Mp Hedger

Intragonadal regulation of immune system functions.

Immune responses within the mammalian gonads, and in particular the testis, are deficient in spite of adequate lymphatic drainage and the presence of lymphocytes and MHC II+ macrophages. There is considerable evidence from in vivo and in vitro studies that this suppression of the immune system may be due, at least in part, to localized inhibition or regulation of normal lymphocyte and/or macrophage functions within the gonads. In the testis, both steroidal and non-steroidal products of the Leydig cells, including androgens, endorphins, and inhibin-related proteins, have been implicated in mediating this activity. In turn, a number of immune cell cytokines affect steroidogenic cell function in vitro. The studies described in this paper indicated that [3H]-thymidine incorporation by adult rat thymocytes in vitro was inhibited by conditioned medium collected from short-term incubations of Percoll-purified adult rat Leydig cells, but stimulated by testicular interstitial fluid and by conditioned medium collected from short-term incubations of adult rat seminiferous tubules. The factors responsible for these effects on thymocyte function appeared to be of large molecular weight, as they were retained by ultrafiltration membranes with exclusion limits of 10,000 or 30,000 daltons. It is hypothesized that an immunosuppressive mechanism, principally mediated by non-steroidal factors secreted by the steroidogenic cells of the gonadal interstitial tissue, exists within the gonads in order to prevent activation of the immune system by germ cell antigens and growth factors associated with germ cell proliferation and differentiation. This mechanism probably acts in parallel with normal antigen-specific tolerance mechanisms operating at the gonadal level. As immune responses to germ cells are believed to be a significant causative factor in infertility, particularly in men, this represents an important area for further study.

Degradation of luteinizing hormone-releasing hormone (LHRH) and an LHRH agonist by the rat testis

Degradation of LHRH and [D-Ser(tBu)6,des-Gly-NH10(2)]LHRH ethylamide (LHRH-A), during incubation with high-speed supernatants of rat testes, as assessed by reversed-phase (RP)-HPLC fractionation of the iodinated peptides and by radioimmunoassays for LHRH or LHRH-A, was principally due to a neutral 43 000 Da peptidase with apparent Km values at 25 degrees C of 0.15 microM for LHRH and 1.19 microM for LHRH-A. The peptidase was inhibited by sulphydryl reagents, TLCK, 1,10-phenanthroline, EDTA, bacitracin, other LHRH analogues, oxytocin, [Lys8]vasopressin and somatostatin. It was predomantly located in seminiferous tubule supernatants (98% of recovered activity), with much lower levels in interstitial fluid (2%), interstitial tissue or testicular particulate fractions (less than 0.8%). Extracts of cultured immature Sertoli cells produced LHRH- and LHRH-A-degradation profiles, as assessed by RP-HPLC, that were identical to those produced by testicular supernatants. Similar levels of peptidase activity/mg protein were observed in immature and adult rat testes. These studies indicate that the principal LHRH-peptidase in the rat testis is produced by cells of the seminiferous epithelium, chiefly the Sertoli cell, and may play an important role in regulating the activity of LHRH and other peptide hormones in the testis.

259. Identification of elevated levels of apoptosis among T-cells isolated from the rat testis

Protection of the developing gametes from attack by the immune system is essential for reproductive success. Autoimmune infertility represents a failure of this protection. Specific T-cell apoptosis is the main mechanism for control of antigen-specific immune responses. Studies were undertaken to investigate this regulatory process in adult rat testes. Flow cytometry was employed in conjunction with annexin-V/propidium iodide dual staining to identify apoptotic cells concurrent with CD3 staining to identify T-cells. CD3-positive cells isolated from the testicular interstitial tissue were shown to be 34.12xa0±xa03.0% apoptotic (meanxa0±xa0s.e.m., nxa0=xa03) at collection. This was consistently greater than the numbers of apoptotic CD3-positive cells isolated from lymph nodes (4.04xa0±xa01.95%, nxa0=xa02), spleen (16.77xa0±xa04.73%, nxa0=xa04) and peripheral blood (9.64xa0±xa01.44%, nxa0=xa02). These results also were confirmed by using T-cells purified with MACS microbeads against the pan T-cell marker OX52 to improve sample purity: 40% of isolated testicular T-cells and 3% lymph node T-cells were found to be undergoing apoptosis. The level of apoptosis among T-cells isolated from another non-lymphoid organ, the liver, was only 6%. It is hypothesised that the immunosuppressive milieu of the testis induces an increased level of apoptotic deletion among T-cells that gain entry into the testis and potentially threaten gamete viability. Further studies of the mechanism responsible for this elevated level of T-cell apoptosis in the testis will significantly enhance our knowledge of how testicular immune tolerance is maintained.