Study design
This is an observational study of infertile women before and during IVF treatment at the International Peace Maternity and Child Health Hospital (IPMCH), Shanghai, China. The Institutional Ethics Committee of the IPMCH approved the acquisition protocol. Follicular fluid and cumulus granulosa cells on the day of oocyte retrieval were obtained from 27 patients undergoing IVF at the reproductive center. The subjects were recruited with male factor infertility or tubal factor infertility. Subjects with endometriosis and PCOS were excluded. The age of the patients ranged from 25 to 40 years, and the size of the follicles ranged from 19 to 24 mm.
Collection of GCs and follicular fluid
The size of the follicle was estimated by ultrasound at the time of oocyte retrieval. Follicular aspirates contain mural GCs and oocytes surrounded by cumulus GCs. After the oocytes were removed by the embryologist, the remaining material was centrifuged at 3000 g for 15 mins to isolate the GCs. The fluid from the first aspirated follicle was used to measure its hormone and neurotransmitter metabolic levels.
Quantification of neurotransmitters in follicular fluid by liquid chromatography–tandem mass spectrometry (LC-MS/MS)
Pretreated human follicular fluid was prepared for targeted metabolomics experiments. For this purpose, 12-point standard calibration curves in the respective range with reference material were measured, and the limit of detection (LOD) and limit of quantification (LOQ) for each neurotransmitter under investigation were calculated. LC-MS/MS was conducted by an ultra-performance liquid chromatography method using a Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm, Waters, USA). The Waters acquisition UPLC was coupled to an API 4000 triple quadrupole mass spectrometer (AB SCIEX, USA). The mobile phase A buffer was 10% methanol in water (containing 0.1% formic acid), and the mobile phase B buffer was 50% methanol in water (containing 0.1% formic acid). The LC gradient elution was at a flow rate of 0.3 mL/min at 0-8.0 min and 0.4 ml/min at 8.0-17.5 min, then 10%-30% B at 0-6.5 min; 30%-100% B at 6.5-7 min; 100% B at 7-14 min; and 100%-10% B at 14-17.5 min. The injection volume was 5 µl and the column temperature was set to 40 °C. Twenty-three metabolites (Ach: Acetylcholine chloride; Gln: Glutamine; Glu: Glutamate; His: L-Histidine; NE: norepinephrine; Tyr: Tyrosine; Trp: Tryptophan; Kyn: Kynurenine; GABA: 4-Aminobutyric acid; HisA: Histamine; PA: Picolinic acid; TyrA: Tyramine; DA: Hydroxytyramine hydrochloride; TrpA: Tryptamine; 5-HT: Serotonin hydrochloride; E: Adrenaline hydrochloride; KynA: Kynurenic acid; 5-HIAA: 5-Hydroxyindole-3-acetic acid; DOPA: Levodopa; XA: Xanthurenic acid; VWA: Vanillymandelic Acid; 5-HTTP: 5-Hydroxytryptophan; MT: Melatonine) were simultaneously reported in LC-MS/MS multiple reaction monitoring (MRM) mode. Data analysis was performed with Analyst 1.6 software.
RNA Extraction and qRT-PCR
Granulosa cells were collected in TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA was extracted according to the manufacturer’s instructions. A total of 1 mg RNA was converted to cDNA with a Takara kit (Applied Biosystems Foster City, CA, USA; Takara, Shiga, Japan). The genes of interest were amplified with a 7900HT fast real-time PCR system (Applied Biosystems) and SYBR Green Real-time PCR Master Mix (Applied Biosystems/Takara). The PCR primers were designed according to the cDNA sequences in the NCBI database. The primer sequences used are shown in Table 1. The cycling conditions used for the PCR analysis were as follows: 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s (40 cycles). 18S was used as an internal control. The 2-DDCt method was employed to determine the relative mRNA expression level.
Hormone metabolic levels in the follicular fluid
The concentrations of twenty kinds of hormones in the follicular fluid were detected by high-performance liquid chromatography–mass spectrometry (HPLC-MS/MS). An Agilent 1200 series high-performance liquid chromatography (HPLC) instrument (Agilent, USA) was utilized. A PAL autosampler (CTC, Swiss) and a Gemini-NX-C18 column (2.0 mm×50 mm, 3 μm, Waters, USA) were used. The ion source was an API-4000 quadrupole electrostatic field orbit trap high-resolution mass spectrometer (Applied Biosystems, USA). The scanning mode was multiple reaction monitoring (MRM) (Agilent-1200 LC system coupled to an API400 mass spectrometer).
Testosterone concentration in the follicular fluid
Follicular fluid samples were drawn into prechilled tubes containing sodium heparin (143 USP/10 ml). The samples were vortexed and centrifuged immediately at 4 °C for 20 min at 3000 g and then stored at -80 °C until they were assayed. The chemiluminescence method was used to detect the level of testosterone in the follicular fluid.
KGN cell culture and treatment
The human ovarian granulosa cell line KGN was kindly gifted by Dr. Zuwei Yang (IPMCH, School of Medicine, Shanghai Jiao Tong University, Shanghai, China). KGN was initially cultured in Dulbecco minimal essential medium (DMEM)-F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum and antibiotics (100 IU/mL penicillin, 100 μg/ml streptomycin) in a 5% CO2 atmosphere at 37 °C. After deprivation of glutamine for 24 h, the cells were treated with the corresponding conditioned medium for 48 h.
Enzyme-linked immunosorbent assay (ELISA)
Cell culture supernatant samples were collected and centrifuged for 20 min at 2500 rpm to acquire the supernatant at -4 °C. The supernatant was stored at -80 °C until further use.
Cell culture supernatant concentrations of 17b-estradiol (LOD, 0.5-120 pg/mL, CV <10%, Shanghai Xinle Biological Technology, Shanghai, China) and progesterone (PROG) (LOD, 0.05-6.4 ng/mL, CV <10%, Shanghai Xinle Biological Technology, Shanghai, China) were measured according to procedures provided by the manufacturers using an enzyme-linked immunosorbent assay (ELISA) kit.
Western blotting
Proteins were extracted from cultured cells with RIPA lysis buffer (Yeasen, Shanghai, China) supplemented with protease inhibitor cocktail (Yeasen, Shanghai, China) and phosphatase inhibitor (Yeasen, Shanghai, China) on ice. Cell lysates were cleared with centrifugation at 13000 rpm for 10 min. The protein concentration was measured via a BCA kit (Thermo Scientific, Rockford, IL, USA). The protein was separated on a 10% SDS polyacrylamide gel (Yamei, Shanghai, China), transferred to PVDF membranes (Millipore, Billerica, MA, USA), and then blocked in a blocking solution at room temperature for 1 h. The following primary antibodies were used: rabbit antibodies against β-tubulin (1:1000, CST, MA, USA) and anti-PCNA antibody (1:1000, CST, MA, USA). After incubation with primary antibodies at 4 °C overnight, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2500, CST, MA, USA) for 1 hour at room temperature. The bands were detected by electrochemiluminescence (ECL, Millipore, Billerica, MA, USA). The relative intensity of the target proteins was normalized to β-tubulin using ImageJ software (NIH, Bethesda, MD, USA).
EdU Staining
EdU (RiboBio, Guangdong, China) was added to the KGN cell culture medium at a concentration of 10 µM. KGN cells were cultured for 24 hours and stained with Alexa 594 and DAPI. The detailed and complete steps were performed as recommended by the manufacturer of the EdU detection kits (RiboBio, Guangzhou, China) and observed under a microscope (Leica, WetzlaR, Germany). The number of EdU-positive cells per 100 cells was evaluated.
Statistical analysis
All statistical analyses were performed using Prism software, and a p value of <0.05 was considered statistically significant. Means and standard error were used as descriptive statistics. T-tests were used to compare different variables between the low-level and high-level glutamine groups. Linear correlation analysis was used to compare the expression of the IDH1 gene and steroidogenesis in luteinized granulosa cells. In the current study, when a power analysis was performed with 80% power and an a value of 0.05, the number of samples in each group needed to be 13 to confirm statistical significance.