Effect of epidermal growth factor (EGF) on steroid and cyclic nucleotide secretion, proliferation and ERK-related MAP-kinase in cultured rabbit granulosa cells

Summary

The aim of our in vitro experiments was to study the effects of EGF on rabbit ovarian cells, as well as the possible mechanisms of these effects. The influence of EGF on steroidogenesis, proliferation, cyclic nucleotides and MAP-kinase in rabbit granulosa cells were studied. Results of RIA showed, that EGF stimulated the release of progesterone (1-100 ng/ml), cAMP (at 100 ng/ml), cGMP (1-100 ng/ml). EGF effect on estradiol output was biphasic: at dose 1 ng/ml it inhibited, whilst at 100 ng/ml it strongly increased estradiol secretion. Immunocytochemical study demonstrated an EGF-induced (10 ng/ml) increase in the proportion of cells revealing proliferating cell nuclear antigen (41% vs 24.7% in control, p < 0.01). EGF (10 ng/ml) increased the proportion of cells with immunoreactivity to ERK-1 (more than two-fold) and ERK-3 (three-fold) members of the MAP-kinase family. Moreover, EGF induced the translocation of ERK-1 to the nucleus, whilst preferentially cytoplasmic localization of ERK-3 was not changed after EGF addition. This can indicate regulation of ERK-1 and -3 by EGF, as well as differential patterns of ERK-1 and ERK-3 expression in response to EGF in cultured granulosa cells. - These results indicate that EGF can be a stimulator of proliferation, steroidogenesis and cyclic nucleotide release by rabbit granulosa cells. Stimulation of cAMP and cGMP release, and activation of ERK-related MAP kinase in granulosa cells after EGF addition indicates the involvement of these intracellular messengers in mediating the EGF action on the ovary.

Key words:

Progesterone - Estradiol - cAMP - cGMP - ERK-MAP kinase

References

• 1 Ackland J F, Schwartz A B, Mayo K E, Dodson R E. Nonsteroidal signals originating in the gonads.  Physiol Rev. 1992;  72 731-787
• 2 Apostolakis E M, Garai J, Lohmann J E, Clark J H, O'Malley B W. Epidermal growth factor activates reproductive behavior independent of ovarian steroids in female rodents.  Mol Endocrinol. 2000;  14 1086-1098
• 3 Assarsson B, Nilsson I, Selstam G. Ovarian levels of epidermal growth factor receptor mRNA in the rat - a postovulatory decrease.  Acta Physiol Scand. 1995;  154 177-183
• 4 Boland N I, Gosden R G. Effects of epidermal growth factor on the growth and differentiation of cultured mouse ovarian follicles.  J Reprod Fert. 1994;  101 369-374
• 5 Bramley T A, Menzies G S. Specific binding sites for ¹²⁵I-labelled epidermal growth factor in the ovine corpus luteum and human placenta.  J Endocrinol. 1992;  135 5-16
• 6 Buck P A, Schomberg D W. [¹²⁵]iodo-epidermal growth factor binding and mitotic responsiveness of porcine granulosa cells and modulated by differentiation and follicle-stimulating hormone.  Endocrinol. 1988;  122 28-33
• 7 Budnik L T, Mukhopadhyay A K. Epidermal growth factor, a modulator of luteal adenylate cyclase.  J Biol Chem. 1991;  266 13908-13913
• 8 Budnik L T, Mukhopadhyay A K. Modulatory action of epidermal growth factor on differentiated human granulosa lutein cells: cross-talk between ligand activated receptors for EGF and gonadotropin.  Mol Cell Endocrinol. 1996;  124 141-150
• 9 Cameron M R, Foster J S, Bukovsky A, Wimalasena J. Activation of mitogen-activated protein kinases by gonadotropins and cyclic adenosine 5′-monophosphates in porcine granulosa cells.  Biol Reprod. 1996;  55 111-119
• 10 Franchimont P, Hazee-Hagelstein M T, Charlet-Renard Ch, Jaspar J M. Effect of mouse epidermal growth factor on DNA and protein synthesis, progesterone and inhibin production by bovine granulosa cells in culture.  Acta Endocrinol. 1986;  111 122-127
• 11 Hiramatsu S, Maruo T, Matsuo H, Mochizuki M. Effects of epidermal growth factor on the proliferation and differentiation of porcine granulosa cells cultured in vitro.  Nippon Sanka Fujinka Gakkai Zasshi. 1992;  44 55-61
• 12 Keel B A, Hildenbrandt J M, May J V, Davis J S. Effects of epidermal growth factor on the tyrosine phosphorylation of mitogen-activated protein kinases in monolayer cultures of porcine granulosa cells.  Endocrinol. 1995;  136 1197-1204
• 13 Lavandero S, Ocaranza M P, Sapag-Hagar M. Epidermal growth factor increases cyclic AMP levels in pregnant rat mammary gland explants.  Res Com Chem Path Pharm. 1990;  69 317-323
• 14 Leung P, Steele G. Intracellular signalling in the gonads.  Endocrine Rev. 1992;  13 476-498
• 15 Makarevich A, Sirotkin A, Chrenek P, Bulla J, Hetenyi L. The role of IGF-I, cAMP/protein kinase A and MAP-kinase in the control of steroid secretion, cyclic nucleotide production, granulosa cell proliferation and preimplantation embryo development in rabbits.  J Steroid Biochem Mol Biol. 2000;  73 123-133
• 16 Maruo T, Ladines-Llave C A, Samoto T, Matsuo H, Manalo A S, Ito H, Mochizuki M. Endocrinology. 1993;  132 924-931
• 17 Mathews M B, Bernstein R M, Franza B R, Garrels J I. Identity of the proliferating cell nuclear antigen and cyclin.  Nature. 1984;  309 374-376
• 18 Morbeck D E, Flowers W L, Britt J H. Response of porcine granulosa cells isolated from primary and secondary follicles to FSH, 8-bromo-cAMP and epidermal growth factor in vitro.  J Reprod Fert. 1993;  99 577-584
• 19 Moreno-Cuevas J, Khan-Dawood F S. Epidermal growth factor receptors in rat ovarian tissue.  Tissue Cell. 1997;  29 55-62
• 20 Nair B G, Rashed H M, Patel T B. Epidermal growth factor produces inotropic and chronotropic effects in rat hearts by increasing cyclic AMP accumulation.  Growth Factors. 1993;  8 41-48
• 21 Paton A C, Collins W P. Differentiation processes of granulosa cells.  Oxford Rev Reprod Biol. 1992;  14 169-223
• 22 Richardson M C, Gadd S C, Masson G M. Augmentation by epidermal growth factor of basal and stimulated progesterone production by human luteinized granulosa cells.  J Endocrinol. 1989;  121 397-402
• 23 Roy S, Greenwald G. In vitro effects of epidermal growth factor, insulin-like growth factor-I, fibroblast growth factor, and follicle-stimulating hormone on hamster follicular deoxyribonucleic acid synthesis and steroidogenesis.  Biol Reprod. 1991;  44 889-896
• 24 Scaramuzzi R J, Downing J A. The in vivo effects of fibroblast growth factor and epidermal growth factor on the secretion of estradiol, androstenedione and progesterone by the autotransplanted ovary in the ewe.  J Endocrinol. 1995;  146 301-311
• 25 Schaeffer H-J, Sirotkin A V. The release of insulin-like growth factor-I by luteinized human granulosa cells in vitro: regulation by growth hormone, oxytocin, steroids and cAMP-dependent intracellular mechanisms.  Exp Clin Endocrinol Diabetes. 1995;  103 361-366
• 26 Schams D, Koll R, Li C. Insulin-like growth factor-I stimulates oxytocin and progesterone production by bovine granulosa cells in culture.  J Endocrinol. 1988;  116 97-100
• 27 Seger R, Krebs E G. The MAPK signaling cascade.  FASEB J. 1995;  9 726-735
• 28 Singh B, Rutledge J M, Armstrong D T. Epidermal growth factor and its receptor gene expression and peptide localization in porcine ovarian follicles.  Mol Reprod Dev. 1995;  40 391-399
• 29 Steinkampf M P, Mendelson C R, Simpson E R. Effects of epidermal growth factor and insulin-like growth factor I on the levels of mRNA encoding aromatase cytochrome P-450 of human granulosa cells.  Mol Cell Endocrinol. 1988;  59 93-99
• 30 Tekpetey F, Singh B, Barbe G, Armstrong D. Localization of epidermal growth factor (EGF) receptor in the rat corpus luteum, and EGF and transforming growth factor-a stimulation of luteal cell steroidogenesis in vitro.  Mol Cell Endocrinol. 1995;  110 95-102
• 31 Tilly J L, Billig H, Kowalski K I, Hsueh A JW. Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanisms.  Mol Endocrinol. 1992;  6 1942-1950
• 32 Transduction Laboratory, antibodies catalogue .76-78 1997
• 33 Trzeciak W H, Duda T, Waterman M R, Simpson E R. Effects of epidermal growth factor on the synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells in primary culture.  Mol Cell Endocrinol. 1987;  52 43-50
• 34 Urban R J, Veldhuis J D. Endocrine control of steroidogenesis in granulosa cells.  Oxford Rev Reprod Biol. 1992;  14 225-261