Animals
This study was approved by the Ethics Committee of Tongji University (#TJBG03621101; Shanghai, China) and carried out following the Guide for the Care and Use of Laboratory Animals of Tongji University. A total of 40 C57BL/6N female mice (6–8-week old; Beijing Vital River Laboratory Animal Technology, China) were maintained at the Animal Research Center of Tongji University under a 12/12-h light/dark cycle with free access to water and food.
POI mouse model
Mice were randomly assigned to control, low-dose LPS, high-dose LPS, and CTX groups (n = 10/group). To induce POI, mice were administered 0.5 mg/kg LPS (L2630; Sigma-Aldrich, St. Louis, MO, USA) once daily for 14 days, 2.5 mg/kg LPS twice weekly for 2 weeks, or 150 mg/kg CTX (C0768; Sigma-Aldrich) once weekly for 2 weeks via i.p. injection. At 2 weeks after treatment, mice were anesthetized with 1% sodium pentobarbital via i.p. injection, followed by blood sample collection through cardiac puncture. After cervical dislocation, the ovaries were immediately collected and fixed with 4% paraformaldehyde or stored at − 80°C until use.
Monitoring the estrous cycle
The vaginal epithelial cells were collected daily by vaginal lavage to monitor the estrous cycle. Cells were observed using a light microscope (Eclipse E100; Nikon, Japan) under the bright field. Images were acquired at 10× magnification using a Nikon DS-U3 imaging system. Representative cell morphologies at corresponding cycle phases are shown in Supplementary Fig. 1.
Histopathological examination
The ovary tissue samples were fixed in 4% paraformaldehyde overnight, dehydrated, paraffin-embedded, and sliced into 4-µm-thick sections. After dewaxing and rehydration, the sections were subjected to hematoxylin & eosin (H&E), immunohistochemical (IHC), or Masson staining following standard methods. H&E staining was performed to count primordial follicles as previously described(20). For each mouse, the primordial follicles in three consecutive sections were counted. For IHC staining, the sections were immersed in sodium citrate solution (0.01 M) for 20–30 min for antigen retrieval, followed by incubation with 3% hydrogen peroxide to quench the endogenous peroxidase. Then, the section was blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature, followed by an overnight incubation with primary anti-α-smooth muscle actin (α-SMA) antibody (Servicebio, Wuhan, Hubei, China) at 4 ºC and phosphate buffered saline (PBS) rinses. After an incubation with the secondary antibody for 50 min at room temperature, the section was stained using a DAB detection kit (DAKO, Agilent Technologies, Santa Clara, CA, USA). Images were acquired using an XSP-C204 microscope (COIC, Chongqing, China). The results were assessed by two independent pathologists in a blinded manner. The intensity of the staining was scored as previously described(21). For Masson staining, the collagen fibers in ovarian tissue samples were stained using a Masson staining kit (Guge Biotechnology, Wuhan, Hubei, China). The blue-stained collagen fibers were observed using an optical microscopy (DS-U3; Nikon, Japan) at magnification 10×. The Masson-positive area was quantified using Image J v1.8.0 (NIH, Bethesda, MD, USA).
TUNEL assay
Ovarian granulosa cell apoptosis was examined using a TUNEL assay kit (Servicebio). Briefly, deparaffinized sections were incubated with 20 mg/mL proteinase K (Guge Biotechnology) at 37°C for 20 min, followed by incubation with permeabilization buffer at room temperature for 20 min. After a 10-min equilibration at room temperature, sections were incubated with terminal deoxynucleotidyl transferase and deoxyuridine triphosphate at 37°C for 2 h in a moist chamber. Images were acquired using a Nikon Eclipse Ti-SR microscope.
Enzyme-linked immunosorbent assay (ELISA)
The serum levels of anti-Müllerian hormone (AMH), E2, FSH, and IL-1β were measured using the corresponding ELISA kit (Elabscience, Wuhan, Hubei, China) following the manufacturer’s protocols.
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from ovarian tissue samples using Trizol (RNAiso Plus; Takara, Japan). cDNA was synthesized using a PrimeScript™ RT reagent kit (Takara). Amplification was performed using TB Green® Premix EX TaqTM II (Takara) and gene-specific primers (Table 1; Sangon, Shanghai, China) on a qRT-PCR device (QuantStudio5, Thermo Fisher Scientific, Waltham, MA, USA). The relative mRNA levels of the genes were quantified using the 2−ΔΔCT method and normalized to GAPDH mRNA level.
Table 1
Primers for quantitative real-time PCR.
Gene
|
Forward (5'–3')
|
Reverse (5'–3')
|
GAPDH
|
GACTGGATAAGCAGGGCGG
|
CCCAATACGGCCAAATCCGT
|
IL-1β
|
GAAATGCCACCTTTTGACAGTG
|
CTGGATGCTCTCATCAGGACA
|
COL1A1
|
TGACCTTCCTGCGCCTAATG
|
AAGTTCCGGTGTGACTCGTG
|
BAX
|
GGCCTTTTTGCTACAGGGTTTC
|
CAGCTTCTTGGTGGACGCAT
|
BCL-2
|
GCTGGGTAGGTGCATGTCTG
|
CAGGGGAGCAAAGCTACAAACT
|
Western blot analysis
The ground ovarian tissue samples were lysed using RIPA buffer (Biotechwell, Shanghai, China). Total proteins were isolated by centrifuging the lysates at 12,000 rpm for 5 min. Protein concentrations were measured using a bicinchoninic acid kit (Biotechwell). The proteins were separated using gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked using 5% BSA for 2 h at room temperature, followed by an overnight incubation with anti-TLR (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2 (1:1000; Affinity, Scoresby, Victoria, Australia), anti-collagen type I alpha 1 chain (Col1A1; 1:1000; Affinity), anti-α-SMA (1:1500; Servicebi), anti-BAX (1:1500; ImmunoWay Biotechnology, Plano, TX, USA), anti-P-P65 (1:1000; ImmunoWay Biotechnology), anti-MyD88 (1:1000; ImmunoWay Biotechnolog), anti-P65 (1:1000; Affinity), or anti-GAPDH (1:2000; Biotechwell) at 4°C. After a 2-h incubation with a secondary antibody (1:2000; Jackson Immuno, West Grove, PA, USA) at room temperature, the protein bands were developed using an enhanced chemiluminescence kit (Biotechwell).
Statistical analysis
Data were presented as the mean ± standard error of the mean. Statistical analysis was carried out using the SPSS software (V18.0; IBM, Armonk, NY, USA). Intergroup differences were compared using one-way analysis of variance. Statistical significance was analyzed using the Student’s t-test. A P value less than 0.05 was considered statistically significant.