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.

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.

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.

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.

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.

Focal orchitis in undescended testes: discussion of pathogenetic mechanisms of tubular atrophy.

OBJECTIVE: To evaluate seminiferous epithelium lesions in adult cryptorchid testes showing lymphoid infiltrates in seminiferous tubules and interstitium (i.e., focal orchitis). Also, to consider the possible role of this lesion in the etiology of tubular atrophy. METHODS: We performed a histopathologic study of the cryptorchid testes and adjacent epididymides removed from 50 adult men who had not been previously treated for cryptorchidism. The study included morphologic and semiquantitative evaluation of seminiferous tubule pathology (according to germ cell numbers), Sertoli cell morphology, tubular lumen dilation, rete testis pattern (normal, hypoplastic, or cystic), and epididymal pattern (normal or epididymal duct hypoplasia). The study also included immunohistochemical evaluation of immune cell markers. The results were compared with clinical and laboratory findings. RESULTS: Focal lymphoid infiltrates (mainly lymphocytes) in seminiferous tubules and interstitium were found in 22 patients (44%), all of whom had unilateral cryptorchidism. The course of orchitis was asymptomatic, and laboratory data were normal. According to the seminiferous tubule pathology, a variety of histopathologic diagnoses, were made: (1) mixed atrophy consisting of Sertoli cell-only tubules intermingled with tubules showing maturation arrest of spermatogonia (11 testes, 4 of which also showed hyalinized tubules); (2) Sertoli cell-only tubules plus hyalinized tubules (4 testes); (3) Sertoli cell-only tubules (3 testes); (4) intratubular germ cell neoplasia (2 testes, 1 of which also showed hyalinized tubules); (5) complete tubular hyalinization (1 testis); and (6) tubular hyalinization plus some groups of tubules with hypospermatogenesis (all germ cell types were present although in lower numbers, 1 testis). Dysgenetic Sertoli cells, that is, Sertoli cells that had undergone anomalous, incomplete maturation, were observed in all nonhyalinized seminiferous tubules with inflammatory infiltrates. Tubular ectasia was observed in 13 cases. The rete testis was hypoplastic and showed cystic transformation in 18 testes, and the epididymis was hypoplastic in 15 testes. CONCLUSIONS: The causes of these focal inflammatory infiltrates are unknown. It is possible that tubular ectasia and Sertoli cell dysgenesis are involved and that these alterations cause a disruption of the blood-testis barrier and allow antigens to enter the testicular interstitium, giving rise to an autoimmune process.

Expression and regulation of the CXC-chemokines, GRO/KC and IP-10/mob-1 in rat seminiferous tubules.

Testicular inflammation is classically observed in the pathogenesis of viral and bacterial infection or tumoral invasion. In these situations, leukocyte infiltration is generally encountered. GRO/KC (growth-related oncogene) and IP-10/mob-1 (IFN-gamma-inducible protein) are two CXC-chemokines which attract neutrophils and activated T lymphocytes, respectively, have been studied for their ability to participate to testicular inflammation (orchitis). In the present work, using Northern blot and immunocytochemistry, we aimed to investigate whether GRO/KC and IP-10/mob-1 are produced within the seminiferous tubules of the testis and if these chemokines are induced by a number of pro-inflammatory cytokines and lipopolysaccharides (LPS). Our results show that GRO/KC and IP-10/mob-1 mRNAs were never found in germ cells, whether they were stimulated or not. In contrast, GRO/KC mRNA was expressed by isolated peritubular cells when stimulated by interleukin-1 alpha and beta (IL-1 alpha and IL-1 beta) or LPS and to a lesser extent by tumor necrosis factor-alpha (TNF-alpha) and by Sertoli cells when the latter were stimulated by rIL-alpha and rIL-1 beta and to a lesser extent by TNF-alpha and LPS. Moreover, IP10/mob-1 transcripts were strongly induced in peritubular cells by interferon-alpha (IFN-alpha) and IFN-gamma, whereas, in isolated Sertoli cells, INF-alpha and TNF-alpha were the only potent inducers. The kinetics of GRO/KC and IP-10/mob-1 mRNA expression by peritubular and Sertoli cells (significant stimulation as early as 1 hour and 4 hours post-exposure to the stimuli, respectively) are compatible with the hypothesis of a rapid mobilisation of these cells in an inflammatory process. Moreover, the dose-dependent effects of pro-inflammatory cytokines to induce a chemokine response were compatible with a high sensitivity of peritubular and Sertoli cells in orchitis. In conclusion, this present study shows that 2 CXC-chemokines, GRO/KC and IP10/mob-1, are produced by testicular somatic cells of seminiferous tubules, strongly indicating a likely role of these chemokines in the accumulation of neutrophils and T lymphocytes during orchitis of various origins.

TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium.

Sertoli cells have long been considered to be involved in the regulation of the immune response in the testis. More recently, the Fas system has been implicated in the maintenance of the immune privilege in the testis as well as in the regulation of germ cell apoptosis. However, the control of Fas and Fas ligand (FasL) expression in the testis remains unknown. In the present study, we demonstrate that cultured mouse Sertoli cells constitutively express a low level of membrane-bound Fas protein, but not a soluble form of Fas. Sertoli cells stimulated with TNF-alpha and IFN-gamma markedly increase the expression of both soluble and membrane-bound Fas in a dose-dependent manner. The up-regulated membrane-bound Fas protein is functionally active because it induces a significant level of Sertoli cell death in the presence of Neuro-2a FasL+ effector cells. Interestingly, the soluble form of Fas, which is induced by the same cytokines but has an antiapoptotic effect, is also functional. In fact, conditioned media from TNF-alpha-stimulated Sertoli cell cultures inhibit Neuro-2a FasL+-induced cell death. Taken together, our data suggest a possible regulatory role of TNF-alpha and IFN-gamma on Fas-mediated apoptosis in the testis through disruption of the balance between different forms of Fas.

Sertoli cells, but not tumor cells, of seminoma in situ express ICAM-1.

Tumor and surrounding testicular tissue from six seminomas and one combined seminoma/embryonal carcinoma were examined for the expression of ICAM-1, VCAM-1 and ELAM-1. This was done by immunohistochemical staining of frozen samples using monoclonal antibodies and the avidin-biotin/ peroxidase or alkaline phosphatase staining method. ICAM-1 was expressed by Sertoli cells of intratubular germ cell neoplasia, but not by any of the cells in normal seminiferous epithelium, or by neoplastic germ cells whether invading or not. In addition inflammatory cells and endothelium expressed ICAM-1. VCAM-1, and also occasionally ELAM-1, was expressed only on endothelial cells in and outside the tumors. These results are discussed in relation to lymphocytic infiltration and immune surveillance of seminomas and T-cell tolerance to the antigens of the immunologically privileged seminiferous epithelium.

Autoantigenicity of the basal compartment of seminiferous tubules in the rat.

The expression of testicular autoantigens has been thought to be restricted to the luminal compartment of the seminiferous tubules. In the present study it was observed that germ cells in the basal compartment of seminiferous tubules and lamina propria of the seminiferous tubule bound antibodies in anti-testis immunosera in vivo after passive transfer of the sera by intra-peritoneal injection. The anti-testis immunosera were prepared in orchiectomised rats to avoid pre-collection adsorption of antibodies in the basal compartment of the seminiferous tubules. When testis-immune sera collected from non-orchiectomised rats or normal sera were transferred no such binding of antibodies to the basal germ cell surface or the lamina propria occurred. In Western blot analysis the anti-testis immune sera prepared in orchiectomised rats defined polypeptides from the adult rat testis with relative MWs of 19-23, 26-28, 30, 34, 38, 40-43, 45-47, 51-52, 56-57, 68, 78 and 97 kDa. The 40-43 kDa band was not detected by sera prepared in non-orchiectomised rats, suggesting that this autoantigen was expressed in the basal compartment of the seminiferous tubules. These observations suggest that the segregation of testicular autoantigens in the luminal compartment of seminiferous tubules is not complete for all of them in the rat and emphasize the role of more dynamic mechanisms in prevention of anti-germ cell autoimmune disease.

HLA class I antigens are expressed by Sertoli cells of intratubular germ cell neoplasia.

Five frozen samples from seminomas and one from a combined seminoma/embryonal carcinoma and surrounding tissue were examined for the presence of HLA class I molecules using an antibody directed against a monomorphic epitope of the 45 kDa long chain of HLA-A, -B and -C molecules. Only tubules with intratubular germ cell neoplasia stained positively, while normal seminiferous tubules and invasive tumor cells stained negatively. The immunohistology with anti-HLA class I antibody was compared to that with anti-beta 2-microglobulin antibody which reacts with the light nonpolymorphic chain of all HLA class I molecules. Immunohistology with these two antibodies was indistinguishable. This result combined with immunohistology using anti-PLAP indicates that the stained cells are Sertoli cells. The results are discussed in relation to lymphocytic infiltration and immune surveillance of seminomas.

Fixation of testicular tissue for immunohistochemical and ultrastructural examination.

The fixation of testicular tissue with glutardialdehyde destroys the antigenicity of cell epitopes in many cases. To obtain both, morphological and immunohistochemical examination, a fixation which could preserve the antigenicity and condition of the testicular structure was sought. The solution obtained was a mixture of 3.7% formalin with 0.2% glutardialdehyde and 0.05% saponin in a phosphate buffer of pH 7.4. Blocks of human tests were immersed in this fixative, embedded in Epon 812 or paraffin for conventional light and electron microscopy and immunohistochemical staining. The immunohistochemical examination was focused on the lamina propria of the human seminiferous tubules. Good preservation of the structure was observed both in light and electron microscopy. Cytological details were seen by light microscopy, especially the recognition of single tumour cells. Electron microscopically, all cells of the seminiferous tubules and the lamina propria showed a well-preserved internal structure. At the same time the peritubular cells of the lamina propria exhibited a well-expressed vimentin and desmin immunoreactivity, thus providing evidence that the corresponding epitopes retain their antigenicity under these fixation conditions. The applied fixation procedure provides comparable results in preservation of the structure to glutardialdehyde but does not destroy the antigenicity of epitopes.

Expression of the proliferation-associated Ki-67 antigen in bovine testicular tubular cells during the seminiferous epithelial cycle, demonstrated with the MIB-1 antibody.

Ki-67 expression in the seminiferous tubule of the bovine testis was studied by immunohistochemistry during the seminiferous epithelial cycle using the monoclonal antibody MIB-1. Spermatogonial proliferation is most obvious in stages 5-7, and 8, when B-spermatogonia divide. A lower rate of spermatogonial propagation is observed preceding or during meiosis in stages 1-4. The MIB-1 antibody also gives positive results with some post-spermatogonial tubular cells. Preleptotenes passing through S-phase in stage 1 reveal positive nuclei. During prophase of meiosis I pachytenes react strongly, diplotenes react in an attenuated manner, while leptotenes and zygotenes stay negative. Secondary spermatocytes seen in stage 4 are positive as are the chromosomes during meta- and anaphase of the meiotic divisions. Post-meiotic spermatids are also decorated but stop Ki-67 expression abruptly at the end of stage 4. Sertoli cells are negative.

Secretion of tumor necrosis factor alpha by testicular macrophages.

While it has been shown that culture medium from testicular macrophages can influence testosterone production when added to Leydig cells, the identity of the factor(s) responsible for this activity remains unknown. Since tumor necrosis factor alpha (TNF alpha) has been shown to be capable of influencing testosterone production by Leydig cells, a series of studies was conducted to determine if testicular macrophages produce TNF alpha. It was found that testicular macrophages from adult rats produce a factor that is capable of lysing L929 cells, which are used as a traditional bioassay for TNF alpha. The TNF alpha activity in the macrophage-conditioned medium could be neutralized by the addition of anti-murine TNF alpha but not with the addition of preimmune IgG. While lipopolysaccharide (LPS) slightly increased the release of TNF alpha, neither follicle-stimulating hormone (FSH) nor testosterone had a similar effect. It was not determined if the isolation procedure had artificially 'activated' the macrophages. Medium from cultured Sertoli cells, Leydig cells and peritubular cells did not contain TNF alpha activity. These studies are consistent with the hypothesis that the paracrine interaction between testicular macrophages and Leydig cells is mediated in part by TNF alpha.

Testosterone immunoreactivity in the seminiferous epithelium of rat testis: effect of treatment with ethane dimethanesulfonate.

Androgens drive spermatogenesis by processes that are largely unknown. Direct effects on germ cells and indirect effects mediated via testicular somatic elements are currently under consideration, and specific localization of androgens in seminiferous tubules may provide information as regards this. Adult male rats were injected with ethane dimethanesulfonate (EDS; 75 mg/kg body weight) or vehicle. Testes were fixed and paraffin-embedded for localization of testosterone immunoreactivity 1 and 2 weeks after treatment, using the unlabeled antibody (PAP) technique. Plasma testosterone dropped from a pre-treatment level of 2.3 ng/ml to below 0.2 ng/ml 3 days after EDS injection and remained at low levels until the end of observation, accompanied by a progressive decrease in testicular weight. In the seminiferous tubules of vehicle-injected males, testosterone immunoreactivity was found in nuclei of spermatocytes and spermatids and in nuclei and the cytoplasm of Sertoli cells, and showed typical variations according to the stage of spermatogenesis. One week after EDS treatment, immunoreactivity had disappeared from the seminiferous epithelium. Two weeks after treatment, staining of germ cells was detected in two out of four males. The disappearance and reappearance of immunoreactivity coincided with the time course of EDS effects on rat Leydig cells, and we conclude that it corresponds to androgen specifically localized in fixed, paraffin-embedded tissue. Because staining of germ cell nuclei varied with the stage of spermatogenesis, the technique may detect a physiologically relevant androgen fraction; its location suggests that androgens may also directly affect certain germ cell stages.