Both FSH and germ cells interact with Sertoli cells by different pathways to cause a rapid activation of protein kinases and/or phosphatases. Because the mechanism of germ cell activation of Sertoli cell protein phosphorylation appears to involve increased levels of intracellular calcium, the effect of hormone or germ cell treatment on calcium levels in cultured Sertoli cells will be studied using the fluorescent calcium indicators, Fura2 and Indo1. Patterns of protein phosphorylation also suggest that the mechanism by which germ cells affect Sertoli cells involves the phosphatidylinositol (PI) pathway. Therefore, studies of Sertoli cell PI metabolism will be conducted. The factor from germ cells which stimulates Sertoli cell protein phosphorylation will be isolated, characterized and used to produce antibodies in order to learn by which germ cell stage it is produced. Two proteins whose phosphorylation is specifically affected by germ cells will be purified by conventional liquid chromatography, metal chelate chromatography, affinity, chromatography and HPLC. These phosphoproteins will be isolated in order to develop antibodies to the phosphoproteins. The antibodies will then be used to localize the phosphoproteins at the light and electron microscope levels with Sertoli cells and the testis and to study the correlation of each phosphoprotein with the cycle of the seminiferous epithelium. Antibodies will also be used to screen a Sertoli cell cDNA lambda gtll expression library in order to determine the sequences of the phosphoproteins. It is expected that localization of the phosphoproteins and determination of their amino acid sequences will lead to identification of the proteins and their functions. The studies should contribute to understanding Sertoli-germ cell interaction and hormone-mediated response mechanisms of Sertoli cells during spermatogenesis.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD017121-07
Application #
3314190
Study Section
Reproductive Biology Study Section (REB)
Project Start
1983-01-01
Project End
1994-03-31
Budget Start
1991-04-01
Budget End
1994-03-31
Support Year
7
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Hew, K W; Ericson, W A; Welsh, M J (1993) A single low cadmium dose causes failure of spermiation in the rat. Toxicol Appl Pharmacol 121:15-21
Hew, K W; Heath, G L; Jiwa, A H et al. (1993) Cadmium in vivo causes disruption of tight junction-associated microfilaments in rat Sertoli cells. Biol Reprod 49:840-9
Redmond, T; Brott, B K; Jove, R et al. (1992) Localization of the viral and cellular Src kinases to perinuclear vesicles in fibroblasts. Cell Growth Differ 3:567-76
Pittenger, G L; Gilmont, R R; Welsh, M J (1992) The low molecular weight heat shock protein (hsp27) in rat Sertoli cells: evidence for identity of hsp27 with a germ cell-responsive phosphoprotein. Endocrinology 130:3207-15
Welsh, M J; Ireland, M E (1992) The second messenger pathway for germ cell-mediated stimulation of Sertoli cells. Biochem Biophys Res Commun 184:217-24
Van Eldik, L J; Barger, S W; Welsh, M J (1992) Antisense approaches to the function of glial cell proteins. Ann N Y Acad Sci 660:219-30
Sweet, S C; Gnegy, M E; Welsh, M J (1991) Presence of matrix-specific antibodies in affinity-purified polyclonal antibodies. J Immunol Methods 136:31-6
Selinfreund, R H; Barger, S W; Welsh, M J et al. (1990) Antisense inhibition of glial S100 beta production results in alterations in cell morphology, cytoskeletal organization, and cell proliferation. J Cell Biol 111:2021-8
Sanchez, E R; Hirst, M; Scherrer, L C et al. (1990) Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90. J Biol Chem 265:20123-30
Redmond, T; Sanchez, E R; Bresnick, E H et al. (1989) Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells. Eur J Cell Biol 50:66-75

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