Identification of primary ovarian granulosa cell by H & E staining and immunofluorescence
The culture of GCs in the quail ovary is presented in supplementary materials (Supplementary 1). All procedures were in accordance with the Institutional Animal Care and Use Committee of Northeast Agricultural University. The steps of HE staining were as follows: first, fixed with 4% paraformaldehyde for 20 min, then hematoxylin staining for 5 min, eosin staining for 6 min, ethanol gradient dehydration, and finally observed under the fluorescence microscope. Specific expression steps of ovarian GCs, after 48h of cell culture, cells were washed with PBS for 3 times, fixed with paraformaldehyde for 30 min, again washed with PBS for 3 times, permeated with Triton for 10 min, washed for 3 times, and treated at 4 ℃. The follicle stimulating hormone receptor antibody FSHR (1:250) was incubated overnight, the secondary antibody (1:3000), DAPI was stained, and the specific binding status was observed under a fluorescence microscope (Motic Company).
Determination of half Inhibitory concentration and cellular activity
To determine the half-inhibitory concentration (IC50) of ATR and DACT on quail ovarian GCs, different concentrations at 10, 20, 50, 100, 250, 500, 750, 1000 µM of ATR and DACT were added into the culture medium of ovarian GCs respectively, after 24 h of culture, OD value measured by CCK-8 method. The GCs of ovary were inoculated in 96-well plates, medium with concentrations of 20 (A20), 100 (A100), 250 (A250) ATR and 20 (D20), 100 (D100), and 200 (D200) DACT were added, and culture was continued for 24 h, CCK-8 operating steps and cell activity calculation was performed according to the instructions (Shanghai Dong ren Chemical Technology Co, Ltd.).
A large number of GCs were digested with trypsin from Petri dishes. The sample is processed according to the previous method (Baumgärtner and Krakowka. 1988) and GCs were observed by transmission electron microscopy (TEM, H-7650, Hitachi Limited, Tokyo, Japan) (n = 5). The damage criteria of cell mitochondria were based on the previous scoring criteria (Schiering, et al. 2017).
Measurement of oxidative stress
Oxidative stress-related factors, including total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), (CAT), (GSH), (GSH-Px) activities, and malondialdehyde (MDA), GSH and ROS were detected according to the manufacturer’s instructions. Determination of ROS in cells was performed by chemical fluorescence method (Nanjing Jian Cheng Institute of Biological Engineering).
Ovarian GCs were digested with trypsin and collected by centrifugation at 1000 rpm/min for 5 min. Next, ovarian GCs were washed twice with PBS. 1−10 × 105 cells/mL were collected and the specific operation steps were based on the previous operation method(Cui, et al. 2021). Indicators of ovarian GCs apoptosis were then analyzed using Flowjo 7.6 software.
RNA extraction and real-time PCR analysis
Total RNA was isolated from ovarian GCs with RNAout reagent (Beijing Tian gen, P.R. China) The mRNA levels of Nrf2 and apoptosis-related factors were analyzed by qRT-PCR according to the previous method (Wei, et al. 2018, Su, et al. 2019). The related gene primers were designed with Oligo 7 software and shown in supplementary materials (Table S1). qRT-PCR was performed by LineGene 9600 (Bioer Technology Co. Ltd). To calculate the mRNA relative expression, the mRNA relative expression levels were calculated using the 2-△△CT method and normalized to the mean of β-actin1 and 2.
Nrf2 immunofluorescence analysis
The cells were fixed with 4% paraformaldehyde for 30 minutes, 0.1% Triton™X-100 penetrating fixation for 10 min, sealed with 5% skimmed milk for 1 h and incubated overnight with primary anti-Nrf2 (1:2000) (Quail Nrf2 Polyclonal Antibody in Li jin long Laboratory). Petri dishes were washed with PBS 3 times and incubated with Alexa Fluor 488 (1:2000) for 1 h. Finally, Antifade Mounting Medium with DAPI was added and observed under a fluorescence microscope. The DAPI stain (blue) and the secondary Alexa Fluor 488 (red). (Antifade Mounting Medium with DAPI).
Our results were statistically analyzed using GraphPad Prism 5.1, Origin 2018, SPSS 22.0 software. All results were applied to analyze the effect of cytotoxicity under ATR and DACT exposure in ovarian GCs. The data were expressed as the mean ± standard deviation (SD). P value of < 0.05 was considered significant.