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Basement Membrane Increases G-Protein Levels and Follicle-Stimulating Hormone Responsiveness of Sertoli Cell Adenylyl Cyclase Activity^*

MARTIN DYM, SUPANYA LAMSAM-CASALOTTI, MENG-CHUN JIA, HYNDA K. KLEINMAN and VASSILIOS PAPADOPOULOS

Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes of Health (M.D., H.K.K.) Bethesda, Maryland 20892
Department of Anatomy and Cell Biology, Georgetown University Medical Center (M.D., S.L.-C.,M.-C.J., V.P.) Washington, D.C. 20007

Address correspondence to: Dr. Vassilios Papadopoulos, Department of Anatomy and Cell Biology, Georgetown University School of Medicine, 3900 Reservoir Road NW, Washington, DC 20007.

Abstract

On a basement membrane substrate, Sertoli cells in culture have been shown to assume a phenotype similar to that of the in vivo differentiated cells. Sertoli cells from 10-dayold rats were cultured on plastic and on different extracellular matrix substrates [laminin, a reconstituted basement membrane (Matrigel), and a synthetic laminin peptide containing the arginine- glycine-aspartic acid (RGD) tripeptide sequence] to investigate the effects of the extracellular matrix on FSH responsiveness. Both laminin and Matrigel markedly enhanced, the cAMP response to FSH and cholera toxin, indicating modifications at the level of guanine nucleotide-binding regulatory (G) proteins. Furthermore, Sertoli cells grown on either of theses two substrates responded to physiological levels of FSH (25–50 ng/ ml), whereas pharmacological levels of FSH (500 ng/ml) were required for cells grown on either plastic or on the RGDcontaining laminin peptide. Immunoblotting of Sertoli cell plasma membranes with antibodies directed against the -subunit of the stimulatory G-protein (Gsa) of adenylyl cyclase indicated that Sertoli cell culture on either laminin or Matrigel increased the amounts of Gsa. These results were further confirmed by immunoprecipitating the Gsa protein from the particulate fraction of [³⁵S]methionine metabolically labeled Sertoli cells. However, Northern blot analysis using a cDNA probe for Gsa did not demonstrate changes in gene expression when Sertoli cells were grown on the various substrates. Immunofluorescent studies revealed that the Gs complex of adenylyl cyclase was preferentially located at the base of the Sertoli cells at the site of contact with the extracellular matrix. These data suggest that culture of epithelial Sertoli cells on basement membrane substrates enhances the Gs complex of adenylyl cyclase and the cAMP response to FSH, consistent with the more differentiated morphology and function of the cells. (Endocrinology 128: 1167–1176, 1991)

Footnotes

^* This work was supported by Grant HD-16260 from the NIH and a grant from the Mellon Foundation.

WHO research training grant recipient.

Received August 30, 1990.

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