Involvement of the transcription factor STAT1 in the regulation of porcine ovarian granulosa cell functions treated and not treated with ghrelin.
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, 100 ng/ml), transfection-induced overexpression of transcription factor STAT1, and their combination on apoptosis (expression of apoptosis-related peptides caspase-3, BAX and anti-apoptotic peptide BCL2), proliferation (expression of proliferating cell nuclear antigene 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, immunocytochemistry and 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 PGF and OXT release. 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 directly affects 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.
Benco
A
A
Konstantin the Philosopher University, 94974 Nitra, Slovakia.
Sirotkin
A V
AV
Vasícek
D
D
Pavlová
S
S
Zemanová
J
J
Kotwica
J
J
Darlak
K
K
Valenzuela
F
F
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D015870:Gene Expression / Q000502:physiology; D054439:Ghrelin / Q000494:pharmacology*; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D006728:Hormones / Q000378:metabolism; D010053:Ovary / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013552:Swine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D054439:Ghrelin; D006728:Hormones; D050794:STAT1 Transcription Factor