title: 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.
authors.affiliation: Research Institute of Animal Production, Hlohovská 2, 949 92 Nitra, Slovakia. sirotkin@scpv.sk
authors.forename: A V
authors.initials: AV
authors.lastname: Sirotkin
authors.forename: R
authors.initials: R
authors.lastname: Grossmann
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002645:Chickens; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D005260:Female; D054439:Ghrelin / Q000031:analogs & derivatives* / Q000494:pharmacology; D006107:Granulosa Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D007124:Immunoenzyme Techniques / Q000662:veterinary; D007150:Immunohistochemistry / Q000662:veterinary; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D006080:Ovarian Follicle / Q000166:cytology / Q000187:drug effects; D011374:Progesterone / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D054440:Receptors, Ghrelin / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D014668:Vasotocin / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D054439:Ghrelin; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D054440:Receptors, Ghrelin; D051028:bcl-2-Associated X Protein; D013739:Testosterone; D011374:Progesterone; D004958:Estradiol; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D014668:Vasotocin