Advertisement

Studies on Regulation of Ovarian Steroidogenesis in Vitro: The Need for a Serum-Free Medium

  • Joseph Orly
  • Patricia Weinberger-Ohana
  • Yigal Farkash
Conference paper
Part of the Proceedings in Life Sciences book series (LIFE SCIENCES)

Summary

Granulosa cells from rat ovary can be readily grown in serum-free medium. The defined medium consists of a basal nutrient mixture of DMEM and Ham’s F-12 media, supplemented by insulin, transferrin, hydrocortisone and fibronectin (4F medium). In 4F medium the granulosa cells expressed all their known differentiated functions: in response to gonadotropic hormone such as follitropin (FSH), the cells produced progestins and estrogen as well as expressed new receptors to lutropin (LH). In contrast to the cells’ responsiveness in 4F medium, the presence of serum in the culture medium clearly suppressed the expression of all three hormonally-induced functions. As low as 0.2% serum was sufficient to block 90% of the progestin production in response to FSH.

Since cyclic AMP is now accepted to be the intracellular mediator of all the gonadotropin-induced functions, we tested whether serum impairs the accumulation of cAMP triggered in the granulosa cells by FSH. It was evident that cells grown in the presence of serum accumulated 17 times less cAMP compared with cells exposed to FSH in 4F medium. However, addition of a phosphodiesterase inhibitor (IBMX) to the serum-containing medium, practically abolished the serum inhibition of cAMP accumulation. These results ruled out the possibility that serum suppresses FSH-induced functions merely by inhibition of FSH binding to its receptor or by impairing, by as yet an unknown mechanism, the FSH induced activation of adenylate cyclase. Alternatively, these findings suggest that serum diminishes the rising intracellular cAMP pool by apparently activating a phosphodiesterase enzyme which rapidly degrades the freshly generated cyclic nucleotide. Although there is still room for further study of phosphodiesterase effect in both intact granulosa cells and cell-free lysates, the concept seems attractive regarding the possibility that serum can modulate the cultured cell responsiveness to hormones by activating the cyclic nucleotide catabolizing enzyme, rather than directly inhibiting the cAMP generating system, namely, adenylate cyclase.

Keywords

Granulosa Cell Cyclic Nucleotide Adenylate Cyclase Assay Isolate Granulosa Cell Fetal Calf Seron 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Coon, H.G. (1966) Proc. Natl. Acad. Sci. USA 55, 66–73.PubMedCrossRefGoogle Scholar
  2. 2.
    Seeds, N., Gilman, A., Amano, J. and Nirenberg, M. (1970) Proc. Natl. Acad. Sci. USA 66, 160–167.PubMedCrossRefGoogle Scholar
  3. 3.
    Bottenstein, J.E. and Sato, G.H. (1979) Proc. Natl. Acad. Sci. USA 76, 514–517.PubMedCrossRefGoogle Scholar
  4. 4.
    Darmon, M., Bottenstein, J. and Sato, G. (1981) Develop. Biol. 85, 463–473.PubMedCrossRefGoogle Scholar
  5. 5.
    Darmon, M., Stallcup, W. and Pittman, Q. (1982) Exp. Cell Res. 138, 73–78.PubMedCrossRefGoogle Scholar
  6. 6.
    Yaffe, D. and Saxel, O. (1977) Differentiation 7, 159–166.PubMedCrossRefGoogle Scholar
  7. 7.
    Orly, J., Sato, G.H. and Erickson, G.F. (1980) Cell 20, 817–827.PubMedCrossRefGoogle Scholar
  8. 8.
    Ruoslahti, E., Vuento, M. and Engvall, E. (1970) Biochim. Biophys. Acta 534, 210–218.Google Scholar
  9. 9.
    Nimrod, A., Tsafriri, A. and Lindner, H.R. (1977) Nature 267, 632–633.PubMedCrossRefGoogle Scholar
  10. 10.
    Hiller, S.G., Zeleznik, A.J. and Ross, G.T. (1978) Endocrinology 102, 937–946.CrossRefGoogle Scholar
  11. 11.
    Erickson, G.F., Wang, C. and Hsueh, A.J. (1979) Nature 279, 336–338.PubMedCrossRefGoogle Scholar
  12. 12.
    Dorrington, J.H., Moon, Y.S. and Armstrong, D.J. (1975) Endocrinology 97, 1328–1331.PubMedCrossRefGoogle Scholar
  13. 13.
    Erickson, G.F. and Hsueh, A.J.W. (1978) Endocrinology 102, 1275–1282.PubMedCrossRefGoogle Scholar
  14. 14.
    Zeleznik, A.J., Midgley, A.R., Jr. and Reichert, L.E., Jr. (1974) Endocrinology 95, 818–826.PubMedCrossRefGoogle Scholar
  15. 15.
    Richards, J.S., Ireland, J.T., Rao, M.C., Bernath, G.A., Midgley, A.R., Jr. and Reichert, L.E., Jr. (1976) Endocrinology 99, 1562–1570.PubMedCrossRefGoogle Scholar
  16. 16.
    Schulster, D., Orly, J., Seidel, G. and Schramm, M. (1978) J. Biol. Chem. 253, 1201–1206.PubMedGoogle Scholar
  17. 17.
    Humes, J.L., Rounbehler, M. and Kuehl, F. (1969) Anal. Biochem. 32, 210–217.PubMedCrossRefGoogle Scholar
  18. 18.
    Wang, C., Hsueh, A.J.W. and Erickson, G.F. (1979) J. Biol. Chem. 254, 11330–11336.PubMedGoogle Scholar
  19. 19.
    Channing, C.P. and Seymour, J.F. (1970) Endocrinology 87, 165–169.PubMedCrossRefGoogle Scholar
  20. 20.
    Nimrod, A. (1981) FEBS Lett. 131, 31–33.PubMedCrossRefGoogle Scholar
  21. 21.
    Knecht, M., Amsterdam, A. and Catt, K. (1981) J. Biol. Chem. 256, 10628–10633.PubMedGoogle Scholar
  22. 22.
    Hsueh, A.J.W., Wang, C. and Erickson, G.F. (1980) Endocrinology 106, 1697–1705.PubMedCrossRefGoogle Scholar
  23. 23.
    Orly, J., Farkash, Y., Hershkovits, N., Mizrahi, L. and Weinberger, P. (1982) In Vitro, in press.Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1983

Authors and Affiliations

  • Joseph Orly
    • 1
  • Patricia Weinberger-Ohana
    • 1
  • Yigal Farkash
    • 1
  1. 1.Department of Biological Chemistry, Institute of Life SciencesThe Hebrew University of JerusalemJerusalemIsrael

Personalised recommendations