Sertoli cells play an important role in the bioregulation of spermatogenesis by providing mechanical support and chemical signals to spermatogenic cells. Work of this laboratory has shown that the intermediate filament protein, vimentin is phosphorylated in a FSH-dependent manner in cultured Sertoli cells. We now want to determine the mechanisms regulating the phosphorylation of vimentin and other cytoskeletal proteins of Sertoli cells during different stages of rat spermatogenesis. Two-dimensional polyacrylamide gel electrophoresis will be used to resolve [32P]orthophosphate and [Gamma-32P]ATP-labeled proteins following activation of cAMP- and Ca2+/calmodulin-dependent protein kinases. Preliminary studies have shown that Sertoli cells secrete specific proteins that are also spermatogenic stage-dependent. We propose the identification of factors that control the synthesis of these proteins during rat spermatogenesis. Seminiferous tubular segments corresponding to distinct spermatogenic stages identified by their transillumination pattern and histology will be labeled with [35S]methionine and secretory proteins resolved by gel electrophoresis. Sertoli cell-specific secretory proteins designated S80, S45 and S35 will be purified on DEAE-cellulose column chromatography and characterized. Antibodies against these proteins will be produced. The effect of growth factors on the regulation of the mitogenic and non-mitogenic function of Sertoli and peritubular cells will be evaluated using the complementation test of Stiles et al. and gel electrophoresis to define autocrine or paracrine effects. Factors involved in the modulation of Sertoli cell function induced by peritubular cells will be studied using a coculture method that allows cell-cell functional interaction but prevents cell-cell contact. We will use gel electrophoresis to determine the effect of peritubular cell-specific proteins, designated P95, P40 and P38, on the synthesis and secretion of [35S]methionine-labeled Sertoli cell-specific proteins, S80, S45 and S35. These Sertoli cell-specific secretory proteins are of interest because they have been shown to be expressed in a spermatogenic stage-dependent manner and, therefore, they may play an important role in spermatogenesis. Knowledge of the regulatory mechanisms of spermatogenesis are relevant to the development of acceptable methods of contraception and treatment of infertility of the male.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD011884-09
Application #
3311709
Study Section
Reproductive Biology Study Section (REB)
Project Start
1978-04-01
Project End
1988-06-30
Budget Start
1987-04-01
Budget End
1988-06-30
Support Year
9
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Mochida, K; Tres, L L; Kierszenbaum, A L (1998) Isolation of the rat spermatid manchette and its perinuclear ring. Dev Biol 200:46-56
Rivkin, E; Cullinan, E B; Tres, L L et al. (1997) A protein associated with the manchette during rat spermiogenesis is encoded by a gene of the TBP-1-like subfamily with highly conserved ATPase and protease domains. Mol Reprod Dev 48:77-89
Tres, L L; Kierszenbaum, A L (1996) Sak57, an acidic keratin initially present in the spermatid manchette before becoming a component of paraaxonemal structures of the developing tail. Mol Reprod Dev 44:395-407
Tres, L L; Rivkin, E; Kierszenbaum, A L (1996) Sak 57, an intermediate filament keratin present in intercellular bridges of rat primary spermatocytes. Mol Reprod Dev 45:93-105
Kierszenbaum, A L; Rivkin, E; Fefer-Sadler, S et al. (1996) Purification, partial characterization, and localization of Sak57, an acidic intermediate filament keratin present in rat spermatocytes, spermatids, and sperm. Mol Reprod Dev 44:382-94
Kierszenbaum, A L (1994) Mammalian spermatogenesis in vivo and in vitro: a partnership of spermatogenic and somatic cell lineages. Endocr Rev 15:116-34
Tres, L L; Cahn, F; Kierszenbaum, A L (1992) Permeable collagen-glycosaminoglycan cross-linked copolymers for the study of biological responses of cocultured Sertoli and spermatogenic cells. Cell Biol Toxicol 8:61-71
Zakeri, Z; Curto, M; Hoover, D et al. (1992) Developmental expression of the S35-S45/SGP-2/TRPM-2 gene in rat testis and epididymis. Mol Reprod Dev 33:373-84
Smith, F F; Mertz, J R; Krebs, I et al. (1992) Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein. Mol Reprod Dev 33:363-72
Kierszenbaum, A L; Ueda, H; Tres, L L (1991) Immunogold electron microscopic localization of antigenic sites in the outer dense fiber region of rat sperm tail obtained by using antisera to two Sertoli cell secretory proteins. J Electron Microsc Tech 19:261-8

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