Effects of ghrelin and its analogues on chicken ovarian granulosa cells

Abstract

The aim of these in vitro experiments was (1) to examine the effects of ghrelin on the basic functions of ovarian cells (proliferation, apoptosis, secretory activity); (2) to determine the possible involvement of the GHS-R1a receptor and PKA- and MAPK-dependent post-receptor intracellular signalling cascades; (3) to identify the active part of the 28-amino acid molecule responsible for the effects of ghrelin on ovarian cells. We compared the effect of full-length ghrelin 1–28, a synthetic activator of GHS-R1a, GHRP6, and ghrelin molecular fragments 1–18 and 1–5 on cultured chicken ovarian cells. Indices of cell apoptosis (expression of the apoptotic peptide bax and the anti-apoptotic peptide bcl-2), proliferation (expression of proliferation-associated peptide PCNA), and expression of protein kinases (PKA and MAPK) within ovarian granulosa cells were analysed by immunocytochemistry. The secretion of progesterone (P 4), testosterone (T), estradiol (E 2) and arginine–vasotocin (AVT) by isolated ovarian follicular fragments was evaluated by RIA/EIA. It was observed that accumulation of bax was increased by ghrelin 1–28, GHRP6 and ghrelin 1–18, but not by ghrelin 1–5. Expression of bcl-2 was suppressed by addition of ghrelin 1–28, GHRP6 and ghrelin 1–5, but promoted by ghrelin 1–18. The occurrence of PCNA was reduced by ghrelin 1–28, GHRP6, ghrelin 1–18 and ghrelin 1–5. An increase in the expression of MAPK/ERK1, 2 was observed after addition of ghrelin 1–28, GHRP6 and ghrelin 1–18, but not ghrelin 1–5. The accumulation of PKA decreased after treatment with ghrelin 1–28 and increased after treatment with GHRP6 and ghrelin 1–18 but not ghrelin 1–5. Secretion of P 4 by ovarian follicular fragments was decreased after addition of ghrelin 1–28 or ghrelin 1–5 but stimulated by GHRP6 and ghrelin 1–18. Testosterone secretion was inhibited by ghrelins 1–28 and 1–18, but not by GHRP6 or ghrelin 1–5. Estradiol secretion was reduced after treatment with ghrelin 1–28 but stimulated by ghrelins 1–18 and 1–5; GHRP6 had no effect. AVT secretion was stimulated by ghrelin 1–28, GHRP6 and ghrelin 1–18, but inhibited by ghrelin 1–5. The comparison of the effects of the four ghrelin analogues on nine parameters of ovarian cells suggest (1) a direct effect of ghrelin on basic ovarian functions—apoptosis, proliferation, steroid and peptide hormone secretion; (2) that the majority of these effects can be mediated through GHS-R1a receptors; (3) an effect of ghrelin on MAPK- and PKA-dependent intracellular mechanisms, which can potentially mediate the action of ghrelin at the post-receptor level; (4) that ghrelin residues 5–18 may be responsible for the major effects of ghrelin on the avian ovary.