ABSTRACT
The aim of our in vitro studies was to understand the role of leptin in controlling proliferation, apoptosis, and protein kinase A (PKA) in human ovarian cells. We analyzed the in vitro effects of leptin (0, 1, 10 or 100 ng/ml) on the accumulation of proliferation-related peptides (PCNA, cyclin B1), apoptosis-associated peptide (Bax) and the intracellular signaling molecule PKA in cultured human granulosa cells using immunocytochemistry and Western immunoblotting. It was observed that leptin stimulated in a dose-dependent manner the accumulation of PCNA (at doses 1-100 ng/ml), cyclin B1 (at doses 10 or 100 ng/ml), Bax (at doses 10 or 100 ng/ml) and PKA (at doses 1-100 ng/ml) in cultured human ovarian cells. These observations suggest the ability of leptin to control directly human ovarian cell functions: proliferation, apoptosis, and intracellular messenger PKA.
Subject(s)
Apoptosis , Cell Proliferation , Cyclic AMP-Dependent Protein Kinases/metabolism , Granulosa Cells/enzymology , Leptin/metabolism , Adult , Blotting, Western , Cell Cycle , Cells, Cultured , Cyclin B/metabolism , Cyclin B1 , Female , Granulosa Cells/immunology , Granulosa Cells/pathology , Humans , Immunohistochemistry , Proliferating Cell Nuclear Antigen/metabolism , Recombinant Proteins/metabolism , Signal Transduction , bcl-2-Associated X Protein/metabolismABSTRACT
AIMS: The aim of our in vitro studies was to understand the role of leptin and the insulin-like growth factor I/insulin-like growth factor protein (IGF/IGFBP) system in controlling human ovarian function. METHODS: We studied the action of leptin (0, 1, 10, or 100 ng/ml) and immunoneutralization of IGF-I using specific antiserum (0.1%) on the release of progesterone (P), estradiol (E), oxytocin (OT), IGF-I, IGFBP-3, and prostaglandins F (PGF) by these cells using radioimmunoassay/immunoradiometric assay. RESULTS: It was found that leptin stimulated the secretion of OT, IGFBP-3, and PGF. It suppressed the secretion of E and IGF-I, but not P, into the medium. The addition of antiserum against IGF-I decreased IGF-I output, increased P, OT, IGFBP-3, and PGF secretion, and had no effect on E release. Immunoneutralization of IGF-I also prevented or reversed the effects of leptin on P, E, IGF-I, IGFBP-3, PGF, but not on OT. CONCLUSIONS: These observations (1) demonstrate that leptin directly controls the secretory activity of human ovarian cells, (2) confirm the involvement of IGF-I in the regulation of ovarian cells, and (3) suggest an inter-relationship between leptin and the IGF/IGFBP system in the control of these functions and the involvement of IGF/IGFBP system in mediating leptin action on the ovary.
Subject(s)
Granulosa Cells/metabolism , Insulin-Like Growth Factor Binding Proteins/physiology , Insulin-Like Growth Factor I/physiology , Leptin/physiology , Cells, Cultured , Culture Media, Conditioned , Estradiol/metabolism , Female , Humans , Immune Sera/pharmacology , Insulin-Like Growth Factor Binding Protein 3/metabolism , Insulin-Like Growth Factor I/immunology , Insulin-Like Growth Factor I/metabolism , Leptin/administration & dosage , Oxytocin/metabolism , Progesterone/metabolism , Prostaglandins F/metabolismABSTRACT
Thrombopoietin (TPO) is known to be involved in megakariocytopoesis, but its role in the control of ovarian function is unknown. The aims of this study were to determine whether TPO can regulate the proliferation, apoptosis and secretory activity of ovarian cells, to identify possible intracellular mediators of TPO action, especially protein kinase A (PKA), and to define their interrelationships within ovarian cells. We investigated the effect of TPO treatment (0, 1, 10 or 100 ng/ml) on the following characteristics of cultured porcine ovarian follicles, determined using SDS-PAGE and Western blotting, immunocytochemistry, RIA and ELISA: the expression of intracellular peptides associated with proliferation (PCNA), apoptosis (Bax), tyrosine kinase (TK, phosphotyrosine), Cdc2/p34 kinase, PKA and the transcription factor CREB-1, and the secretion of progesterone, androstenedione, estradiol-17beta, oxytocin, inhibin A, inhibin B, IGF-I, transforming growth factor-2beta (TGF-2beta) and IGF-binding protein 3 (IGFBP-3). The involvement of PKA-dependent pathways was examined by evaluating the effect of a PKA blocker (KT5720, 1 microg/ml), either alone or in combination with TPO, on the parameters listed above. A TPO-induced increase in expression of PCNA, Bax, PKA, TK, Cdc2/p34 and CREB was observed. Furthermore, TPO was able to inhibit androstenedione, estradiol, TGF-2beta and IGFBP-3 secretion, and to stimulate oxytocin, inhibin A, inhibin B and IGF-I secretion. Progesterone secretion was not stimulated. The PKA blocker KT5720, when given alone, reduced the expression of Bax and TGF-2beta, augmented the expression of PKA, CREB and oxytocin, but did not influence the secretion of progesterone, androstenedione, estradiol, IGFBP-3, inhibins A and B or IGF-I. When given together with TPO, the PKA blocker prevented or reversed the action of TPO on PKA, CREB, androstenedione, estradiol, IGFBP-3, oxytocin, but not its effect on Bax, TGF-2beta or inhibin B. On the other hand, treatment with KT5720 augmented the effect of TPO on progesterone, inhibin A and IGF-I. These results provide the first evidence that TPO may be a potent regulator of ovarian function (e.g. proliferation, apoptosis and the secretion of peptide hormones, steroids, growth factors and growth factor-binding protein, as well as of the expression of some intracellular messengers). Furthermore, they demonstrated the importance of PKA in controlling these functions and in mediating the effects of TPO on ovarian cells. It remains possible that other (TK- and Cdc2/p34-dependent) intracellular mechanisms are also involved in mediating TPO action on the ovary.
