Objective:We determined the relative effects of insuling and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3β-hydroxysteroid dehydrogenase (3βHSD) in human luteinized granulosa cells.
Methods:Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treatd with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3βHSD were evaluated.
Results:Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/μL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3βHSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02) Insulin (greater than 100 nmol/L) alone increased 3βHSD protein content as measured by Western analysis 1.8-2fold over basal levels, whereas FSH alone increased protein contetn 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3βHSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3βHSD 3.2-fold. In the presence of insulin plus FSH, 3βHSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3βHSD.
Conclusion:Insulin is a regular of both 3βHSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.
1. Carr BR. Disorders of the ovary and female reproductive tract. In: Wilson JD, Foster DW, eds. Williams textbook of endocrinology. 8th ed.Philadelphia: WB Saunders, 1992:733-798.
2.
2. Poretsky L, Kalin MF. The gonadotropic function of insulin. Endocr Rev1987;8:132-141.
3.
3. Barbieri R, Smith S, Ryan KJ. The role of hyperinsulinemia in the pathogenesis of ovarian hyperandrogenism. Fertil Steril1988;50:197-212.
4.
4. Amsterdam A, May JV, Schomberg DW. Synergistic effect of insulin and follicle-stimulating hormone on biochemical and morphological differentiation of porcine granulosa cells in vitro. Biol Reprod1988;39:379-390.
5.
5. Goonerate AD, Thacker PA, Laarveld B, Murphy BD, Rajkumar K. Comparative effects of insulin and insulin-like growth factor-1 on follicle-stimulating hormone-induced responses in porcine granulosa cells. Steroids1990;55:105-108.
6.
6. Davoren JB, Hsueh A. Insulin enhances FSH-stimulated steroidogenesis by cultured rat granulosa cells. Mol Cell Endocrinol1984;35:97-105.
7.
7. Saragueta P, Krimer A, Charreau EH, Tesone M. Insulin regulation of steroidogenic activity in rat culture luteal cells. Steroid Biochem1989;32:393-397.
8.
8. Diamond MP, Webster BW, Carr RK, Wentz AC, Osteen KG. Human follicular fluid insulin concentrations. J Clin Endocrinol Metab1985;61:990-990.
9.
9. Garzo VG, Dorrington JH. Aromatase activity in human granulosa cells during follicular development and the modulation by follicle-stimulating hormone and insulin. Am J Obstet Gynecol1984;148:657-657.
10.
10. Steinkampf MP, Mendelson CR, Simpson ER. Regulation by follicle-stimulating hormone of the synthesis of aromates cytochrome P-450 in human granulosa cells. Mol Endocrinol1987;1:465-471.
11.
11. Steinkampf MP, Mendelson CR, Simpson ER. Effects of epidermal growth factor and insulin-like growth factor I on the levels of mRNA encoding aromatase cytochrome P-450 of human ovarian granulosa cells. Mol Cell Endocrinol1988;59:93-99.
12.
12. Doody KJ, Lorence MC, Mason JI, Simpson ER. Expression of messenger ribonucleic acid species encoding steroidogenic enzymes in human acid species encoding steroidogenic enzymes in human follicles and corpora lutea throughout the menstrual cycle. J Clin Endocrinol Metab1990;70:1041-1045.
13.
13. Milewich L, Shaw CE, Mason JI, Carr BR, Blomquist CH, Thomas JL. 3β-Hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5α-androstane-3β, 17β-diol and dehydroepiandrosterone as substrates. J Steroid Biochem Mol Biol1993;45:525-537.
14.
14. Milewich L, Shaw CE, Doody KM, Rainey WE, Mason JL. 3β-Hydroxysteroid dehydrogenase activity in glandular and extraglandular human fetal tissues. J Clin Endocrinol Metab1991;73:1134-1140.
15.
15. Naville D, Rainey WE, Milewich L, Mason JI. Regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5 → 4-isomerase expression by adrenocorticotropin in bovine adrenocortical cells. Endocrinology1991;128:139-145.
16.
16. Rainey WE, Naville D, Cline N, Mason JI. Prostaglandin E2 is a positive regulator of adrenocorticotropin receptors, 3 beta-hydroxysteroid dehydrogenase, and 17 alpha-hydroxylase expression in above adrenocortical cells. Endocrinology1991;129:1333-1339.
17.
17. Doody KM, Carr BR, Rainey WE, et al. 3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland. Endocrinology1991;126;2487-2492.
18.
18. Sasano H, Mori T, Sasano N, Nagura H, Mason JI. Immunolocalization of 3 beta-hydroxysteroid dehydrogenase in human ovary. J Reprod Fertil1990;89:743-751.
19.
19. Maruo T, Hayashi M, Matsuo H, Ueda Y, Morikawa H, Mochiczuki M. Comparison of the facilitative roles of insulin and insulin-like growth factor I in the funnctional differentiation of granulosa cells: In vitro studies with the porcine model. Acta Endocrinol1988;117:230-240.
20.
20. Saumande J. Culture of bovine granulosa cells in a chemically defined serum-free medium: The effect of insulin and fibronectin on the response to FSH. J Steroid Biochem Mol Biol1991;38:189-196.
21.
21. McArdle CA, Kohl C, Riege I, Groner I, Wehrenbert U. Effects of gonadotropins, insulin and insulin-like growth factor I on ovarian oxytocin and progesterone production. Mol Cell Endocrinol1991;78:211-220.
22.
22. Adashi EY, Rasnick CE, Svoboda ME, Vanwyk H. Somatomedin-C synergizes with follicle-stimulating hormone in the acquisition of progestin biosynthetic capacity by cultured rat granulosa cells. Endocrinology1985;116:2135-2135.
23.
23. Hill GA, Osteen KG. The effect of insulin on in vitro progesteron production of human granulosa cells. Int J Fertil1992;37:93-98.
24.
24. Hurwitz A, Laufer N, Keshet I, et al. The effect of insulin on progresterone production and cellular growth in long-term cultures of human granulosa lutein cells. Fertil Steril1987;48:71-71.
25.
25. Diamond MP, Lavy G, Polan ML. Progesterone production from granulosa cells of individual human folliclkes derived from diabetic and nondiabetic subjects. Int J Fertil1989;34:204-208.
26.
26. Holst N, Bertheussen NH, Burhol PG, Forsdahl F. Medium-associated luteinization expressed as progesterone release in granulosa-luteal cells isolated from patients undergoing in-vitro fertilization. Hum Reprod1991;6:1343-1348.
28. Adashi EY, Resnick CE, D'Ercole JA, Svoboda ME, Van Wyk JJ. Insulin-like growth factors as intraovarian regulator of granulosa cell growth and function. Endocr Rev1985;6:400-400.
29.
29. Amiel SA, Sherwin RS, Simonson DC, Lauritano AA, Tamborland WV. Impaired insulin action in puberty. N Engl J Med1986;315:215-219.
30.
30. Erickson GF, Magoffin D, Garzo VG, Cheung AP, Chang R. Granulosa cells of polycystic ovaries: Are they normal or abnromal?Hum Reprod1992;7:292-299.
