Mouse grouping and modeling

A total of 40 ICR mice (gender, female; age, 5 wk old; HFK Bio-Technology, Beijing, China) were randomly divided into the following four groups (n = 10): the control group; the OHSS group; the OHSS + 16 IU VD3 group; and the OHSS + 24 IU VD3 group. To establish an OHSS mouse model, ovarian induction and VD3 administration were carried out as previously described by Jiang et al. and Burjiah et al. (2022) respectively. Mice in the control group were first subcutaneously injected with 10 IU pregnant mare serum gonadotrophin (PMSG). Following 48 h, mice were then injected subcutaneously with 10 IU hCG (both from Solarbio, Beijing, China). Both hormones were dissolved in normal saline. Mice in the OHSS group were daily injected with 20 IU PMSG for three consecutive days, followed by injection of 30 IU hCG. Finally, mice in the OHSS + VD3 group received daily 16 or 24 IU VD3 (Solarbio) orally via a gastric probe for 20 d after hCG injection. All mice were euthanized by cervical dislocation following anesthesia with 1.5% pentobarbital sodium. Blood was collected by cardiac puncture. In addition, the ovaries were removed, weighted, and photographed.

Permeability assay

Permeability assay was performed as previously described (Huang et al. 2022). Briefly, mice were injected with 0.1 mL Evans blue (5 mM; MilliporeSigma, Merck, Darmstadt, Germany) through the caudal vein after anesthesia. Following Evans blue injection for 30 min, 2 mL sterile saline was injected into the abdominal cavity and the entire abdomen was gently massaged for 1 min. Peritoneal fluid was extracted and centrifuged at 160 × g for 5 min. The supernatant was transferred into a 96‐well plate and the optical density (OD) was measured at wavelengths of 405 and 620 nm.

Cell isolation and treatment

The ovaries were first placed in pre-cooled PBS, and following excess tissue removal, they were then incubated in pre-cooled DMEM-F12 (Gibco; Thermo Fisher Scientific, Shanghai, China). To release ovarian granulosa cells into the culture medium, the follicles were punctured with a 1-mL syringe needle. Granulosa cells were then transferred into T25 culture flasks and cultured in DMEM-F12 supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific) and 1% penicillin–streptomycin (Biosharp, Hefei, China) at 37 °C in an incubator with 5% CO2. When confluence reached ~ 90%, the cells were collected after trypsinization and seeded at a passage ratio of 1:3 into a 6-well plate at a density of 106 cells/well. Cells at passages 2 ~ 4 were used in this study. For the subsequent mechanistic studies, granulosa cells were transfected with a PTX3 overexpression plasmid. For cell transfection, pcDNA3.1 plasmids purchased from GenePharma Co., Ltd. (Shanghai, China) were mixed with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Shanghai, China) and were then incubated for an additional 24 h prior harvesting. In the indicated groups, granulosa cells were treated with 10 IU hCG or VD3 (0, 50, 100 or 200 nM) for 24 h.

Hematoxylin–eosin (H&E) staining

Ovarian tissues were fixed in 4% paraformaldehyde overnight, dehydrated with gradient alcohol, embedded in paraffin, and cut into 4-μm-thick sections. Subsequently, the deparaffinized tissue sections were stained with H&E solution (Sangon, Shanghai, China) for 5 min and 2 min, respectively, at room temperature. The sections were then dehydrated with gradient alcohol and became transparent using xylene. The tissue specimens were observed under a microscope (Olympus Corporation, Tokyo, Japan).

Assessment of inflammatory factors

Ovarian tissues were rinsed with pre-cooled PBS (0.01 M; pH = 7.4), and after weighing, they were cut into small sections. Subsequently, the tissues were fully ground on ice and the resulted homogenate was centrifuged at 5000 × g for 10 min to obtain the supernatant. The levels of TNF-α, IL-1β, and IL-6 were measured in the cell supernatant using the corresponding commercially available ELISA kits (Elabscience, Wuhan, China). The OD values at a wavelength of 450 nm were recorded and the cytokine levels were calculated according to the standard curve.

Serum hormone and growth factor detection

The serum and tissue hormone estradiol (E2) and progesterone (Prog) levels and those of VEGFA were measured with the corresponding commercially available ELISA kits (Elabscience). The OD was recorded at a wavelength of 450 nm and the levels of E2, Prog, and VEGFA were calculated based on the standard curve.

Western blot analysis

Ovarian tissues and cells were lysed in RIPA buffer and the protein extracts were then separated by SDS-PAGE. The separated proteins were then transferred onto PVDF membranes, followed by blocking with 5% skim milk. The membranes were first incubated with primary antibodies against VEGFA (sc-7269; 1:500), PTX3 (sc-373951; 1:200), and GAPDH (sc-365062; 1:200; all from Santa Cruz Biotechnology, Shanghai, China) at 4 °C overnight and then with the corresponding HRP-conjugated goat anti-mouse IgG secondary antibody (31,430; 1:20,000; Thermo Fisher Scientific, Shanghai, China) for 1 h at room temperature. The blots were visualized using an ECL reagent (Affinity, Changzhou, China) and were semi-quantified using ImageJ software.

Immunohistochemistry (IHC) and immunofluorescence (IF) assays

For antigen retrieval, paraffin-embedded ovarian sections were deparaffinized and microwaved in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Subsequently, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 20 min. Sections and cells were blocked with 5% goat serum for 30 min, followed by incubation with primary antibodies against CD31 (ab281583; 1:4000; Abcam, Shanghai, China) or VEGFA (sc-7269; 1:300; Santa Cruz Biotechnology) at 4 °C overnight. The following day, the cells and tissue sections were incubated with biotinylated goat anti-rabbit (31,820; 1:2000) or FITC-conjugated anti-mouse (F-2761; 1:2000) secondary antibodies (Thermo Fisher Scientific). The specimens were counterstained with hematoxylin and the positive immunostaining was assessed under a microscope (Olympus Corporation). Cells were stained with DAPI and observed under a fluorescence microscope (Olympus Corporation).

Molecular docking

The molecular structure of PTX3 (PDB ID:7zl1) was downloaded from RCSB PDB (www.rcsb.org). Water molecules and excess ligands around PTX3 protein were deleted, the structure of VD3 was hydrogenated, and systematic three-dimensional structural optimization was then performed. Docking was run in AutoDock software (version 4.2; Scripps Research Institute, San Diego, CA) and the composite structure was displayed.

Statistical analysis

All data are expressed as the mean ± SD. The differences between multiple groups were compared with one-way ANOVA followed by Tukey’s post hoc test. All data were analyzed using SPSS software. p < 0.05 was considered to indicate a statistically significant difference.