Growth hormone activates PI3K/Akt signaling and inhibits ROS accumulation and apoptosis in granulosa cells of patients with polycystic ovary syndrome

From November 2018 to November 2019, patients with PCOS (aged 22–36 years) diagnosed according to the Rotterdam criteria [26] were recruited, then further according to computer-generated random numbers randomly assigned into two groups: one group took GH treatment during controlled ovarian stimulation (COS) as PCOS-GH group; the other group had no GH treatment as PCOS-C group. Written informed consent was obtained from each participant. The study also conforms to the Declaration of Helsinki for Medical Research involving Human Subjects (2013 revision). Age-matched women of tubal infertility (aged 25–37 years) who underwent in vitro fertilization and embryo transfer (IVF-ET) were recruited as non-PCOS controls. Patients with hydrosalpinx, systemic lupus erythematosus, or sicca syndrome; uncontrolled endocrinopathy such as diabetes, hyperthyroidism, hypothyroidism, and hyperprolactinemia; or currently taking anti-OS medicine such as vitamin E, vitamin C, and Coenzyme Q10 were excluded from the study.

Medical history such as menstrual cycle regularity, duration of infertility, and treatment was collected from all the participants. Physical examinations included measurements of height, body weight, waist circumference, and hip circumference. Body weight index (BMI) was calculated as weight divided by height squared (kg/m²). The waist-to-hip ratio (WHR) was calculated as the waist circumference divided by the hip circumference. Androgen-related symptoms of hirsutism and acne were evaluated as previously reported [27, 28]. Plasma glucose, estradiol (E₂), progesterone (P), total testosterone (TT), luteinizing hormone (LH), follicle-stimulation hormone (FSH), sex hormone binding globulin (SHBG), and fasting insulin (FINS) levels were measured as reported previously. The free androgen index (FAI) was calculated as TT (nmol/L)/SHBG (nmol/L) × 100. The homeostasis model assessment (HOMA-IR) index was calculated as fasting glucose (mmol/L) × fasting insulin (mIU/L)/22.5. The intra- and inter-assay coefficients of variation for these values were < 5 and < 10%, respectively.

COS regimen was gonadotropin-releasing hormone antagonist protocol for all participants. Recombined follicular stimulation hormone (rFSH) (Gonal-F; Merck-Serono KGaA., Darmstadt, Germany) was administered starting from day 2 of the menstrual cycle. The dose of rFSH was adjusted according to follicular growth. In the PCOS-GH group, patients were subcutaneously administered with 4 IU/d of recombinant human GH (Jintropin, Changchun GeneScience Pharmaceutical Co., Ltd., Changchun, Jilin, China) until the trigger day. Cetrorelix (Cetrotide; Merck-Serono KGaA.) was administered when one of the below criteria was matched: serum E₂ > 300 pg/mL, leading follicle diameter reached 13–14 mm, LH > 10 IU/L. Recombinant human chorionic gonadotropin (Ovitrelle®; Merck-Serono KGaA., Darmstadt, Germany) was administered as the trigger when the diameters of at least two follicles reached ≥18 mm. After 36 h, oocytes were retrieved under transvaginal ultrasound guidance. During oocyte retrieval, primary GCs in follicle fluid (FF) were collected.

During oocyte retrieval, FF was collected from follicles with a diameter ≥ 16 mm measured on the retrieval day, then immediately separated by centrifugation at 700×g for 5 min at room temperature. The precipitates were suspended in left 2 ml of FF and gently layered into 3 mL of 50% lymphocyte separation medium (Solarbio Science and Technology Corporation, Beijing, China). After centrifugation at 700×g for 10 min at room temperature to remove red blood cells and debris, GCs layered at the interface of the gradient were collected and washed twice with 5 mL of phosphate-buffered saline (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, Jiangsu, China). The residual red blood cells were further removed using red blood cell lysis buffer (Solarbio Science and Technology Corporation). GCs from each patient were collected separately and considered as one sample. One portion of GCs was immediately examined intracellular ROS levels, mitochondrial membrane potential (MMP), and apoptosis; the remaining GCs were stored at − 80 °C refrigerator.

ROS generation in GCs was measured using 2′,7′-diclorodihydrofluorescein di-acetate (H2-DCFDA) method by ROS assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). Briefly, GCs were resuspended in PBS and incubated with 10 μM H2-DCFDA in the dark for 25 min at 37 °C, and then incubated with 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) (NeoFroxx, Frankfurt, Germany) for 5 min. After washed three times with PBS, GCs suspensions were added to glass slides, and examined by fluorescence microscopy (Olympus Corporation, Tokyo, Japan). The examination wavelength was 488 nm, and the emission wavelength was 525 nm.

Similar with the above protocol but without DAPI, NanoDrop UV-Vis spectrophotometry (Thermo Scientific, MA, USA) was used to measure the intracellular ROS level of GCs. The relative ROS levels were presented as the fluorescence intensity of the PCOS group relative to that of non-PCOS controls.

Apoptosis of GCs were detected using the Annexin V-FITC apoptosis detection kits (KeyGEN Bio TECH Co., Ltd.). Briefly, 1 × 10 ⁵/ Test of GCs were resuspended in 500 μL binding buffer, then labeled with Annexin V-FITC (5 μL) and propidium iodide (PI) (5 μL) for 15 min in the dark at room temperature. After 1 h, the green (Annexin V-FITC) and red (PI) fluorescence were examined by flow cytometry (MilliporeSigma Co., Ltd., Burlington, MA, USA). The examination wavelength was 488 nm, and the emission wavelength was 530 nm.

The MMP of GCs was examined using JC-1 Apoptosis Detection Kits (KeyGEN Bio TECH Co., Ltd.). In brief, 1 × 10 ⁵/ Test of GCs were resuspended and incubated with 500 μL JC-1 reagent solution at 37 °C in the dark for 15 min. JC-1 accumulates in functional mitochondria with high mitochondrial membrane potential (ΔΨm) and forms aggregates that emit red fluorescence. When mitochondrial transmembrane potential is depolarized with low ΔΨm, JC-1 releases from the mitochondria and forms monomers that emit green fluorescence. After washed two times with incubation buffer, the green and red fluorescence were examined by flow cytometry (MilliporeSigma Co., Ltd., Burlington, MA, USA). The examination wavelength was 488 nm, and the emission wavelength was 530 nm.

Frozen GCs were rapidly thawed and total RNA was isolated using the RNAprep Pure Micro Kit (Tiangen Biotech Co., Ltd., Beijing, China). The quality of RNA was checked at an absorbance of 260 nm/280 nm by Nanodrop-2000 (ThermoFisher Scientific, Waltham, MA, USA). Total RNA was reverse transcribed to cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Tokyo, Japan). Polymerase chain reaction (PCR) was performed using TB Green™ Premix Ex Taq™ II (TaKaRa) on a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 65 °C for 5 s. The PCR system (20 μL) comprised RNase free dH₂O (6.4 μL), cDNA (2 μL), forward primer (0.8 μL), reverse primer (0.8 μL) and 2 × TB Green Premix Ex Taq II (10 μL). All PCR reactions were conducted in triplicate. Each experiment was repeated at least three times. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as the internal control as indicated and fold changes were calculated by the 2^-ΔΔCt method. Primers were designed and synthesized at Beijing Tsingke Biological technology (Beijing, China). Primers used in the RT-qPCR are shown in Table 1.

Total protein was isolated from GCs using the RIPA lysis buffer (KeyGen Biotech. Co., Ltd.) containing Halt™ Protease Inhibitor Cocktail (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. Total protein concentration was determined using a quantitative BCA protein kit (Thermo Scientific). The total proteins (60 μg/lane) were subsequently subject to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked with Tris-buffered saline with Tween-20® that contained 5% bovine serum albumin (Bio-Rad) for 1 h at room temperature and subsequently incubated with a primary antibody according to the manufacturer’s instructions at 4 °C overnight. Specific primary antibodies included PI3K (1:2000; ab140307, Abcam, Cambridge, MA, USA), p-PI3K (Tyr607, 1:1000; ab182651, Abcam), Akt (1:10000; ab179463, Abcam), p-Akt (Ser473, 1:2000; ab81283, Abcam), FOXO1 (1:1000; 2880, Cell Signaling, Beverly, MA, USA), p-FOXO1 (Ser 256, 1:1000; 9461, Cell Signaling), Bax (1:1000; 5023, Cell Signaling), Bcl-2 (1:500; 01556, Wanleibio, Shenyang, China), caspase-9 (1:1000; 9502, Cell Signaling), cleaved caspase-9 (Asp330, 1:1000; 7237, Cell Signaling), caspase 3 (1:1000; ab32351, Abcam), cleaved caspase-3 (Asp175, 1:1000; 9661, Cell Signaling), and GAPDH (1:2000; 2188R, Bioss, Beijing, China). On the day after washing, the membranes were incubated with secondary antibodies for 2 h at room temperature. SuperSignal® West Pico Trial Kit (ThermoFisher Scientific) was used for signal detection and the protein bands were visualized using a GelDoc XR densitometer (Bio-Rad). The relative intensities of each protein band were determined using the GAPDH band as an internal reference.

The cleaved caspase-9 and caspase-3 have bioactivity to induce apoptosis. The concentrations of active caspase-9 and caspase-3 in GCs lysates were determined using human caspase-9 ELISA kit and caspase-3 ELISA kit (Elabscience Biotechnology Co., Ltd., Hubei, China), respectively, and a 450-nm Perlong DNM-9602G microplate spectrophotometer (Beijing Perlong New Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The amount of protein loaded in each well was the same (100 μg/well) and each sample was detected in duplicate. The intra- and inter-assay coefficients of variation for these values were < 5% and < 10%, respectively. The sensitivities of the caspase-9 and caspase-3 assays were 0.99 ng/mL and 0.19 ng/mL, respectively.

All data were statistically analyzed using SPSS 17.0 software (SPSS Inc., Chicago IL, USA). Continuous variables are expressed as means ± standard deviation. The normality of data distribution was assessed using Kolmogorov–Smirnov tests. Between-group comparisons were assessed using one-way ANOVA with post-hoc Bonferroni tests. Categorical data were compared using Chi-squared tests. Two-tailed P values < 0.05 were considered statistically significant.

The prevalence of irregular menstrual cycles, hirsutism, and acne in patients with PCOS was more common than in non-PCOS controls. The anthropometrics, endocrine, and metabolic parameter such as WHR, LH/FSH ratio, TT, SHBG, FAI, FINS, and HOMA-IR were significantly different in patients with PCOS when compared to non-PCOS controls (P < 0.05). The clinical, endocrine, and metabolic characteristics were not significantly different between the PCOS-GH and PCOS-C groups (P > 0.05). (Table 2).

The green fluorescence intensity visualized by fluorescent microscopy of ROS in GCs of the PCOS-GH group was weaker compare to that in the PCOS-C group, but similar to that of non-PCOS controls. Quantitative detection using a spectrophotometer indicated that the ROS intensity in the PCOS-GH group (1.10 ± 0.21) was significantly lower than that in the PCOS-C group (2.78 ± 0.35) (P < 0.05), but no difference with non-PCOS controls (1.00 ± 0.21) (P > 0.05). The fluorescence intensity of ROS was expressed as the fold change relative to the control (Fig. 1).

MMP in the PCOS-GH group was significantly higher than that in the PCOS-C group (0.94 ± 0.26 vs. 0.22 ± 0.18) (P < 0.05), but no difference when compared to non-PCOS controls (0.94 ± 0.26 vs. 0.79 ± 0.21) (P > 0.05). (Fig. 2 a, 2 b).

The early (7.20% vs. 28.18%) and late apoptosis rate (9.37% vs.19.01%) in GCs of the PCOS-GH group was significantly lower than those in the PCOS-C group (P < 0.05), but similar to those in non-PCOS controls (11.07% and 11.48%, respectively) (P > 0.05). (Fig. 2 c, 2 d).

To study the mechanisms of GH for alleviating OS and mitochondrial dysfunction in GCs, candidate genes and proteins involved in PI3K/Akt signaling were determined by RT-qPCR and western blotting, respectively. In the GCs of the PCOS-GH group, the mRNA and protein levels of FOXO1 were significantly lower than those in the PCOS-C group (P < 0.05); the protein levels of p-PI3K/PI3K, p-Akt/Akt, and p-FOXO1 were significantly higher than those in the PCOS-C group (P < 0.05). The above parameters were no difference between the PCOS-GH and non-PCOS groups (P > 0.05). (Fig. 3).

Figure 4 showed the significantly decreased both mRNA and protein levels of Bax, and increased Bcl-2 in GCs of the PCOS-GH group compared with those in the PCOS-C group (P < 0.05). Furthermore, both mRNA and protein levels of Bax and Bcl-2 were no difference between the PCOS-GH and non-PCOS groups (P > 0.05).

Upon apoptotic stimulation, caspase-9 and its down signal caspase-3 is activated and cleaved, resulting in apoptosis. Figure 4 showed significantly decreased caspase-9 and caspase-3 mRNA levels, and decreased cleaved caspase-9/caspase-9 and cleaved caspase-3/caspase-3 protein levels in GCs of the PCOS-GH group compared with those in the PCOS-C group (P < 0.05). The mRNA and protein levels did not differ between the PCOS-GH and non-PCOS groups (P > 0.05).

Fig. 4 B showed that the protein bands of cleaved caspase-9 and cleaved caspase-3 were decreased to almost undetectable levels in the PCOS-GH and non-PCOS groups. For quantitative analysis, we measured the concentration of active caspase-9 and active caspase-3 in the cell lysate by ELISA. In Fig. 4 D, the concentrations of active caspase-9 (7.11 ± 1.31 ng/mL vs. 22.39 ± 2.79 ng/mL) and active caspase-3 (5.90 ± 1.42 ng/mL vs. 15.88 ± 2.11 ng/mL) were significantly lower in GCs of the PCOS-GH group compared with those in the PCOS-C group (P < 0.05). The concentration of active caspase-9 (7.11 ± 1.31 ng/mL vs. 6.99 ± 1.08 ng/mL) and active caspase-3 (5.90 ± 1.42 ng/mL vs. 5.35 ± 1.06 ng/mL) did not significantly differ between the PCOS-GH and non-PCOS groups (P > 0.05)