Female neonatal CD-1 mice (1.8-2.0g; 13 pregnant mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were orally dosed on days 2 through 5 after birth(birth day was set as day 1) with 2.7 µmol/kg tamoxifen (Shanghai Fudan Forward Science & Technology Co., Ltd, Shanghai, China) suspended in a peanut oil/lecithin/condensed milk mixture (2:0.2:3,v/v) at a dose volume of 5 µL/g body weight . All mice were housed under controlled conditions of temperature (22–26°C) and light (12-/12-h light/dark cycle) with ad libitum access to food and water. After 75 days, tamoxifen-treated mice were randomly divided into five groups: Group I, adenomyosis model group; Group II, melatonin 5 mg/kg body weight (Me 10); Group III, melatonin 10 mg/kg body weight (Me 20); Group IV, melatonin 15 mg/kg body weight (Me 30); and Group V, melatonin 25 mg/kg body weight (Me 50). Melatonin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in a small volume (0.02 mL) of absolute ethanol and diluted in 0.9%NaCl to a final concentration of 1 mg/ml. Mice from Groups II, III, IV, and V were intraperitoneally (i.p.) injected with melatonin every 12h at various doses (5, 10, 15 and 25 mg/kg body weight) for 4 weeks. Mice in the control group and adenomyosis model group received vehicle treatment of melatonin (NaCl + Ethanol). During days 110-125, all mice were mated with male mice from 19:00 to 07:00 and then checked for the presence of a vaginal plug at 08:00-9:00 the following day. The vaginal plug day was defined as gestation day 0.5. The animals were killed by cervical dislocation at day 8.5 for implantation site count. The optimal concentration of melatonin was determined by the number of implantation sites and used for the subsequent study. In a second experiment, another group of female mice received the same treatments described, after which the animals were euthanized at gestation day 4.5 (implantation window) of pregnancy, the uterus were removed for morphometric and endometrial receptivity analyses, and the oviducts were collected for blastocyst retrieval. Flow chart of the experiments can be seen in supplementFigure. All animal experiments were approved by the Ethics committee of the Tong-ji University (No. TJBG01321101) and complied with international guidelines for the care and use of laboratory animals.
Uterine Section and Morphometric Analysis
Uterine tissues from the experimental mice were harvested at night on day 4.5. Approximately 4/5 fractions of the uterus were used for collecting endometrial tissues, immediately dropped into liquid nitrogen, and stored at -80℃ for subsequent gene and protein analyses. Another 1/5 fraction of the uterine tissues were formalin-fixed and paraffin-embedded and cut into routine 5-µm-thick sections using a microtome for hematoxylin and eosin (H&E) staining and immunohistochemical(IHC) analysis. Images were observed under a Motic EasyScanner (Motic China Group Co., Ltd., Xiamen, China) and photographed. Six sections from each uterus were analyzed, and morphometric parameters were measured using Image Pro-Plus software (v.6.0; Media Cybernetics, Silver Spring, MD, USA). The interface between the myometrial and endometrial layers (P1) and the inner luminal surface area (P2) were traced, and the latter was subtracted from the former as the endometrial area (EA). The endometrial thickness index (ETI) was used to determine the thickness of endometrium, which was calculated using the formula [24, 25]:
Two investigators independently completed the measurement on different occasions.
RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After the measurement of RNA concentration and quality, RNA was reverse transcribed to cDNA using PrimeScript RT reagent Kit (TaKaRa Bio, Shiga, Japan) according to manufacturer’s instructions. The primers were designed and purchased from Sangon Biotechnology (Shanghai,China). qRT-PCR was performed using the TB Green Premix Ex Taq kit (TaKaRa Bio) and the Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. We detected expression levels of glyceraldehyde 3-phosphate dehydrogenase (Gapdh), Lif, integrin-β3, Hoxa10, Hoxa11, estrogen receptor α(Era), progesterone receptor α(Pra),P53,glutathione Peroxidase1(Gpx1), super Oxide Dismutase(Sod1), B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). PCR conditions included 10µL TB Green, 0.4µL Dye II, 0.8µL forward and reverse primers, 2µL template, and ddH2O to a total volume of 20µL. The procedure was as follows: 95°C for 30s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s, a melting curve from 60 to 95°C with incremental increases of 0.5°C every 5 s. GAPDH was used an the standard control, and primer sequences are listed in Table1. Relative quantification of gene expression was performed using the 2-△△CT method.
Approximately 20mg of mouse endometrium was ground (Cebo Biotechnology, Shanghai, China) and lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Jiangsu, China) with a protease inhibitor cocktail (Thermo Fisher Scientific) on ice for 30 min. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of total protein (30µg/lane) were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA, USA) according to standard procedures. The membranes were then blocked using 5% non-fat milk (Sangon Biotech) or 5% bovine albumin (Sangon Biotech) in Tris-buffered saline with 0.05% Tween-20 detergent for 1.5 h on a shaker then incubated with primary antibody against GAPDH, Lif, Integrin-β3, Hoxa10, Hoxa11, p44/42 mitogen-activated protein kinase[MAPK; extracellular signal-regulated kinase (ERK) 1/2], phosphorylated(p)‐p44/42MAPK(ERK1/2)(Thr202/Tyr204), p‐p38 MAPK(Thr180/Tyr182), p38 MAPK, TNF‐α, IL‐1β, IκB‐α, p-NF-κB p65(ser536), and NF-κB p65 at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies at room temperature for 1.5 h. Images were obtained using a chemiluminescent imaging system (Tanon Science & Technology Co., Ltd., Shanghai, China), with GAPDH was used as the internal control. Antibody information is presented in Table 2. The intensities of the protein bands were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
Formalin-fixed, paraffin-embedded uteri were subjected to routine 5µm thickness sectioning for IHC analysis. All IHC experiments were performed using a standard protocol. Briefly, sections were deparafﬁnized by incubating with xylene for 30 min and rehydrated in an ethanol series, followed by antigen retrieval and endogenous peroxidase blockage. The sections were then incubated with respective primary antibodies, including anti-α-smooth muscle actin (α-SMA), -Lif, - Integrin-β3, -Hoxa10 and -Hoxa11, overnight at 4°C in a humidified chamber, followed by incubation with the corresponding secondary antibodies. Phosphate-buffered saline was used as the negative control instead of the primary antibody. Antibody information is presented in Table 2. Image Pro-Plus software (Iv.6.0; Media Cybernetics) was used to evaluate the reactivity of the endometrial glands and luminal surface epithelium of the uterus (average positively-stained area percentage) using two independent analyzers.
All experiments were carried out at least three times, data represent the as mean ± standard deviation (SD). Statistical analyses were conducted using one-way analysis of variance, followed by Turkey’s or Bonferroni post hoc analysis for multiple comparisons. All analyses were performed using GraphPad Prime software (GraphPad Software, LaJolla, CA, USA) and SPSS (v.18.0; SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.