Konstantin the Philosopher University, 94974 Nitra, Slovakia.
Reproduction. 2009 Sep;138(3):553-60. doi: 10.1530/REP-08-0313. Epub 2009 Jun 15.
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.
我们的体外实验旨在研究转录因子 STAT1 和激素 ghrelin 对控制猪卵巢功能的作用。我们用不同浓度（0、1、10、100ng/ml）的 ghrelin 处理、转染诱导转录因子 STAT1 过表达以及它们的组合处理，评估对培养的猪卵巢颗粒细胞凋亡（凋亡相关肽 caspase-3、BAX 和抗凋亡肽 BCL2 的表达）、增殖（增殖细胞核抗原 PCNA、增殖相关蛋白激酶 MAPK/ERK1,2 的表达）和激素孕酮（P4）、前列腺素 F（PGF）和催产素（OXT）释放的影响，采用放射免疫分析、免疫细胞化学和 SDS-PAGE-免疫印迹法。结果发现，ghrelin 单独作用时，增加了增殖相关 PCNA 和 MAPK/ERK1,2 的表达，减少了凋亡相关物质 caspase-3、BAX、BCL2 的积累，降低了 P4 的释放，增加了 PGF 和 OXT 的释放。ghrelin 倾向于促进对照组和转染组中 STAT1 的积累，尽管在转染组中，ghrelin 在 1ng/ml 时减少了 STAT1 的积累。通过基因构建体转染猪颗粒细胞促进了 STAT1 的表达和凋亡相关 BAX 的表达，但 BCL2 的表达没有增加，并且减少了增殖相关 MAPK/ERK1,2 的积累，但没有增加 PCNA 的积累。它还促进了 PGF 和 OXT 的释放，但没有促进 P4 的释放。STAT1 的过表达逆转了 ghrelin 对 STAT1、PCNA、PGF、OXT（从刺激变为抑制）、BCL2、P4（从抑制变为刺激）的作用，阻止了 ghrelin 对 caspase-3 和 BAX 的作用，但不影响 ghrelin 对 MAPK/ERK1,2 表达的作用。这些结果表明，ghrelin 直接影响猪卵巢细胞的功能-刺激增殖，抑制凋亡并影响分泌活性。此外，它们还证明了转录因子 STAT1 参与控制这些功能，促进了一些凋亡标志物（BAX），抑制了一些增殖标志物（MAPK/ERK1,2），并刺激了 PGF 的释放。最后，获得的数据未能证明 STAT1 参与介导 ghrelin 对卵巢细胞功能的作用。
