Past studies from our laboratory have shown that, during rat testicular development, Sertoli cells synthesize and secrete a protein (S45-S35 heterodimeric protein; synonymous names: SGP-2, clusterin, TRPM-2) which then acquires a spermatogenic stage-dependent expression pattern. The purpose of this renewal application is to continue our study of S45-S35 function in spermatogenesis. During the previous funding periods, we have shown that components of the head and tail of developing spermatids and epididymal sperm contain proteins with antigenic homology to S45-S35. We have also examined the expression of S35-S45 mRNA during testicular development and spermatogenesis. During the current funding period we have found that pachytene and spermatids also express S35-S45 mRNA. However, while in Sertoli cells the somatic-type S35-S45 encoded precursor gives rise to the disulfide linked S45-S35 heterodimer, the spermatogenic-type S35-S45 gene generates a similar precursor but its post-translational processing yields monomers. We have also shown that proteins associated with both outer dense fiber (ODF) polymers of sperm tail and sperm protamine-DNA chromatin are antigenically homologous to the S45-S35 precursor. As a result of these studies we have identified structural components in sperm with a role in the morphogenesis of ODF polymers and packaging of sperm chromatin in an orderly fashion. Therefore, we are in a unique situation to analyze molecular aspects of spermiogenesis, in particular the marking genes which may be required during spermiogenesis and, eventually, at fertilization and during early embryogenesis. During the next funding period, we will address specific aspects of the structure of the spermatogenic-type S35-S45 gene and its function in sperm formation through five specific aims: (1) to determine the structure and functional regulation of the spermatogenic-type S35-S45 gene, in particular, the characterization of alternative polyadenylation and splicing sites and identification of nuclear regulatory elements controlling the in vitro transcription of somatic- and spermatogenic-type S35-S45 genes. (2) To characterize molecular aspects of the coiled-coil subunits of ODF polymers and ODF-associated protein(s) and to examine more rigorously the molecular basis for the antigenic homology of ODF-associated proteins with the common somatic and spermatogenic precursor. (3) To elucidate early stages of the assembly of the ODF polymer by in vitro reconstitution and chemical crosslinking of ODF subunits and ODF-associated protein(s). (4) Isolate sperm nuclear matrix proteins bound to a distinct portion of the sperm DNA loop, define DNA molecular characteristics and determine the primary structure of a 38.5 kD nuclear matrix protein antigenically homologous to the somatic/spermatogenic common precursor. (5) Isolate and identify DNA sequences specifically associated with segments of sperm DNA loops bound to or free of nuclear matrix proteins and approach the question of gene marking during spermiogenesis. Results from these studies are significant for understanding molecular mechanisms whereby anomalous transcripts can lead to the formation of abnormal and unfertile sperm as well as testicular malignancies.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD011884-14
Application #
3311707
Study Section
Reproductive Endocrinology Study Section (REN)
Project Start
1978-04-01
Project End
1995-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
14
Fiscal Year
1992
Total Cost
Indirect Cost
Name
City College of New York
Department
Type
Schools of Medicine
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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