Animals and treatments

A total number of 36 C57BL/6 female wild-type mice were purchased from Beijing HFK Bioscience Co. Ltd (Beijing, China). In addition, four Csnk1α1 floxed mice (Csnk1a1flox/flox) (Stock #025398)[31] and two Amh-Cre transgenic mice (Stock #033376) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and bred in the laboratory. All the animals were housed in an environment with a temperature of 22 ± 2 ºC, relative humidity of 60%–65%, and a light/dark cycle of 12/12 h and were given basal diet and pure water.

The mice were anesthetized using pentobarbital sodium and subsequently euthanized by cervical vertebral fracture, and the uterus, ovaries, and oviducts were collected and stored at -80 ○C for protein detection or fixed in 4% paraformaldehyde (PFA) diluted in phosphate-buffered saline (PBS, pH7.4) for immunohistochemistry (IHC). In addition, the blood serum was collected to measure estrogen levels.

Fertility test

Sexually mature females of Csnk1α1 knockout mice (Cre ± ; loxp + / + or cKO) and controls (Cre-/-; loxp + / + or Con) (10–12 weeks) were used for the fertility test. Female mice were mated with wild-type C57BL/6 males (16 weeks) for three months. Female vaginal plugs were checked every morning before pregnancy, and the number of pups in each cage was recorded for each group.

Determination of each phase of the estrous cycle

The phase of each estrous cycle was determined by the examination of vaginal smears stained with Wright's dye (Solarbio Life Sciences, Beijing, China). Estrous cycle phases were defined as follows [50, 51]: proestrus (100% intact epithelial cells), estrus (100% cornified epithelial cells), metestrus (∼50% cornified epithelial cells or exfoliated epithelial cells and 50% leukocytes), and diestrus (cell debris, some cornified epithelial cells or leukocytes).

Isolation and culture of primary ovarian GCs

Mice were given pregnant mare serum gonadotropin (PMSG,10 IU). Ovarian primary granulosa cells (GCs) were isolated and collected by follicle puncture as previously described [52]. Primary ovarian GCs were incubated in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) containing 10% fetal bovine serum (FBS) supplemented by 100 U/mL penicillin and 100 mg/mL streptomycin, in a 5% CO2 incubator with saturated humidity and a constant temperature of 37 ○C.

In vitro experiments

In vitro experiments were divided into three stages. E2 content and protein detection were measured after each step.

(1)

Primary ovarian granulosa cells were isolated from mice (Fig. 5), and inoculated in 6-well plates (1 × 107/well) for 12 h. Then, the cells were trypsinized, and hormone levels in the supernatant were analyzed.

(2)

Twenty adult WT were selected for this experiment. Primary ovarian GCs from the WT mice were collected and seeded in a 6-well plate at a density of 1 × 106 cells in a standard culture medium. After attaching to the bottom of the dish, the culture medium was replaced with a medium without FBS, and cells were cultured 6 h. D4476 was dissolved in DMSO and further diluted in complete medium and cells were then treated with D4476 (50 μM) for an additional 6 h; the equivalent volume of DMSO was used as D4476 vehicle control.

(3)

Ten adult WT were selected for this experiment. Primary ovarian GCs from the WT mice were collected and seeded in a 6-well plate at a density of 1 × 106 cells in a standard culture medium. After attaching to the bottom of the dish, the culture medium was replaced with a medium without FBS, and cells were cultured 6 h. Cells were then treated with RAF1 inhibitor RAF709 (5 nM) for 6 h as previously reported [45].

Real-time quantitative PCR (RT-qPCR)

The tissue and cell samples were dissected and extracted by grinding under liquid nitrogen. Total RNA was extracted from the tissue and Trizol reagent (TaKaRa Biotechnology, Dalian, China) following the manufacturer’s instructions. Purified RNA (1 µg) was used as a template for cDNA synthesis. Samples were mixed with Oligo (dT) at 72 ○C for 5 min, cooled to 0–4 ○C for 5 min, and then mixed with MLV reverse transcriptase, dNTP, and RNA safe (Promega, Madison, WI, USA) for 1 h at 42 ○C. RT-qPCR was performed using a standard Takara SYBR Premix Ex Taq protocol (Vazyme Biotech Co., Ltd, Nanjing, China) on an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific Corp., Waltham, MA, USA). The mixture was heated to 95 ○C for 10 min, followed by 40 cycles of 95 ○C for 15 s and 60 ○C for 1 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels were used for data normalization, and the relative abundance of genes was determined using ABI PRISM 7500 equipped software (©2009–2017, Analytik, Jena AG). The relative product levels were quantified using the 2–∆∆Ct method; the primers for RT-qPCR analyses are presented in Table 1 (Additional file 13). Genotyping identification of mice was performed by PCR using primer sets published on the Jackson Online website at https://www.jax.org/strain/025398 and https://www.jax.org/strain/033376.

Western Blotting (WB)

Ovaries and GCs (stage 1, 2, and 3; see in vitro experiments) were lysed with RIPA buffer (C1053, Applygen, Beijing, China). The protein concentration of each sample was quantitated by the BCA assay reagent (HX18651, Huaxingbio, Beijing, China). Samples were electrophoresed on SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, Billerica, MA, USA). The membrane was soaked in methanol, and the target proteins were transferred to a PVDF membrane and incubated with CK1α antibody (1:2000, ab64939, Abcam), RAF1 antibody (1:2000, ab137435, Abcam), CYP19A1 antibody (1:1000, BA3704, Boster), P-ERK1/2 antibody (1:1000, 4370T, Cell Signaling Technologies), ERK1/2 (1:1,000, A4782, ABclonal), Frizzled (1:1000, M003762, Abmart), LPR6 (1:1000, M026228, Abmart), β-catenin (1:1000, 8480, Cell Signaling Technologies), GAPDH (1:2000, PA5-85074, Ambion) and α-tubulin antibody (1:1000, T40103, Abmart) at 4 ○C overnight. The membrane was then washed in TBST (0.1% Tween-20 in TBS, Sigma-Aldrich, P1379) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:4000,115–035-062, Jackson Immuno Research)or HRP conjugated goat anti-rabbit IgG (1:4000, 111–035-003, Jackson Immuno Research) for 2 h at room temperature. After 30 min in TBST, the membrane was treated with ECL Western blotting substrate (32209; Thermo Scientific, Waltham, MA, USA) at room temperature and assessed using the ImageJ software (1.4.3, ©1993–2006, Wayne Rasband), GAPDH and α-tubulin was used as the endogenous control.

Immunohistochemical analysis

Tissue sections in paraffin were dewaxed in ethanol and soaked in 3% H2O2 (vol/vol) for 20 min to eliminate endogenous peroxidase activity. Samples were microwaved in 0.01 M sodium citrate buffer on high power for 15 min, washed with PBS, and then incubated with normal goat serum (10%) for 1 h at room temperature to eliminate background nonspecific coloring. The sections were then incubated with CK1α antibody (1:200, ab64939, Abcam) overnight at 4○C and biotinylated goat anti-rabbit IgG (1:200; 11–065-14, Jackson) was incubated for 2 h at 37 ○C. HRP-conjugated streptavidin (1:200; 016–030-084, Jackson) was used for incubating at room temperature, and diaminobenzidine was added for color rendering, with the color development degree controlled under the microscope. The appearance of brown staining was considered a positive reaction, which was analyzed using VENTANA Image Viewer (©2017, Ventana Medical Systems, Inc).

Immunofluorescence (IF) assay

After reaching 85% confluence, GCs grown on glass slides were fixed in 4% paraformaldehyde (PFA) for 15 min and then treated with cold methanol for 15 min. GCs and tissue sections were then washed with PBS for 10 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787), then blocked using 10% normal goat serum in TBS for 1 h at room temperature. The slides were incubated with RAF1 antibody (1:150; HY-100510, Abcam), FSH receptor (FSHR) antibody (1:100, sc-13935, Santa Cruz), DDX4 antibody (1:100, ab13840, Abcam), cleaved caspase-3 (1:200, 9664S, Cell Signaling Technologies), FOXL2 antibody (1:200, 19,672–1-AP, Proteintech) overnight at 4 ○C. Then, the slides were washed with PBS and incubated for 2 h at room temperature with the FITC-labeled goat anti-rabbit IgG (GAR-FITC, ZF-0311, Zhongshan) for 50 min. Then the nuclei were dyed with 4',6-diamidino-2-phenylindole (DAPI, 1:1,000, D8417; Sigma) for 10 min. Nonimmune rabbit IgG was used as a negative control. The signals were collected by using a fluorescence microscope photograph system (Leica Microsystems, Buffalo Grove, USA).

ELISA

E2 content from blood serum mice in proestrus and culture liquid supernatant from GCs were determined (n = 3 per experimental group) using an ELISA kit (Quanzhou Ruixin Biological Technology Co., Ltd, Quanzhou, China), following the manufacturer’s instructions. Briefly, blood was collected from mouse eyeballs, after which the serum was separated by centrifugation. For primary cell supernatant, 1 × 106 cells were grown in a 6-well plate, after which the cell supernatant was collected when the cell density was approximately 70% (24 h after cell walling).

Oocyte collection

Four Control mice and four cKO mice aged 10–12 weeks (four mice with similar body weight were selected for the research object in each group) were superovulated with 10 IU (intraperitoneal) pregnant mare serum gonadotropin (PMSG; Ningbo Hormone Products Co., Ltd, Ningbo, China). After 48 h, 10 IU of human chorionic gonadotropin (hCG; Ningbo Hormone Products Co., Ltd, Ningbo, China) was injected. Thirteen h after the hCG injection, cumulus-oocyte complexes were recovered in an M2 medium supplemented with 4 mg/mL bovine serum albumin from the ampulla of the oviducts. Cumulus cells were removed from oocytes with hyaluronidase (300 IU/mL) for 5 min in the M2 medium. Only oocytes with normal morphology were used for statistics and IF assay.

Flow cytometry

For apoptosis detection, primary granulosa GCs (adherent and floating) were harvested and analyzed according to the manufacturer’s protocol (Beijing Biosea, Biotechnology, Co., Ltd, Beijing, China). Briefly, 1–5 × 105 GCs were resuspended in 500 µL of 1X Annexin V binding buffer and then in annexin V-FITC (5 µL) and propidium iodide (5 µL) for 5 min in the dark. Early and late cell apoptosis were analyzed by flow cytometer (BD, Biosciences, San Jose, CA, USA).

The maximum absorption and emission wavelengths of FITC were 490 and 525 nm, while those of PI-DNA complexes were 535 and 615 nm. Two control groups (PI staining and Annexin V-FITC staining) were set up. Data analysis was performed using the FlowJo software (14.0.0, ©2012, Flexera Software LLC), following the manufacturer’s instructions. FITC on the abscissa and PI on the ordinate. Live cells (Annexin V-/PI-), early apoptotic cells (Annexin V + /PI-), late apoptotic cells (Annexin V + /PI +).

Coimmunoprecipitation assay

Ovarian tissues and primary ovarian GCs were collected in precooled NP40 buffer. Coimmunoprecipitation assay was performed using protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's instructions. Briefly, 500 μg sample was incubated with 2 μg of anti-CK1α antibody or anti-IgG antibody for 2 h, followed by incubation with 20 μL of protein A/G agarose beads overnight at 4 ○C. The samples were washed 5 times with precooled NP40 buffer, boiled for 5 min and analyzed via WB.

Transcriptome profiling

A total number of six ovarian tissue samples (three ovaries for each group) were randomly selected from the control and cKO groups. Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample were quantified using NanoDrop (ND-1000, Wilmington, DE, USA). The mRNA was reverse transcribed into first-strand cDNA using the Super Script III reverse transcriptase kit (Invitrogen, 18,080–085), followed by second-strand cDNA synthesis using the Second Strand cDNA synthesis kit (Bio Wavelet, BWR001). The cDNA library preparation was performed as described previously [53]. The average insert size for the final cDNA library was 300 bp (± 50 bp), and all libraries were sequenced using the Illumina HiSeq platform [54].

Identification of differentially expressed genes and gene functional enrichment analyses

Genes with an adjusted P-value (Q-value) < 0.01 and an absolute value of fold change > 1.5 were considered differentially expressed genes. The threshold for the significantly enriched gene sets was here set as P-value < 0.05. The gene expression signatures and relevant biological information were performed by online tools such as https://www.omicstudio.cn/tool and https://hiplot.com.cn.

Computation protein–protein docking

Docking of CK1α (PDB ID: 5fqd) with RAF1 (PDB ID: 6kkn) was performed using ZDOCK and RDOCK from Discovery Studio 2016 [55]. The ZRANK algorithm ranked the docking poses, and the best-docked pose was selected based on the evaluation of binding interface residue. The top-ranked structures were further visualized by Discovery Studio (DS) and Ligplot + v.2.2.

Statistical analysis

All experimental data were processed and analyzed by GraphPad Prism software (8.0.2, ©1992–2019, GraphPad Software Inc., San Diego, CA, USA) and SAS software (©2001, Indigo Rose, Corporation). All quantitative data are presented as means ± standard deviation. One-way analysis of variance (ANOVA) and Duncan’s tests were used to analyze the main effects of treatments, and a P-value < 0.05 was considered to indicate statistically significant differences. WB grayscale images were converted to peak and their size was measured by ImageJ software (1.4.3, ©1993–2006, National Institutes of Health, Rockville Pike, Bethesda, MA, USA). Each experiment was repeated at least three times.