Rat ovarian follicles are composed of small and large granulosa cells (GC). The present studies demonstrate that small GCs undergo insulin- or phorbol ester-dependent mitosis in vitro. In order to examine the cellular and molecular events that account for insulin's mitogenic action, small GCs were cultured with either insulin, phorbol ester (TPA), or both insulin and TPA. Insulin and TPA increased GC numbers by 21 +/- 3% and 20 +/- 2% over control values, respectively (p < 0.05). Simultaneous addition of insulin and TPA increased GC numbers by 20 +/- 3% (p < 0.05). In a second experiment, small GCs were exposed to control medium, insulin, staurosporine (a protein kinase C [PKC] inhibitor), or both insulin and staurosporine. These studies revealed that insulin induced a 21 +/- 5% increase in GC numbers and that staurosporine blocked insulin's mitogenic action. These observations suggest that insulin mediates its mitogenic action through a PKC-dependent mechanism. Since the proto-oncogenes, c-fos and c-jun, are expressed during GC mitosis, studies were undertaken to determine whether or not the expression of these two proto-oncogenes products was enhanced by insulin. The expression of c-fos and c-jun proteins was assessed by immunocytochemistry. These studies showed that after 5 h, insulin increased the percentage of cells that stained for c-fos and c-jun by 15 +/- 2% and 19 +/- 4, respectively (p < 0.05). The expression of these proto-oncogenes was blocked by staurosporine. Both progesterone and 8-br-cAMP, which block insulin-dependent GC mitosis, also inhibited the expression of c-fos and c-jun. Finally, small GCs were cultured with insulin in the presence of sense and antisense oligonucleotides specific for c-fos and c-jun. Under these conditions, insulin induced a 32 f 10% and 24 2 5% increase in cell numbers in the presence of c-fos and c-jun sense oligonucleotides, respectively. Insulin-dependent GC proliferation was completely prevented in the presence of either c-fos or c-jun antisense oligonucleotide. Taken together, these experiments support the concept that insulin stimulates small GC mitosis by activating PKC, which induces the expression of both c-fos and c-jun. Both of these genes play essential roles in GC mitosis.

Cellular and molecular mechanisms that mediate insulin-dependent rat granulosa cell mitosis / J. J. Peluso, A. M. Luciano, A. Pappalardo, B. A. White. - In: BIOLOGY OF REPRODUCTION. - ISSN 0006-3363. - 52:1(1995 Jan), pp. 124-30-130.

Cellular and molecular mechanisms that mediate insulin-dependent rat granulosa cell mitosis

A. M. Luciano
Secondo
;
1995

Abstract

Rat ovarian follicles are composed of small and large granulosa cells (GC). The present studies demonstrate that small GCs undergo insulin- or phorbol ester-dependent mitosis in vitro. In order to examine the cellular and molecular events that account for insulin's mitogenic action, small GCs were cultured with either insulin, phorbol ester (TPA), or both insulin and TPA. Insulin and TPA increased GC numbers by 21 +/- 3% and 20 +/- 2% over control values, respectively (p < 0.05). Simultaneous addition of insulin and TPA increased GC numbers by 20 +/- 3% (p < 0.05). In a second experiment, small GCs were exposed to control medium, insulin, staurosporine (a protein kinase C [PKC] inhibitor), or both insulin and staurosporine. These studies revealed that insulin induced a 21 +/- 5% increase in GC numbers and that staurosporine blocked insulin's mitogenic action. These observations suggest that insulin mediates its mitogenic action through a PKC-dependent mechanism. Since the proto-oncogenes, c-fos and c-jun, are expressed during GC mitosis, studies were undertaken to determine whether or not the expression of these two proto-oncogenes products was enhanced by insulin. The expression of c-fos and c-jun proteins was assessed by immunocytochemistry. These studies showed that after 5 h, insulin increased the percentage of cells that stained for c-fos and c-jun by 15 +/- 2% and 19 +/- 4, respectively (p < 0.05). The expression of these proto-oncogenes was blocked by staurosporine. Both progesterone and 8-br-cAMP, which block insulin-dependent GC mitosis, also inhibited the expression of c-fos and c-jun. Finally, small GCs were cultured with insulin in the presence of sense and antisense oligonucleotides specific for c-fos and c-jun. Under these conditions, insulin induced a 32 f 10% and 24 2 5% increase in cell numbers in the presence of c-fos and c-jun sense oligonucleotides, respectively. Insulin-dependent GC proliferation was completely prevented in the presence of either c-fos or c-jun antisense oligonucleotide. Taken together, these experiments support the concept that insulin stimulates small GC mitosis by activating PKC, which induces the expression of both c-fos and c-jun. Both of these genes play essential roles in GC mitosis.
Granulosa Cells; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Tetradecanoylphorbol Acetate; Progesterone; Oligonucleotides, Antisense; Gene Expression; Amino Acid Sequence; Insulin; Rats; Alkaloids; Base Sequence; Mitosis; Staurosporine; Rats, Wistar; Molecular Sequence Data; Genes, fos; Protein Kinase C; Proto-Oncogene Proteins c-jun; Female; Proto-Oncogene Proteins c-fos; Genes, jun
Settore VET/01 - Anatomia degli Animali Domestici
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/184235
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