The supernatant was assayed for protein content and subjected to Western blot analysis to detect anti phospho Akt and anti total Akt. Samples containing equal amounts of pro tein were separated by 10% acrylamide SDS PAGE. The relevant proteins were detected on blots using their specific antibodies. Determination of androstenedione levels Androstenedione levels were determined using EIA at the end of the stimulation. Protein was quantified using the Bradford method. RNA extraction and RT PCR Total RNA was isolated using TRIzol according to the manufacturers instruc tions. The RNA pellets were ethanol precipitated, washed, and resuspended in sterile ribonuclease free water. Qual ity of the RNA was assessed by fractionating it on 1% aga rose gel and observing the presence of the typical 28S and 18S rRNA under UV light.

RT PCR analyses for bovine CYP17A1, StAR, and 36B4 were performed on total RNAs from cultured theca cells using specific primers. Primers used for bovine CYP17A1 were respectively. In each case, RNAs were describes it reverse transcribed in a final volume of 40 l solution con taining 1× first strand buffer, 500 M each deoxynucleotide triphosphate, 10 mM dithiothreitol, 200 U SuperScript III RNase H free reverse transcriptase, 200 ng random hexamers, and 2 g total RNA. The target cDNAs were amplified for 30 cycles and 25 cycles, respectively, in a thermal cycler using deoxynucleotide triphosphate and 1. 5 U of TaKaRa Ex Taq. Aliquots of PCR products were electrophoresed on 1. 5% agarose gels and stained with ethidium bromide.

The relative integrated density of each band was scanned and digitized using FluorChem, the ratios of densitometric read ings of the amplified target cDNA and internal control, 36B4, DNA were analyzed. Statistical analysis All experiments were selleck repeated at least three times using theca cells obtained from separate groups of bovines. Data were subjected to ANOVA. Group means were contrasted using Tukeys post hoc multiple comparison test. P 0. 05 was considered significant. All values are expressed as mean SEM. Results Experiment 1 LH increases phospho Akt content in bovine theca cells Total Akt was present in theca cells at 0 h and remained constant during culture with LH. During the 5 min to 8 h of culture, Akt was not phosphorylated by LH. However, the amount of phospho Akt began to increase at 12 h and reached its highest level at 24 h after addition of LH.

Experiment 2 Effects of the PI3K inhibitors on LH induced androgen production in theca cells Results show that LH significantly increased androstene dione production in bovine theca cells. Addition of the PI3K inhibitors wortmannin and LY294002 significantly decreased LH induced androstenedione production in theca cells. Experiment 3 Effects of the PI3K inhibitors on CYP17 and StAR mRNA expressions in theca cells Results show that LH significantly increased CYP17A1 mRNA level in the theca cells.