Spermatogenesis is a complex cyclic process involving the interaction of many different cell types. The process of spermatogenesis can be described in terms of recurring patterns of cellular associations. These associations define the cycle of the seminiferous epithelium which has 14 distinct stages in the rat. All 14 stages appear in the rat testes at any given time and spermiation which occurs only in stage VIII takes place continuously in various parts of the tubules. The experiments described in this proposal are designed to identify and characterize unique gene products associated with testicular cells which are expressed only at specific stages of the cycle. These experiments are possible because of 2 major technical advances: First, by feeding young rats a vitamin A deficient diet for several weeks and then restoring the missing vitamin to the diet, spermatogenesis ceases and then reinitiates. In the reinitiated testes only 2 to 4 stages of the cycle are present at a given time and spermatogenesis proceeds in a synchronous fashion. Second, a novel technique for subtractive hybridization which utilizes biotinylated RNA and streptavidin has been developed and has proven to be very effective. The proposed experiments are to obtain mRNA from testes synchronized to stages 11-V (early), VII-VIII (middle), and XII-XIV (late) in the cycle. Subtractive hybridization procedures will be used to generate libraries of sequences which are unique or enriched in each stage. After appropriate screening techniques, transcripts will be characterized as to cellular origin, DNA sequence and derived amino acid sequence. It is possible that the data we obtain will be important in: a) providing a more complete understanding of the development and maintenance of the spermatogenic cycle including insights into the function of the components, b) providing cell specific and stage specific markers for future studies, c) delineation of limiting or regulatory factors in spermatogenesis, d) providing molecular explanations for a variety of pathologies associated with spermatogenesis, e) providing a potential for the techniques of genetic engineering to be used to alleviate these pathologies, f) suggesting new factors which may be important for the eventual success of in vitro spermatogenesis, g) suggesting new approaches for the rationale design of contraceptive or fertility drugs.
