Charles Flickinger - US grants

The objective of the Tissue Specificity Core is to provide initial data on the tissue specificity of putative gamete-specific antigens by assessing the expression of the antigen and of the mRNA for the antigen in a variety of tissues. The Core will also serve as a source of different primate tissues for use in the sub-projects. 1. The presence of mRNA for putative gamete-specific antigens in non- gonadal organs will be sought using Northern analysis. The core will also utilize RT-PCR studies (reverse transcriptase polymerase chain reaction) for more sensitive detection of low abundance mRNAs for selected antigens of special interest. 2. Potential cross-reactivity of antibodies to various gamete antigens in non-gonadal primate tissues will be examined by immunohistochemistry and by competitive ELISA assays. 3. The Core will serve as a source of large numbers of different primate tissues. Tissue samples and isolated RNA will continue to be made available to investigators for use in tissue specificity and related studies carried out in the investigators' laboratories. The data generated from the Tissue Specificity Core will be a first step in assessing the gonadal specificity of gene products which are candidates for contraceptive vaccines. In addition, analysis of cross-reactivity of sera from fertility trials will aid in assessing the performance of the immunogens. The studies performed by this core form the basis for further immunopathologic studies to be carried out as needed in the individual projects to further investigate tissue cross-reactivity.

The overall goal of this project is to determine the effects of obstruction of the male reproductive tract on the structure and function of its components, with emphasis on the development of germ cells in the seminiferous tubules, induction of antisperm antibodies, and characterization of dominant post-obstruction sperm autoantigens. The studies are aimed at understanding basic responses to obstruction of the vas deferens, which can result from developmental defects, trauma, and vasectomy. The proposed work builds on observations of increased antibodies to specific sperm autoantigens after prepuberal or adult obstruction of the vas deferens and of testicular alterations following vasectomy. The first aim is to determine whether early reversal of vasal obstruction or later postpubertal repair is more consistent with normal development of the testis and epididymis in a rat model system. The second aim is to determine whether obstruction of the vas deferens results in changes in apoptosis in cells of the seminiferous tubules and the epididymal epithelium. The third aim is to identify, isolate, and clone cDNAs to characterize the dominant sperm autoantigens post-obstruction in the rat. Recently, the first post- obstruction sperm autoantigen in the rat model has been successfully cloned, sequenced and expressed. Plasma cells that are producing antibodies to specific sperm antigens also will be localized by a labeled antigen method. The fourth aim is to determine if immunization with a purified recombinant post- obstruction sperm autoantigen results in reproductive tract alterations such as orchitis and epididymitis and to assess how responses to specific autoantigens contribute to post-obstruction chances. The fifth aim is to extend studies of dominant sperm autoaintigens from the rat to humans. The focus will be on species conserved antigens under the principle that conservation of antigens between species reflects conservation of important functions.

women's health; reproductive system; Primates;

DESCRIPTION (provided by applicant): This proposal focuses on the development, regulation and discovery of naturally occurring antimicrobial proteins in the epididymis, highlighting the defensins and cathelicidins. Natural antimicrobial proteins are part of the innate immune system, and they likely protect the reproductive tract from invasion by pathogenic microbes thus helping to prevent diseases such as epididymitis. The first aim is to identify novel antimicrobial epididymal proteins in epididymal luminal fluid and to test their biological activities. The reproductive organs produce a variety of antimicrobial substances and it is likely that some of these natural antibiotics remain to be characterized. A step-wise approach will be used to test for antibacterial activity in luminal fluid from different regions of the epididymis, and subsequently to identify, characterize, and analyze the specificities of antimicrobial proteins. The second aim is to determine the pattern of expression of mRNA and protein for selected defensins and cathelicidin during development of the rat epididymis. These studies will determine the temporal onset of expression, quantify changes in expression levels during development, and localize expression to specific epithelial cell types. The third aim is to determine factors regulating expression of antimicrobial proteins in the epididymis by testing the effects of androgens, luminal fluid, exposure to bacterial products, and obstruction on expression of selected genes. Methods include detection of antimicrobial proteins by a gel overlay method and other bacteriologic assays, northern and western analyses, real time PCR, in situ hybridization, and immunohistochemistry, 2-D gel electrophoresis, microsequencing by mass spectrometry, and standard approaches of molecular biology. A set of antimicrobial proteins known to be present in the rat epididymis will be studied in aims 2 & 3: rat beta defensins 1 and 2 (RBD-1 and RBD-2), Bin1b, a cathelicidin (rCRAMP), and the defensin-like molecule E-3. The proposed studies will increase our knowledge of innate antimicrobial proteins in the male reproductive system, and they also present the opportunity for discovery and characterization of new antibiotic agents.