The goals of this project are to identify the changes in gene expression in various testis lesions induced by known toxicants, determine which cell types and mechanisms are altered after exposure to a toxicant, and define the sequence of changes in gene expression to separate earlier from later effects in testis lesions. We hypothesize that altered patterns of gene expression can be used to identify primary and secondary responses and to identify the initial target cell of a toxicant in the testis, and that the temporal pattern of changes in gene expression can be used to define the mechanisms of toxicity. We are using the NIEHS mouse microarray representing ~8700 genes expressed in a variety of tissues to determine the effects of 4 known male reproductive toxicants. They are tri-o-cresyl phosphate (TOCP), ethylene glycol monomethyl ether (EGME), Boric Acid (BA), and Cadmium (Cd). TOCP initially produces vacuoles in Sertoli cells, followed by death of early pachytene spermatocytes and round spermatids. EGME kills pachytene spermatocytes, but also produces concomitant effects on Sertoli cells. BA inhibits sperm release and is likely to act on Sertoli cells, but differently than TOCP and EGME. Cd produces divergent effects on both Leydig cells and Sertoli cells. These chemicals were chosen because they have relatively specific effects on the testis, but produce these effects at different times after initial exposure and appear to act through different routes and/or target different cell types. The present goal is to determine if changes in patterns of gene expression in the testis in response to these toxicants involves a distinct subset of genes for each toxicant, moderately overlapping subsets of genes, or a particular group of genes that responds to all 4 toxicants. Longer term goals are to determine the specific effects of different toxicants on gene expression in individual cell types of the testis, the time course of changes in gene expression induced by toxicants in different cell types of the testis, the patterns of changes in gene expression with different doses of toxicants, and the similarities and differences in the patterns of change in gene expression induced by other chemicals known or suspected to cause injury to the testis. The NIEHS mouse microarray has a low representation of genes expressed in the male germ cells and it is likely that significant changes in the level of expression were not detected for many genes in the testis. We have begun work on preparing a mouse testis microarray that should be considerably more informative for studies of male reproductive toxicants. Nine cDNA libraries constructed with mRNA from mouse germ cells isolated at different stages of development were sent to the Lawrence Livermore National Laboratory to be arrayed. Three of these libraries were partially sequenced at the Washington University Genome Center. At the present time, 1431 ESTs from a round spermatid library, 2511 ESTs from a pachytene spermatocyte library, and 1840 ESTs from a type B spermatogonial library are listed in GenBank. The clones for these ESTs were deposited with IMAGE associates and are available to all investigators. The ESTs from the 3 libraries currently are being subjected to computer analysis to identify a subset for inclusion on a future microarray. These three libraries need to be subjected to more sequencing and the other libraries also need to be sequenced to identify a significant sampling of genes expressed in the testis. However, the partial sequencing of the 3 libraries was supported by CGAP at NCI and their funding for the mouse EST project at Washington University has ended. Results of these studies will be useful for determining the effects of reproductive toxicants on expression of gene clusters in the testis. The testis has an unusually large number of unique genes, testis-specific homologs and members of gene families, and alternate transcripts. The expression of most, if not all of these genes is developmentally regulated. Most of these genes are expressed in germ cells and are not represented on the current NIEHS mouse microarray. The testis also contains a variety of somatic cells types including Sertoli cells surrounding the germ cells, Leydig cells between the tubules that produce testosterone, peritubular cells that surround the seminiferous tubule and respond to endocrine influences and produce growth factors, and other cell types, which results in a large diversity of genes being expressed. Although the current NIEHS microarray is not ideal for our study, we anticipate that valuable information will be gained and can be supplemented in the future when a more inclusive
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