Luteinizing hormone-induced Akt phosphorylation and androgen production are modulated by MAP Kinase in bovine theca cells

Human LH was provided by the National Institutes of Health and Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA). LY294002 (a PI3K inhibitor) was from Sigma Chemical Co. (St. Louis, MO), and wortmannin (a PI3K inhibitor), H89 (a selective inhibitor of PKA), and U0126 (a potent MEK inhibitor) were purchased from Calbiochem Novabiochem Corp. (San Diego, CA).

Bovine ovaries were collected less than 15 min after slaughter at a local abattoir. The ovaries were placed in an ice-cold buffered salt solution and transferred to the laboratory less than 90 min after collection. The estrous cycle stage was determined morphologically, as described previously by Ireland et al [17]; only those ovaries with a regressing corpus luteum were used for this study. Theca cells were isolated from the ovaries under sterile conditions, as described previously [18]. Briefly, small antral follicles (2-4 mm diameter) with clear surfaces were cut into halves and theca interna removed in situ using fine forceps. Granulosa cells, together with part of the theca cell layer, were removed by scraping with a scalpel under a stereomicroscope. The resultant thin thecal layer was minced and subsequently treated with a Hanks'-HEPES buffer containing collagenase (2150 U/ml, type 1; Sigma) and DNase (100 U/ml; Sigma), 0.4% (vol/vol) BSA, and 0.2% (wt/vol) glucose (pH 7.4). Cell dissociation was allowed to continue for 30-60 min at 37°C with continuous stirring at 80 rpm and 0.25% (wt/vol) pancreatin (Sigma) in a Hanks'-HEPES buffer for 7 min. Dispersed cells were washed three times. Cell viability, as determined using the trypan blue-dye exclusion test, was 90-93%. Purity of the theca cell preparation used in this study was substantiated by the secretion of estradiol; prepared theca cells did not produce estradiol in the presence or absence of forskolin, whereas granulosa cells obtained from the same follicle secret significant (data not shown). Isolated theca cells were plated onto serum-coated dishes with serum-free medium for 36 h. Then they were stimulated with LH (100 ng/ml) for various durations (0, 5 min, 20 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h). Preliminary data indicated that 100 ng/ml of LH is the minimal effective concentration for inducing a significant increase in androgen production and CYP17A1 expression in our culture system.

Western blot analysis was conducted as described previously [12]. Briefly, primary cultures at the end of incubation with the appropriate stimulant or no stimulation as indicated in each experiment were rinsed with ice-cold PBS and once with buffer A [50 mM β-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium vanadate] and were subsequently harvested in buffer A plus proteinase inhibitors. Cell lysates were centrifuged at 20,000 × g for 20 min. 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 protein (40 μg) were separated by 10% acrylamide SDS-PAGE. The relevant proteins were detected on blots using their specific antibodies.

Androstenedione levels were determined using EIA at the end of the stimulation. Protein was quantified using the Bradford method.

Total RNA was isolated using TRIzol (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's instructions. The RNA pellets were ethanol precipitated, washed, and resuspended in sterile ribonuclease-free water. Quality of the RNA was assessed by fractionating it on 1% agarose gel and observing the presence of the typical 28S and 18S rRNA under UV light. RT-PCR analyses for bovine CYP17A1, StAR, and 36B4 (an acidic ribosomal phosphoprotein as an internal control) were performed on total RNAs from cultured theca cells using specific primers. Primers used for bovine CYP17A1 were 5'-TCAGAGAAGTGCTCCGAATCC-3' and 5'-TGCCACTCCTTCTCACTGTGA-3'; those for bovine StAR were 5'-TCGCGGCTCTCTCCTAGGT-3' and 5'-CTGCCGGCTCTCCTTCTTC-3', and those for bovine 36B4 were 5'-GGCGACCTGGAAGTCCAACT-3' and 5'-GGATCTGCTGCATCTGCTTG-3', respectively. In each case, RNAs were reverse transcribed in a final volume of 40 μl solution containing 1× first-strand buffer [3 mM MgCl₂, 75 mM KCl, 50 mM Tris-HCl (pH 8.3)], 500 μM each deoxynucleotide triphosphate, 10 mM dithiothreitol, 200 U SuperScript III RNase H-free reverse transcriptase (Invitrogen Corp.), 200 ng random hexamers, and 2 μg total RNA. The target cDNAs were amplified for 30 cycles (CYP17A1 and StAR) and 25 cycles (36B4, internal control), respectively, in a thermal cycler (94 C for 20 s, 60 C for 30 s, and 72 C for 60 s) using deoxynucleotide triphosphate (0.2 mM) and 1.5 U of TaKaRa Ex Taq (Takara Shuzo Co. Ltd., Kyoto, Japan). 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 (Alpha Innotech Corporation, San Leandro, CA); the ratios of densitometric readings of the amplified target cDNA and internal control, 36B4, DNA were analyzed.

All experiments were repeated at least three times using theca cells obtained from separate groups of bovines. Data were subjected to ANOVA. Group means were contrasted using Tukey's post hoc multiple comparison test. P < 0.05 was considered significant. All values are expressed as mean ± SEM.