressed by other thymocytes. In this way, self-reactive T lymphocytes that would attack peripheral cells producing these proteins are confronted with the self-Ags and, as a consequence, are deleted. In this study, we show that Aire mRNA is also expressed in the testis--another tissue with promiscuous gene expression. Aire protein, however, is expressed only sporadically in spermatogonia and spermatocytes. Transcription of genes that are under Aire control in the thymus is unaffected by Aire in the testis. However, in mice with a disrupted Aire gene, the scheduled apoptotic wave of germ cells, which is necessary for normal mature spermatogenesis, is reduced, and sporadic apoptosis in adults is increased. Because Rag-1 deficiency does not abolish the effect, the adaptive immune system is not involved. We suggest that there is a link between the scheduled and sporadic apoptotic processes and propose that scheduled apoptosis provides a counterselection mechanism that keeps the germline stable.