Luteinizing Hormone-induced Akt Phosphorylation And Its Effect On Androgen Production In Goat Theca Cells

The principal function of ovarian theca cells is steroid hormone production. Theca cells play an important role in controlling ovarian steroidogenesis by providing aromatizable androgens for granulosa cell estrogen biosynthesis. Normal ovarian function requires accurate regulation of steroidogenic activity of theca cells through extraovarian and intraovarian mechanisms. Therefor the accurate regulation play a vital role in follicular development. Androgen production from thecal cells in the gonadotropin-dependent stage is largely under the control of LH from the pituitary. PI3K/Akt pathway play a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, survival, transcription and protein synthesis. Therefore, it has important significance to reveal ovarian physiology and ovarian dysfunction cause by thecal steroidogenic hyperactivity by investigating the intracellular signal transduction mechanisms that LH-induced theca cell steroidogenesis. In this study, theca cells from ovary in goat cultured in vitro for using to examine whether and by what means LH controls PI3K/Akt signaling and androgen production. The results of the studies are sa follows:(1) Ovarian theca layer was striped and cut into small fragments under microscopy in sterile conditions, incubated in DMEM/F12 medium containing 0.1% collagenase Ⅳ at 37℃ for 30 min, and then isolated cells cultured after purified by differential adhesion method. Cell morphology were observed theca cells cultured in vitro, including fibroblast-like phenotypes, growing cells with adherence. Theca cells were grown for 8 times of passages, and aging along with passages, and were grown slowly. The results of cell immunofluorescence indicated that theca cells were vimentin positive(positive rate was more than 95%), and was cytokeration negative.(2) Goat theca cells from small antral follicles were incubated with LH for various durations, and phospho-Akt and total-Akt content were examined using Western blotting indicated that the amount of phospho-Akt reached its highest level at 12 h after treatment with LH. However, theca cells were incubated with LH combined with PI3 K inhibitors for various durations, results show that PI3 K inhibitors inhibited LH-induced Akt phosphorylation in the goat thcea cells.(3) qRT-PCR analyses showed that LH significantly increased CYP11A1 and CYP17A1 mRNA levels in the goat theca cells(P<0.05). Addition of LY294002, significantly decreased LH-induced CYP11A1 and CYP17A1 mRNA expression(P<0.05). However, addition of wortmannin, significantly decreased LH-induced CYP11A1 and CYP17A1 mRNA expression(P>0.05). Neither LH nor the PI3 K inhibitors alter the mRNA levels of HSD3B1 and StAR in the goat theca cells(P>0.05). LH significantly increased LHR mRNA in goat theca cells(P<0.05). Addition of LY294002, but not wortmannin, significantly decreased the expression of LH-induced LHR mRNA(P<0.05). EIA analysis results show that LH significantly increased androstenedione production in goat theca cells(P<0.05). Addition of the PI3 K inhibitors LY294002 and wortmannin significantly decreased LH-induced androstenedione production in goat cells(P<0.05).In conclusion, ovarian theca cells from ovary in goat were isolated and purified in this research, and theca cells cultured in vitro for using to examine whether and by what means LH controls PI3K/Akt signaling and androgen production. This research lays the foundation for further study the intracellular signaling mechanisms that regulate steroidogenesis in theca cells and the role of theca cells in ovarian follicular development.