The overall goal is to obtain from rat Sertoli cells cDNA probes for mRNA sequences that are preferentially stimulated by FSH or testosterone. The technique of subtractive hybridization will be used to generate a library of cloned cDNA that is enriched in hormone responsive sequences. The mRNA from normal Sertoli cells will be reverse transcribed and hybridized to an excess of mRNA from Sertoli cells from hypophysectomized rats. The unhybridized cDNA will be cloned and hormone responsive sequences will be selected via a 3 way screen. The cloned cDNA will be screened by 32P-cDNA prepared from enriched mRNA isolated from Sertoli cells from hypophysectomized rats, hypox. rats treated with FSH and, hypox. rats treated with testosterone. Hormone responsive sequences which are identified by the screen will be characterized as to tissue specificity by Northern blot analysis and size by agarose gel electrophoresis. The probes of interest will be sequenced and the protein coded for will be examined by hybrid-release translation. The characterized probes will be used in studies designed to examine their expression in Sertoli cells in vivo and in culture. Liquid RNA-RNA hybridization techniques will be used to quantify the amount of specific hormone responsive mRNA in cultured Sertoli cells treated with various hormones and in rats which are vitamin A deficient, hypophysectomized, treated with anti-androgens and of various ages. The probes will also be used to quantify the expression of specific mRNA in Sertoli cells associated with different stages of the cycle of the seminiferous epithelium. Once these probes are fully characterized and their expression is well defined they can be used to begin to explore the mechanism(s) by which FSH and testosterone regulate Sertoli cell functions and ultimately the process of spermatogenesis.
| Griswold, M D; Bishop, P D; Kim, K H et al. (1989) Function of vitamin A in normal and synchronized seminiferous tubules. Ann N Y Acad Sci 564:154-72 |
| Roberts, K; Griswold, M D (1989) Testosterone induction of cellular proteins in cultured Sertoli cells from hypophysectomized rats and rats of different ages. Endocrinology 125:1174-9 |
| Morales, C R; Alcivar, A A; Hecht, N B et al. (1989) Specific mRNAs in Sertoli and germinal cells of testes from stage synchronized rats. Mol Endocrinol 3:725-33 |
| Hugly, S; Roberts, K; Griswold, M D (1988) Transferrin and sulfated glycoprotein-2 messenger ribonucleic acid levels in the testis and isolated Sertoli cells of hypophysectomized rats. Endocrinology 122:1390-6 |
