We have found that Sertoli cell secretory protein S70 share antigenic homology with polypeptide components of Sertoli cell secretory protein S45-S35 heterodimeric protein. We have made the unexpected observations that polyclonal antisera raised against these two Sertoli cell secretory proteins crossreact with outer dense fiber polypeptides of sperm tail and that a polyclonal antiserum generated against a major polypeptide of outer dense fibers of sperm tail crossreacts with protein S70 and its breakdown products. These findings suggest a possible product-precursor relationship between S70 and S45-S35 heterodimeric protein and raise important questions concerning the cell origin and molecular organization of outer dense fiber polypeptides of the sperm tail. To determine whether S70 is a precursor for S45-S35 heterodimeric protein, we will analyze the structure and expression of S70 and S45-S35 heterodimeric protein genes and search for regions of genomic homology in spermatogenic cells. Polyclonal antibodies raised against these two Sertoli cell secretory proteins will be used to screen rat testis cDNA expression libraries in lambda gtll phage expression vector. Inserts from positive clones will be subcloned into a multifunctional plasmid vector to determine the complete cDNA sequence and derive protein amino acid sequence. Derived amino acid sequence will be compared with N- terminus amino acid sequence analysis of protein S70 and outer dense fiber polypeptides. cDNA will be used for in situ cytohybridization and Northern blot experiments to determine whether spermatogenic cells, mainly spermatids, contain genome sequences and transcripts complementary to Sertoli cell secretory proteins. To determine whether Sertoli cell secretory proteins are related to outer dense fiber polypeptides, we will raise polyclonal antibodies against purified outer dense fiber polypeptides from sperm tail and search for crossreactive epitopes and N-terminus amino acid sequence homology of Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein and outer dense fiber polypeptides. The subcellular localization and fate of both Sertoli cell secretory proteins and outer dense fiber polypeptides will be determined in rat testis by immunogold electron microscopy. Ultimately, the ontogeny, hormonal and spermatogenic cell dependency of Sertoli cell secretory proteins S70 and S45-S35 heterodimeric protein gene activity and expression will be determined in rat testis during development and in cultured Sertoli cells using an automated cell perifusion system combined with radioimmunoassay.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD011884-11A2
Application #
3311706
Study Section
Reproductive Biology Study Section (REB)
Project Start
1978-04-01
Project End
1992-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
11
Fiscal Year
1989
Total Cost
Indirect Cost
Name
City College of New York
Department
Type
Other Specialized Schools
DUNS #
603503991
City
New York
State
NY
Country
United States
Zip Code
10031
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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