Animals and chemicals
WT C57/BL6 mice were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). Sphk1-/- mice (stock number 019095) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). To generate granulosa cell-specific deletion of Sphk2 mice, exons 4 and 6 of Sphk2 were flanked with LoxP sites (Fig. S4a), and Sphk2 fl/fl mice were crossed with Fshr-Cre mice37. Finally, Sphk2fl/fl; Fshr-Cre mice were crossed into the Sphk1-/- background, yielding Sphk1-/-; Sphk2fl/fl; Fshr-Cre (Sphk1/2gc-/-) mice (Fig. S4b). The primers used for genotyping are shown in Table S3. Female mice (21–23 days old) were injected with 5 IU equine chorionic gonadotropin (eCG) 48 h before use to stimulate follicle development. In some experiments, female mice were treated by 5 IU eCG, followed by 5 IU human chorionic gonadotropin (hCG) 48 h later to induce ovulation. All animal protocols were approved by the Institutional Animal Care and Use Committee of South China University of Technology. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
Isolation and culture of follicles, COCs and denuded oocytes
Large antral follicles (350–400 µm) isolated from eCG-primed mice were cultured on Millicell inserts (Millipore, Billerica, MA, USA) supplemented with LH (1 µg/mL), AG1478 (1 µM) and/or SKI-II (5–20 µM). COCs isolated from eCG-primed mice were cultured in 24-well plates supplemented with NPPC (30 nM), EGF (10 ng/mL), SKI-II, AG1478, S1P (20 µM), W146 (1 µM) and/or JTE-013 (5 µM). Denuded oocytes (DOs) were separated from the COCs by repeatedly drawing the oocytes in and out of a glass pipette slightly smaller in diameter than the oocyte and then cultured in 50 µL drops of M16 medium (M7292) supplemented with EGF, S1P, and/or VPC023019 (5 µM). The follicle and COC culture medium was bicarbonate-buffered MEMα with Earle salts (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 IU/ml penicillin–streptomycin, 0.23 mM pyruvate, and 3 mg/ml bovine serum albumin (BSA). For follicle culture, 1% ITS (I3146) was added. All cultures were carried out at 37℃ in an atmosphere of 5% CO2. At the indicated time points, cumulus cells of COCs and DOs were collected for gene and protein analyses. In some experiments, COCs were collected at 2 h of culture to analyze the levels of calcium, S1P, cGMP, and the binding affinity of NPR2 for NPPC. Oocyte meiotic resumption was assessed at 4 h of culture by scoring the released oocytes for GVB after the removal of cumulus cells. To observe the morphology of cumulus expansion, the follicles were cultured in medium for 8 h, and the COCs were cultured in medium with 5% fetal bovine serum (FBS, Thermo Fisher Scientific) instead of BSA for 15 h. Images were obtained using a Zeiss 7000 microscope (Carl Zeiss, Oberkochen, Germany).
Superovulation, fertilization, and fertility assays
For superovulation analysis, the oocyte-cumulus masses were harvested from the oviducts of WT and Sphk1/2gc-/- female mice at 13 h post-hCG, and the oocytes were categorized and counted. For in vitro fertilization (IVF), COCs isolated from eCG-primed mice were cultured in medium supplemented with or without S1P for 0.5 h and then cultured in S1P-free medium for an additional 15.5 h. DOs obtained by treating COCs with 3 μg/ml hyaluronidase were fertilized with normal sperm isolated from fertile adult male mice in HTF medium. Then, the fertilized oocytes were cultured in KSOM (MR-121-D) medium. The number of 2-cell stage embryos was scored 24 h after IVF, and the number of blastocyst stage embryos was scored on Days 4–5 of the culture. In some experiments, zygotes collected from WT and Sphk1/2gc-/- females were cultured in KSOM medium to continuously observe their embryonic developmental potential. Blastocysts were stained with DAPI to quantify the number of total cells. All cultures were carried out at 37°C in an atmosphere of 5% CO2. Images of embryos at different stages were obtained using a Zeiss 7000 (Carl Zeiss). For the fertility test, 8-week-old WT or Sphk1/2gc-/- female mice were housed with 8-week-old fertile male mice for up to 8 months. The number of pups per litter was recorded at birth, and the average cumulative number of pups per female was calculated at the end of the fertility test.
RNA isolation and analysis
Total RNA was isolated and purified from MGCs, cumulus cells, and oocytes using the RNeasy micro-RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Reverse transcription was performed directly after RNA isolation using the QuantiTek reverse transcription system (Qiagen). qRT–PCR was conducted to quantify the steady-state mRNA levels using a Light Cycler 96 instrument (Roche, Basel, Switzerland). Target gene expression was calculated based on the 2-ΔΔCt method using ribosomal protein L19 (Rpl19) as the endogenous control. For RNA-seq analysis, MGCs and cumulus cells were collected from WT and Sphk1/2gc-/- mice at 2 h post-hCG. RNA was extracted from these samples, and was analyzed by Annoroad Gene Technology Co., Ltd. (Beijing, China). The qRT–PCR primers are shown in Table S3.
Western blotting
Total proteins of MGCs were extracted in WIP buffer (Cell Chip Biotechnology, Beijing, China), and 15–25 μg protein was used for sample loading. Cumulus cells from 50–150 COCs or 50–300 oocytes were lysed in SDS loading buffer. These protein samples were separated by SDS–PAGE and transferred to a PVDF membrane (Millipore). The membranes were blocked in TBST (Tris-buffered saline with Tween 20) buffer containing 5% milk, and then were incubated with primary antibodies (Table S4) overnight at 4℃. After rinsing with TBST buffer, the membranes were incubated with appropriate secondary antibodies (1:5000, Zhongshan Golden Bridge Bio-technology, Beijing, China). Finally, the bands were detected using the SuperSignal West Pico Kit (Thermo Fisher Scientific) and visualized by the Tanon 5200 chemiluminescent imaging system (Tanon, Shanghai, China). The relative intensity of the bands was quantified by ImageJ software (NIH Image, Bethesda, MD, USA). GAPDH or α-tubulin (Tubulin) was used as an internal control.
Immunofluorescence and ovarian histology assays
Ovaries isolated from eCG-primed mice or eCG-primed mice followed by hCG were fixed in 4% paraformaldehyde (PFA), embedded in paraffin and sectioned at 5 μm. The sections were dewaxed, rehydrated and subjected to antigen retrieval using 0.01 M sodium citrate buffer (pH 6.0). After blocking in 10% normal donkey serum, the sections were incubated with primary antibodies (Table S4) followed by Alexa Fluor 488-conjugated secondary antibodies (1:200, Thermo Fisher Scientific). Finally, the sections were counterstained with DAPI. To visualize microtubules and chromosomes, oocytes were fixed in 4% PFA and permeabilized with PBS containing 0.5% Triton X-100 for 30 min. After blocking with 10% normal donkey serum for 1 hour at room temperature, oocytes were incubated with Alexa Fluor 488-conjugated anti-α-tubulin antibodies (1:200, Abcam) and counterstained with DAPI. Digital images were captured using a Zeiss LSM 800 confocal microscope (Carl Zeiss), and the fluorescence intensity was analyzed using Zeiss Zen 3.0 software. The exposure time was kept the same for the control and mutant samples. The fluorescence intensity of MGCs and cumulus cells was calculated by averaging the fluorescence signals of MGCs and cumulus cells in each large antral follicle. The relative fluorescence intensity was analyzed after background subtraction.
For histological analysis, paraffin-embedded ovaries were serially sectioned at 5 μm and stained with periodic acid/Schiff (PAS) reagent and haematoxylin. The numbers of large antral follicles, corpus luteum and oocytes having undergone GVB were counted by examining serial sections through the entire ovary.
Measurement of S1P levels
Ovaries and COCs were isolated from eCG-primed mice or mice at 2 h post-hCG. In some experiments, COCs from eCG-primed mice were collected after different drug treatments for 2 h. More than 1 mg/mL ovarian protein samples or 1500 COC samples per group were used for analyzing S1P levels according to previous reports19,38. Briefly, 12.5 µL of sample suspension, 5 µL of internal standards (1 ng/mL for C17-S1P) and 32.5 µL of methanol were added to a 1.5 mL tube. The mixture was centrifuged at 14,000 × g for 15 min, then 10 µL of the supernatant was injected into the liquid chromatography-tandem mass spectrometry system (LC-MS/MS; LC, Shimazu Nexera X2 LC-30AD, Shimadzu, Japan; MS, AB SCIEX QTRAP 4500, AB SCIEX, MA, USA) equipped with an electrospray ionization (ESI) source and multiple reaction monitoring (MRM) for analysis.
Measurement of the intracellular calcium levels
The intracellular calcium levels were monitored by using Fluo 3-AM (Dojindo Laboratories, Kumamoto, Japan) as reported before9,10. Briefly, COCs with different treatments were collected and then incubated with 5 µM Fluo 3-AM for 20 min. Samples were examined by a Nikon A1 confocal microscope (Nikon, Tokyo, Japan) or a Zeiss LSM 800 confocal microscope (Carl Zeiss). All quantifications were performed using Nikon NIS Elements BR 5.10 software or Zeiss Zen 3.0 software. The fluorescence intensity (pseudocolor) in cumulus cells was analyzed after background subtraction, which represents the intracellular calcium levels.
Measurement of the binding affinity of NPR2 for NPPC
The binding affinity of NPR2 for NPPC was monitored by using mono-5-[and 6]-carboxyfluorescein-labeled NPPC (FAM-NPPC; Phoenix Pharmaceuticals, Belmont, CA) as reported before9. Briefly, COCs with different treatments were collected and incubated with 100 nM FAM-NPPC for 30 min, and then the fluorescence-labeled ligand binding was assessed by a confocal microscope (Nikon A1 or Zeiss LSM 800). The fluorescence intensity (green) in cumulus cells was analyzed after background subtraction, which represents the binding affinity of NPR2 for NPPC.
Measurement of cGMP levels in COCs
COCs from eCG-primed mice were collected after different drug treatments for 2 h. 100 COC samples per group were solubilized in 200 µL HCl (0.1 M) on ice for 10 min, and then were thawed and centrifuged at 12,000 × g for 5 min. The supernatant was collected to a tube and dried in an oven at 60℃. The levels of cGMP were determined using cGMP enzyme immunoassay kit obtained from Cayman Chemicals (Ann Arbor, MI, USA).
P4 level assay
The blood from WT and Sphk1/2gc-/- mice at 48 h post-hCG was collected by cardiac puncture, and the serum progesterone levels were analyzed by Beijing North Biotechnology Institute (Beijing, China).
Statistical analysis
All experiments were performed at least three times independently, and the data are represented as means ± s.d. GraphPad Prism software (v8.4.0, La Jolla, CA, USA) were used to perform the statistical analysis and graph generation. The statistical significance was analyzed either by unpaired two-tailed Student’s t-test (two-group comparison) or by one-way ANOVA followed by Tukey’s Honestly Significant Difference (more than two groups). Values were considered significantly different if P < 0.05.