Molecular identification of lyso-glycerophosphocholines as endogenous immunosuppressives in bovine and rat gonadal fluids.

The ability of the gametes to escape detection by the immune system is vital to successful human reproduction. Furthermore, the observed capacity of the testis in some species to support tissue grafts without rejection (immunological privilege) indicates that spermatogenic cells are protected by local immunoregulatory mechanisms. One of these mechanisms involves targeting T cells for inactivation and destruction within the testicular environment. Although the fluids of the testis and ovary surrounding the developing gametes contain soluble factors that inhibit T cells, the identity of the molecule(s) responsible for this activity has been unknown. Using a specific T-cell proliferation assay to monitor bioactivity, these molecules were purified from bovine ovarian follicular fluid by methanol extraction and sequential reverse-phase HPLC (RP-HPLC). All purified active fractions coincided with the elution position on RP-HPLC of several small molecules ranging in size from 496 to 522 Da. The same molecules were localized to the immunosuppressive fractions of rat testicular interstitial fluid. The active molecules were identified, using capillary electrophoresis electrospray ionization mass spectroscopy, as lyso-glycerophosphocholines (lyso-GPCs), namely, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, a 18:2a/lyso-GPC (putatively, 1-linoleoyl-sn-glycero-3-phosphocholine), and a 20:4a/lyso-GPC (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine). Comparison of the bioactivity and mass spectroscopy profiles of two of the purified molecules with their synthetic standards confirmed the identification. These molecules inhibit T-cell proliferation in response to activation and induce apoptosis of these cells in a time- and dose-dependent manner. The emergence of gonadal lyso-GPCs as potential regulators of critical immune events opens up new avenues of inquiry into the origins of autoimmune infertility and more generally into mechanisms of peripheral immunoregulation and the development of novel immunosuppressives.

Cytokine profiles in the testes of rats treated with lipopolysaccharide reveal localized suppression of inflammatory responses.

Evidence indicates that the testis possesses a reduced capacity to mount inflammatory and rejection responses, which undoubtedly contributes to the ongoing survival of the highly immunogenic germ cells. The contribution of local cytokine expression to this condition was investigated in adult male rats treated with lipopolysaccharide to induce inflammation. Cytokine mRNA and protein expression were determined in tissue extracts and fluids by Northern blot analysis, quantitative PCR, or RNAse protection assay and specific ELISAs. Testicular expression of the proinflammatory cytokines, interleukin (IL)-1beta and tumor necrosis factor-alpha was considerably attenuated compared with the liver (control tissue); in contrast, the testicular IL-6 response was enhanced. Expression of IL-10, a type 2 immunoregulatory cytokine, was similar in both testis and liver, whereas the immunoregulatory/anti-inflammatory cytokines transforming growth factor-beta(1) and activin A were constitutively elevated in both normal and inflamed testes. The IL-1beta and transforming growth factor-beta(1) proteins were present principally in their latent (inactive) forms, indicating that enzymic processing is an important control mechanism for these two cytokines within the testis. These data indicate that inflammatory and regulatory cytokine activity is regulated at both transcriptional and posttranslational levels in a testis-specific manner. It is concluded that a novel pattern of suppression of proinflammatory cytokine responses and normal or elevated expression of immunoregulatory cytokines may be responsible for reduced inflammatory responses and enhanced graft survival in the testis. These data have important implications for the understanding and treatment of male autoimmune infertility, testicular inflammation. and carcinogenesis.

Identification of a novel apolipoprotein, ApoN, in ovarian follicular fluid.

A novel apolipoprotein, designated ApoN, has been identified in bovine ovarian follicular fluid using chromatographic purification methods, amino acid sequence analysis, molecular biology, and bioinformatics. The apolipoprotein is a hydrophobic 12-kDa protein processed from the C terminus of a 29-kDa precursor expressed in a number of tissues, including the ovary, testis, the anterior chamber of the eye, skeletal muscle, uterus, and liver. Bovine, porcine, and murine ApoN display significant homology at the amino acid level across the entire precursor sequence. Surprisingly, there appears to be no orthologous protein in the human, although an APON-like pseudogene is found on chromosome 12. The N-terminal fragment of the ApoN precursor shows significant homology with the N-terminal sequence of the precursor of the cholesterol transport regulatory protein ApoF, but the corresponding C-terminal sequences of ApoN and ApoF possess no homology. ApoN is present in the high-density lipoprotein fraction of bovine serum and both the high-density lipoprotein and low-density lipoprotein fractions of bovine follicular fluid and is found in several tissues that are associated with local immunological privilege. These data suggest that ApoN may play a role in steroidogenesis and/or immunoregulation in the gonads of nonhuman species, as well as similar roles in other tissues.