Expression pattern of circadian genes and steroidogenesis-related genes after testosterone stimulation in the human ovary

Paraffin sections of normal ovarian tissue were obtained from five women aged 29–35 years. All women had undergone bilateral salpingo-oophorectomy with or without hysterectomy for a uterine or unilateral ovarian malignant tumor before chemotherapy or radiotherapy. Informed consent was obtained from all patients. After deparaffinization, antigen retrieval, and blocking in normal goat serum (for CLOCK) or bovine serum (for PER2), the slides were incubated overnight at 4 °C in rabbit anti-human CLOCK polyclonal antibody diluted to 20 μg/mL (catalogue no. ab 65033,Abcam) or goat anti-human PER2 polyclonal antibody diluted to 7.5 μg/mL (catalogue no. ab118489, Abcam), respectively. Slides were washed and incubated with biotin-labeled goat anti-rabbit secondary antibody for CLOCK (CWBIO) or bovine anti-goat secondary antibody for PER2 (Santa Cruz Biotechnology) for 30 min, then washed and incubated with horseradish peroxidase-labeled streptavidin for 10 min. The peroxidase–antibody complex was visualized using 3, 3′-diaminobenzidine (CWBIO). Control experiments included samples treated in the same manner but with omission of the primary antibody. The sections were counterstained with hematoxylin.

For each experiment, human luteinized granulosa cells were obtained from the follicle fluid collected during ovum aspiration of 10 patients undergoing in vitro fertilization. All patients underwent the long protocol of gonadotropin-releasing hormone agonist treatment (1.0 mg tiptorelin acetate; Ipsen Pharma Biotech, France) with human chorionic gonadotropin as the trigger. The follicle fluid was pooled and centrifuged for 5 min at 2500 rpm. Granulosa cells were purified using 50 % Percoll (Sigma) through gradient centrifugation for 10 min at 2000 rpm. Ovarian tissue fragments were then removed from the granulosa cell suspension with a 40-μm cell strainer (Becton-Dickinson). Purified granulosa cells were plated on a 12-well plate (3.0 × 10⁵/well) and cultured in Dulbecco’s modified Eagle medium/Ham’s F12 supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10 % fetal bovine serum (Gibco) in a 37 °C incubator with 5 % CO₂. Cells were cultured for 9 days to reach confluence prior to any treatment. Cells were exposed to 100 ng/mL testosterone (Sigma) dissolved in serum-free medium for 2 h, washed with serum-free medium once, and then cultured further in serum-free medium until harvested. Cells in the control group were cultured in serum-free medium without testosterone treatment. Samples were harvested every 4 h starting at the beginning of treatment and continuing until 48 h.

RNA was extracted with High Pure RNA Isolation Kit (Roche) according to the manufacturer’s instructions, and quantitated by 260/280 UV spectrophotometry (NanoDrop ND-1000, Wilmington, DE, USA). Five hundred nanograms of RNA were subjected to reverse transcription with oligo-dT primers using Roche Transcriptor First-strand cDNA Synthesis Kit. The differential expression of target gene mRNA in granulosa cells was quantified using the following TaqMan® Gene Expression Assays (Applied Biosystems): CLOCK (Hs00231857_m1), PER2 (Hs00256143_m1), BMAL1 (Hs00154147_m1), steroidogenic acute regulatory protein (STAR; Hs00986559_g1), 3-β-hydroxysteroid dehydrogenase type II (HSD3B2; Hs00605123-m1), cholesterol side-chain cleavage cytochrome P450 (CYP11A1; Hs00167984_m1), and cytochrome P450 (CYP19A1; Hs00903413_m1). Quantification was accomplished with an Applied Biosystems 7500 real-time RT-PCR machine using TaqMan® Fast Advanced Master Mix (Invitrogen). The relative mRNA levels were calculated using the comparative cycle threshold method, using ACTB (Hs99999903_m1; Applied Biosystems) as a reference gene.

Data are presented as the least squares mean ± standard error of the mean from three replicate experiments. Least-squares analysis of variance (ANOVA) implemented by SPSS 13.0 was used to analyze all data. Tukey’s multiple-comparison post-hoc test was used if equal variances were validated, or Dunnett’s T3 post-hoc test was used if equal variances were not assumed, when ANOVA returned a value of P ≤ 0.05. P ≤ 0.05 was considered a statistically significant difference.

Immunohistochemistry results showed positive expression of CLOCK in the cumulus and mural granulosa cells in dominant antral follicles and in interstitial cells, but no CLOCK expression was observed in the primordial follicles, primary follicles, and preantral follicles, and in the theca cells of antral follicles. Expression of PER2 was observed in the cumulus cells and mural granulosa cells in the dominant antral follicles and in interstitial cells, was weak in the theca cells of the dominant antral follicle, but was absent in the primordial follicle, primary follicles, and preantral follicles (Fig. 1).

Treatment of testosterone induced oscillations in PER2 expression, with the first peak and bottom occurring at the 24th and 32nd hours, respectively, and the second peak occurring at the 44th hour (P_{4 vs. 24} = 0.028, P_{24 vs. 32} = 0.041, P_{24 vs. 48} = 0.039, P _{4 vs. 44} = 0.024). Expression of CLOCK increased significantly at the 24th hour (P_{4 vs. 24} = 0.04), whereas expression of BMAL1 did not change significantly after testosterone stimulation. In the control group, after changing to serum-free medium, the expression levels of both PER2 (P_{4 vs. 16} = 0.016) and CLOCK (P_{4 vs. 12} = 0.022, P_{4 vs. 16} = 0.031, P_{4 vs. 20} = 0.006, P_{4 vs. 36} = 0.011) increased significantly; however, they did not display an oscillating pattern. The expression of BMAL1 did not change significantly over time (Fig. 2).

Treatment of testosterone induced oscillations in STAR expression, with the first and second peaks occurring at the 24th and 40th hours, respectively (P_{4 vs. 24} < 0.001, P_{24 vs. 32} < 0.001, P_{4 vs. 40} = 0.001). Expression of CYP19A1 in granulosa cells was significantly repressed at the 12th hour (P_{4 vs. 12} = 0.003), whereas the expression levels of HSD3B2 and CYP11A1 did not change significantly after testosterone stimulation. In the control group, changing to serum-free medium significantly stimulated STAR expression (P_{4 vs. 24} = 0.028, P_{4 vs. 36} = 0.001, P_{4 vs. 40} = 0.021), but had no significant effects on the expression levels of CYP11A1, HSD3B2, and CYP19A1 (Fig. 3).