REPRODUCTION RESEARCH Involvement of the transcription factor STAT1 in the regulation of porcine ovarian granulosa cell functions treated and not treated with ghrelin A Benc ˇo 1 , A V Sirotkin 1,2 , D Vas ˇı ´c ˇek 2 , S Pavlova ´ 1,2 , J Zemanova ´ 3 , J Kotwica 4 , K Darlak 5 and F Valenzuela 5 1 Konstantin the Philosopher University, 949 74 Nitra, Slovakia, 2 Department of Genetics and Reproduction, Research Institute of Animal Production, Slovak Agricultural Research Centre, Hlohovska ´ 2, 949 92 Nitra, Slovakia, 3 Slovak University of Agriculture, 949 76 Nitra, Slovakia, 4 Institute of Animal Reproduction and Food Research, 10-747 Olsztyn, Poland and 5 Peptides International Inc., Louisville, Kentucky 40299, USA Correspondence should be addressed to A V Sirotkin at Department of Genetics and Reproduction, Research Institute of Animal Production, Slovak Agricultural Research Centre; Email: sirotkin@scpv.sk Abstract The aim of our in vitro experiments was to study the role of the transcription factor STAT1 and the hormone ghrelin in controlling porcine ovarian function. The effects of treatment with ghrelin (0, 1, 10, 100ng/ml), transfection-induced overexpression of transcription factor STAT1, and theircombination on apoptosis (expression of apoptosis-related peptides caspase-3, BAX and anti-apoptotic peptide BCL2), proliferation (expression of proliferating cell nuclearantigene PCNA, proliferation-associated protein kinase MAPK/ERK1,2) and release of the hormones progesterone (P 4 ), prostaglandin F (PGF) and oxytocin (OXT) in cultured porcine ovarian granulosa cells was evaluated using RIA, immunocytochemistryand SDS-PAGE–western immunoblotting. It was found that ghrelin, when given alone, increased the expression of proliferation-associated PCNA and MAPK/ERK1,2, decreased the accumulation of apoptosis-related substances caspase-3, BAX, BCL2, decreased P 4 , and increased PGFand OXTrelease. Ghrelin tended to promote accumulation of STAT1 in both control and transfected cells, although in transfected cells ghrelin at 1 ng/ml decreased STAT1 accumulation. Transfection of porcine granulosa cells by a gene construct encoding STAT1 promoted the expression of STAT1 and apoptosis-related-BAX but the expression of BCL2 did not, and decreased the accumulation of proliferation-associated MAPK/ERK1,2 but not that of PCNA. It also promoted PGF and OXT but not P 4 release. Overexpression of STAT1 reversed the effect of ghrelin on STAT1, PCNA, PGF, OXT (from stimulatory to inhibitory), BCL2, P 4 (from inhibitory to stimulatory), prevented ghrelin effect on caspase-3 and BAX, but did not affect ghrelin’s effect on MAPK/ERK1,2 expression. These results suggest that ghrelin directlyaffects porcine ovarian cells function – stimulates proliferation, inhibits apoptosis and affects secretory activity. Furthermore, they demonstrated the involvement of the transcription factor STAT1 in controlling these functions, the promotion of some markers of apoptosis (BAX), inhibition of some markers of proliferation (MAPK/ERK1,2) and stimulation of PGF release. Finally, the obtained data failed to demonstrate that STAT1 is involved in mediating the action of ghrelin on ovarian cell functions. Reproduction (2009) 138 553–560 Introduction Searching for new extra- and intracellular regulators of reproductive processes is important for both theoretical and practical reasons. One such extracellular regulator could be the recently described hormone ghrelin, produced by stomach and other tissues. Ghrelin can inhibit secretory activity (Barreiro & Tena-Sempere 2004, Tena-Sempere 2005, 2008) of rat testicular cells and proliferation of rat prostatic cells (Yeh et al. 2005), and may be involved in the control of cell proliferation in ovarian and testicular tumours. Recently it was demonstrated that it can directly control the function of ovarian cells by stimulating proliferation; inhibiting apoptosis; promoting steroid hormone, insulin-like growth factor-I (IGF1) and vasotocin secretion (chicken, Sirotkin & Grossmann 2006; pig, Rak & Gregoraszczuk 2008); and stimulating prostaglandins F (PGF) and PGE (pig, Sirotkin et al. 2008a), progesterone (P 4 ) and IGF1 (rabbit, Sirotkin et al. 2008b) release. Intracellular signalling substances can be involved in controlling ovarian function and in mediating the effects of hormones. Among these substances, the role of some protein kinases, transcription factors and their targets (apoptosis and proliferation-related molecules) have been elucidated (Machino et al. 2003, Mittler et al. 2004). q 2009 Society for Reproduction and Fertility DOI: 10.1530/REP-08-0313 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 01/18/2022 09:23:51AM via free access