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The effects of gonadotropin releasing hormone-I and II in human granulosa cells Hong, In-Sun

Abstract

Gonadotropin-releasing hormone (GnRH) is the key regulator of hypothalamus-pituitary gonadal function. In the anterior pituitary, GnRH stimulates the synthesis and the release of the gonadotropins. GnRH and its receptors have also been found in extra pituitary tissues such as the ovary, breast, and placenta. GnRH has been reported to have antiproliferative effects on granulosa cells from rats, pigs, and humans; however, the underlying mechanisms are unclear. We have successfully established two immortalized human granulosa cell lines by SV40 large T antigen transfection. In the process, we have characterized multiple cellular and molecular features of these immortalized cells and compared these characteristics with those of primary cultured granulosa cells. The features examined include the expression patterns of cytoskeletal proteins (vimentin, cytokeratin 5/6 and desmin), adhesion molecules (Connexin 43 and E-cadherin), steroidogenic enzymes (StAR, p450scc and 3β-HSD, steroid receptors (ER-α,-β and PR-α,-β and gonadotropin receptors (LH and FSH), as well as cell morphology, anchorage independent growth, growth potential, and progesterone production. In addition to its well-established actions on the pituitary–gonadal axis, GnRH-I or II suppressed proliferation and directly induced apoptosis in human granulosa cells. The intracellular signals of apoptosis induced by GnRH I or II, in turn, are mediated by activation of the proteolyic caspase cascade, involving caspase 8,3 and 7. In addition, FSH protected human granulosa cells from undergoing apoptosis induced by GnRH I or II. GnRH I or II also significantly attenuated the stimulatory effect of IGF I on granulosa cell proliferation. Correspondingly, IGF I decreased while GnRH I or II increased cleaved caspases 3 in our granulosa cell line; furthermore, IGF I significantly attenuated the expression of cleaved caspase 3 induced by GnRH I or II. These interactions of IGF I and GnRH I or II in regulating granulosa cell proliferation or apoptosis are mediated through Akt in human granulosa cells. In conclusion, we have established two granulosa cell lines and performed integrated investigations of multiple characteristics to elucidate more clearly the characteristicsof human ovarian granulosa cells. Additionally, the present study demonstrated that GnRH I or II mediates antiproliferate effects via stimulation of granulosa cell apoptosis and inhibition of IGF I activities through Akt signaling pathway in human granulosa cells.

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