Study participants
The study design was approved by the Medical Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (Application ID: [2019]205). All participants gave written informed consent. Human theca cells were isolated from the follicular fluid of patients who underwent IVF/intracytoplasmic sperm injection (ICSI) at our clinic from September 2016 to June 2019. The inclusion criteria were as follows: under 40 years of age, basal serum follicle stimulating hormone (FSH) < 10 IU/L, basal serum anti-Müllerian hormone (AMH) > 1.5 ng/mL, no endocrine disorders, and no history of radiotherapy or chemotherapy. PCOS was diagnosed according to the Rotterdam criteria 2003.
Isolation and purification of human theca cells
For each experiment, follicular fluid was collected during oocyte retrieval from 10–20 patients undergoing IVF/ICSI, followed by centrifugation for 10 min at 1,500 rpm. The precipitate was reconstituted in phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) and filtered with a 100-μm strainer (BD Falcon, BD Biosciences, Franklin Lakes, NJ, USA). The ovarian tissues that remained on the filter membrane were then collected using sterile forceps. The collected ovarian tissues were then digested with 5 mg/mL type I collagenase (Sigma-Aldrich, St. Louis, MO, USA) solution (dissolved and diluted with PBS) at 37°C for around 90 min and were pipetted to accelerate the digestion process. The dispersed cells were collected every 15 min, washed with PBS twice, and resuspended in PBS. The cell suspensions were mixed and filtered with a 40-μm cell strainer (BD Falcon; BD Biosciences) to remove undigested tissue and centrifuged for 5 min at 1,500 rpm. The precipitate was collected and washed with PBS three times, and then resuspended in Dulbecco’s modified Eagle medium/F12 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin (all from Gibco, Thermo Fisher Scientific). The purified cells were counted with a hemocytometer, and the cell viability was determined by trypan blue exclusion (Gibco, Thermo Fisher Scientific). The cell viability was in the range of 90–95%.
Cell culture
Human theca cells were plated on 15 cm culture dishes (6 × 105 viable cells/mL, 20 mL/well) for co-immunoprecipitation (co-IP) analysis, in 6-well plates (3 × 105 viable cells/mL, 2.5 mL/well) for western blotting and enzyme-linked immunosorbent assay (ELISA), 12-well plates (2 × 105 viable cells/mL, 2 mL/well) for RNA extraction and immunofluorescence analysis, and 96-well plates (5 × 104 viable cells/mL, 0.1 mL/well) for Cell Counting Kit-8 (CCK-8) analysis. The plates were cultured at 37°C in a humidified incubator containing 5% CO2, and the medium was changed daily. After the first 48 h of culture, theca cells were treated with various doses (50, 100, 200, 400, 800 ng/mL) of GDF9 (R&D Systems), GDF9 (800 ng/mL) with 5 μM SB525334 (an ALK5 inhibitor; Selleck Chemicals, Houston, TX, USA), or 20 μM kartogenin (an agonist of SMAD4; Selleck) in serum-free culture media for another 48 h. The medium was then collected to determine the concentration of androstenedione and testosterone. RNA was extracted from the cells for quantification of mRNA levels by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total protein was extracted from the theca cells for ELISA, western blotting, or co-IP analysis.
Immunofluorescence staining
Human theca cells were cultured on 15-mm-diameter cover glasses and placed on 12-well plates (Corning, New York, USA) for 48 h. The theca cells were washed three times with PBS (Thermo Fisher Scientific) and fixed with 4% paraformaldehyde (Solarbio) for 15 min at room temperature. Subsequently, 0.5% Triton X-100 (Solarbio) was used to permeate the theca cells for 20 min except for staining for follicle-stimulating hormone receptor (FSHR). Next, 5% donkey serum was added to block non-specific binding sites for 30 min. Rabbit anti-human cytochrome P450 family 17 subfamily A member 1 (CYP17A1) primary antibody (1:100, Abcam, Cambridge, UK), rabbit anti-human anti-FSHR primary antibody (1:100, Abcam, UK), PBS, or 5% donkey serum were then added into respective wells and incubated overnight at 4°C. The following day, theca cells were washed with PBS twice and incubated with goat anti-rabbit secondary antibody (1:100, Bioss, CN) for 1 h at room temperature. After three washes with PBS, the nuclei were stained with DAPI (Solarbio, CN). Finally, processed theca cells were visualized with a laser-scanning confocal microscope (Carl ZEISS, Germany).
Cell viability assay
Cell viability was determined by a CCK-8 assay (Dojindo Laboratories, Japan). After 48 h of culture, theca cells were washed twice with PBS and treated with or without different concentrations of GDF9 (50, 100, 200, 400, 800 ng/mL) in serum-free medium for 24 h. Then, 10 μL CCK-8 solution was added into the culture medium and the cells were incubated for another 1 h. The absorbance at 450 nm was detected by the Sunrise ELISA reader (Tecan Co., Austria).
RNA extraction and quantification
Total RNA was extracted by GeneJET RNA Purification Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. The concentration and purity of extracted RNA were determined by measuring the absorbance at a wavelength of 260 nm and an absorbance ratio of 260/280 nm with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Subsequently, levels of CYP17A1 (cat#Hs01124136_m1), CYP11A1 (cat#Hs00167984_m1), steroidogenic acute regulatory protein (StAR; cat#Hs00986559_g1), 3β-hydroxysteroid dehydrogenase (HSD3B; cat#Hs00605123_m1), and LHCGR (cat#Hs00174885_m1) were quantitatively examined by Taqman Gene Expression Assays (Thermo Fisher Scientific) using an Applied Biosystems 7500 Real-time PCR System (Thermo Fisher Scientific). Relative expression analysis was performed by 2-ΔΔCT method using ACTB (cat#Hs01060665_g1) as a reference gene.
Assays for SMAD2/3 and phospho-SMAD2/3 protein levels
Human SMAD2/3 and phospho-SMAD2/3 protein levels were evaluated with Total SMAD2/3 Sandwich ELISA Kit and phospho-SMAD2/3 Sandwich ELISA Kit (Cell Signaling Technology, Danvers, MA, USA) following the manufacturer’s instructions. Briefly, after treatment with 800 ng/mL GDF9, or 800 ng/mL GDF9 with 5 μM SB525334 for 2 h, total protein was extracted from human theca cells using lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and placed on respective ELISA plates. After incubating with primary and horseradish peroxidase (HRP)-linked secondary antibody, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added to trigger the reaction with HRP. The optical density values at 450 nm were detected by the Sunrise ELISA reader (Tecan Co.).
Assays for steroid concentrations
The concentrations of testosterone and androstenedione in the culture medium were determined by Testosterone Parameter Assay Kit (R&D Systems) and an androstenedione assay kit (Siemens Healthcare Diagnostics Products Limited, UK), respectively, following the manufacturer’s protocol. The detection limits of testosterone and androstenedione were 0.030 ng/mL and 1 nmol/L, respectively.
Western blot analysis
Total proteins were extracted using ice-cold radioimmunoprecipitation assay lysis buffer containing 1 mM PMSF (Solarbio, CN). Protein extracts were quantified using a bicinchoninic acid Protein Assay Kit (Thermo Fisher Scientific) and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Separated proteins were then transferred onto polyvinylidene difluoride membranes (Merck Millipore, Germany). After blocking in Tris-buffered saline with Tween (TBST) with 5% non-fat milk for 1 h at room temperature, the membranes were cut according to the molecular weight of the target proteins. The corresponding membranes were then incubated with primary rabbit antibodies (Cell Signaling Technology) against CYP17A1 (1:1000 dilution), CYP11A1 (1:1000 dilution), StAR (1:1000 dilution), or GAPDH (1:1000 dilution) overnight at 4°C. The membranes were subsequently washed with TBST three times and probed with secondary HRP-linked anti-rabbit antibody (1:1000 dilution, Cell Signaling Technology) for 1 h. Immunoreactive bands were visualized by the ChemiDoc Touch system (Bio-Rad Laboratories Inc., USA) using an enhanced chemiluminescent substrate (Merck Millipore). The target protein expression was determined by ImageJ software and normalized to GAPDH levels.
Co-IP
Co-IP was performed according to the manufacturer instructions of the kit (cat#BES3011, Bersin Bio, CN). Briefly, after treatment with 400 ng/mL GDF9 for 24 h, human theca cells were washed with ice-cold PBS twice and then lysed with immunoprecipitation buffer supplemented with protease inhibitor. Lysates were centrifuged at 16,000 rpm for 15 min at 4°C to remove insolvable debris. Following preclearing, the resultant supernatant was incubated without or with specific antibodies (anti-BMPRII antibody, cat#sc-73752, Santa Cruz Biotechnology; anti-ALK5 antibody, cat#ab31013, Abcam; or rabbit non-specific IgG, cat#A7016, Beyotime, CN) at 4°C overnight. The incubation was then continued for another 2 h after protein-A/G agarose beads were added. After washing with immunoprecipitation wash buffer, immunoprecipitates were eluted with CHAPS eluent buffer, and then co-IP samples were identified by western blotting as described above.
Statistical analysis
Statistical analyses were performed with SPSS 26.0 software. We applied the Kolmogorov–Smirnov test to analyze the distribution of data in two-group comparisons. Normally distributed data are presented as the mean ± standard error of the mean and were compared with two-tailed Student’s t-test. Skewed data are presented as the mean and quartiles and were examined by Mann–Whitney U tests. To identify individual differences between means, differences among more than two groups were assessed by analysis of variance followed by Tukey’s multiple comparison post-hoc tests if the assumption of equal variances was met. Alternatively, Dunnett’s T3 post-hoc tests was used if equal variances were not found. P < 0.05 was considered statistically significant.