Two-month-old female TNFR1−/− C57BL/6 mice, weighing 19–21 g, were used. Animals were housed in the Universidad Nacional de San Luis Animal Facility (San Luis, Argentina) under strict light conditions (12 h light, 12 h darkness), controlled temperature (22 ± 2 °C), sterile water, and ad libitum feeding. All experimental procedures were performed following the Guide for the Care and Use of Laboratory Animals of the National Research Council (8th ed., 2011, Washington, DC) and complied with the ARRIVE guidelines 2.0. This study was reviewed and approved by the Comité Institucional de Cuidado y Uso de Animales (CICUA) of the Universidad Nacional de San Luis (Protocols No. B-304/20 and B-304/21).
Experimental design
Twenty-four TNFR1−/− mice (KO) were randomly divided into three groups: (1) sham-operated mice (KO Sham), (2) EDT-induced mice (KO EDT), and (3) TM-treated EDT-induced mice (KO EDT+TM). The EDT induction consisted of autologous uterine tissue transplantation to the intestinal mesentery5,26. For this, animals were anesthetized with an intraperitoneal injection of ketamine-xylazine: 100 mg/kg of ketamine and 10 mg/kg of xylazine (Holliday Scott, Buenos Aires, Argentina, and Richmond, Buenos Aires, Argentina, respectively). After a mid-ventral incision, the right uterine horn was removed from the animal, divided longitudinally, and then cut into four mm2 pieces. The fragments were sutured in the intestinal mesentery with a 6-0 nylon suture to simulate endometriotic lesions. In Sham mice, three sutures were made without implanting uterine tissue. The mice’s body weight, food consumption, and grooming behavior were monitored daily. One month after inducing EDT, animals were euthanized by cervical dislocation. Immediately, a small mid-ventral orifice was opened, through which 1.5 mL of pH 7.4 phosphate-buffered saline (PBS) was injected into the peritoneal cavity of each animal. Peritoneal lavage fluid was collected and centrifuged at 250 g for 10 min at 4 °C. Supernatants (peritoneal fluid) were collected and stored at − 80 °C for Cu and estradiol determination. Finally, the abdomen of the EDT-induced animals was fully opened to access the endometriotic-like lesions.
Administration of TM
Starting on postoperative day 15 (period required for lesion establishment), each animal in the KO EDT+TM group received 0.3 mg of TM (cat# 323446, Sigma-Aldrich, St Louis, MO, USA) orally, as previously described26. Weekly controls of body weight and hematocrit were performed to ensure that severe Cu deficiency did not occur42,53. Furthermore, no toxicity evidence was observed for the administered dose based on food consumption or grooming behavior compared to the KO Sham and KO EDT groups.
Cu determination
Cu concentration in the peritoneal fluid was determined by electrothermal atomic absorption spectrometry (ETAAS) with a graphite furnace. Peritoneal fluid samples (500 μL) were mineralized with 500 μL concentrated nitric acid and 500 μL hydrogen peroxide. After adding the solutions, transparent samples were obtained by heating at 60 °C for 1 h in a thermostated water bath. All samples and reagents were prepared in 15 mL metal-free polypropylene tubes (Sarstedt, Germany). The reagents used were of trace analysis grade. They included: Ultrapure water with a resistivity of 18.2 MΩ cm produced by an Easy pure RF system from Barnstead (Dubuque, IA, USA), Double distilled acids obtained with a PTFE sub-boiling acid distiller (Distillacid, Berghof Products+Instruments GmbH, Germany) and 30% hydrogen peroxide (Merck, Germany). A Shimadzu Model AA-7000 atomic absorption spectrometer (Tokyo, Japan) was used to perform the measurements, equipped with a GFA-EX7 atomizer and an ASC-6100 autosampler. Integrated platform graphite tubes (L’vov), Shimadzu (Tokyo, Japan), were used in all experiments. A Cu hollow cathode lamp (Hamamatsu, Photonics, K.K., Japan) was employed as a radiation source at 324.8 nm with a 0.5 nm slit. All measurements were performed in duplicate. The results were expressed in µg/L.
Macroscopic analysis of the endometriotic-like lesions
Lesions were identified, counted, and measured with a caliper at two different perpendicular diameters. For the volume calculation, the following equation was used: V = (4/3) π r12 r2 (r1 and r2 are the radiuses and r1 < r2). Subsequently, the lesions were removed and weighed. For each animal, a lesion was fixed in 4% paraformaldehyde in PBS (pH 7.4) for 24 h at 4 °C. Fixed specimens were embedded in paraffin and cut into 4-μm-thick sections. A standard hematoxylin-eosin procedure was used to confirm the presence of endometrial glands and stroma in the ectopic tissue and to count the number of blood vessels. Other 4-μm-sections were prepared for cell proliferation study. Another lesion was placed at − 20 °C in RNAhold® (TransGen Biotech® Co., Ltd., Beijing, China) for RT-qPCR studies. The third lesion was kept at − 80 °C for protein extraction.
Estradiol determination
An electrochemiluminescence immunoassay (ECLIA) kit specific for estradiol (Elecsys Estradiol III, Roche Diagnostics International Ltd., Mannheim, Germany) was used, according to manufacturer’s instructions. The lower and upper detection limits were 5 and 3000 pg/mL, respectively. All measurements were performed in duplicate. Estradiol levels in the peritoneal fluid were expressed in pg/mL.
Immunohistochemistry of PCNA
Proliferating cell nuclear antigen (PCNA) is a nuclear protein involved in cellular DNA replication; therefore, it was evaluated in endometriotic-like lesions of untreated and TM-treated mice by immunohistochemistry. Tissue sections from six different animals per experimental group were deparaffinized in xylene and rehydrated in a graded series of ethyl alcohols. For antigen retrieval, slides were transferred to a glass staining jar filled with 0.01 M sodium citrate buffer (pH 6.0), which was placed in a microwave oven for 10 min on the highest power. Endogenous peroxidase was blocked with 3% H2O2 for 30 min. All the sections were blocked with 4% BSA in 1 × PBS for 2 h at room temperature in a humid chamber and then incubated with a polyclonal rabbit anti-PCNA antibody (1:150; FL-261, Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. For negative controls, the primary antibody was replaced with 1% BSA in 1 × PBS. Subsequently, sections were incubated with a biotinylated goat anti-rabbit IgG antibody (1:500; B8895, Sigma-Aldrich, St Louis, MO, USA) for 1 h at room temperature. Then, they were incubated with HRP-conjugated streptavidin (VectorLabs, Burlingame, CA, USA) for 30 min at room temperature. Diaminobenzidine (DAB) was used as substrate and incubated for 5 min (Cell Marque, CA, USA). Finally, tissue sections were counterstained with hematoxylin, dehydrated with graded alcohols, cleared in xylene, and properly mounted. PCNA-positive cells were identified by the presence of brown nuclear reactivity. Their percentage was established by two different operators at 1000 × under optical light microscopy (Olympus, Japan) by analyzing six representative fields per section. The total positive cell percentage was calculated per slide and was used to obtain the mean of each experimental group.
Mean blood vessel count
Blood vessels of different calibers were counted in sections of endometriotic-like lesions stained with hematoxylin-eosin using an optical light microscope (Olympus, Japan) at 400 ×. Tissue sections from six different animals per experimental group and between 6 and 8 fields per section were observed by two different operators. Blood vessels identified in each section were reviewed and approved by a pathologist. Results were expressed as the mean blood vessel count per field for the KO EDT and KO EDT+TM groups.
Quantitative reverse transcription PCR (RT-qPCR)
RT-qPCR was performed to analyze the gene expression of the following genes: Vegfa, Fgf2, and Platelet-Derived Growth Factor B (Pdgfb). Total RNA was isolated from endometriotic-like lesions using TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNA samples were quantified using an EPOCH™ microplate spectrophotometer (BioTek Instruments, Inc., Vermont, USA). All high-purity and intact RNA samples were treated with RQ1 RNase-Free DNase (Promega). Total RNA (1 μg) was reverse-transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics International Ltd., Mannheim, Germany) and stored at − 20 °C, following manufacturer’s guidelines. For qPCR, cDNA was amplified in an ABI PRISM® 7500 Instrument (Applied Biosystems, USA), using FastStart™ Universal SYBR® Green Master (Roche Diagnostics International Ltd., Mannheim, Germany). The reaction mixture consisted of 2×FastStart™ Universal SYBR® Green Master Mix, cDNA, forward primer (10 μM), reverse primer (10 μM), and nuclease-free water. All primers are described in Table 1. PCR cycling conditions were 95 °C, 10 min; 40 cycles at 95 °C, 15 s; 60 °C, 1 min. The relative expression was calculated using the 2−ΔΔCt method54. All experiments were performed in duplicate. Rn18s (18S ribosomal RNA): internal reference gene.
Antioxidant enzyme activities
Proteins from lesions were extracted in 100 μL RIPA buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and quantified according to Bradford method55, as previously described26. To measure superoxide dismutase (SOD) activity, the pyrogallol autoxidation method was used, monitoring the change in absorbance at 420 nm per min. One unit of the enzyme was expressed as the amount of SOD that inhibits 50% of pyrogallol autoxidation56. The catalase (CAT) activity was determined by measuring the decrease in H2O2 absorption at 240 nm, in which one CAT unit is the amount of enzyme required to decompose 1 μM of H2O2/min57. The glutathione peroxidase (GPX) activity was determined by following NADPH oxidation at 340 nm58. All readings were carried out using a Shimadzu 1800 UV–Visible spectrophotometer. The results were expressed as units of enzyme activity per milligram of protein (U/mg protein). All measurements were performed in duplicate.
Measurement of MDA
The levels of malondialdehyde (MDA), the end product of lipid peroxidation, were determined according to the method described by Draper and Hadley59. This method was based on the reaction of MDA with thiobarbituric acid (TBA). 1,1,3,3-tetraethoxypropane was used as a standard for the calibration curve, and the results were expressed as μmol MDA/mg protein. All measurements were performed in duplicate.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Values were presented as the mean ± SEM. Differences between groups were analyzed using a two-tailed unpaired Student’s t test or one-way ANOVA followed by Tukey’s multiple comparison test (when appropriate). For the correlation analysis, the normality of data was assessed with the Shapiro–Wilk test, and Spearman’s correlation method was applied. Differences were statistically significant when P < 0.05.