E2758

All female mice in this study were ovariectomized and implanted with a silastic capsule of oestradiol as previously described (3 (link)–5 (link)). This ensures uniform hormone concentrations across females and mimics an oestrus hormonal level of estradiol in order to artificially trigger receptivity (3 (link)–5 (link)). Briefly, all females were bilaterally ovariectomized under general anaesthesia with isofluorane. At the same time, a 5-mm-long Silastic capsule (inner diameter: 1.57 mm; outer diameter: 2.41 mm) containing crystalline 17β-oestradiol (E2758, Sigma-Aldrich, USA) (diluted 1:1 with cholesterol (C8667, Sigma-Aldrich, USA)) was inserted under the skin at the nape of the neck to induce oestrous levels of oestradiol. Mice received Carprofen (5 mg/kg) and were allowed to recover for two weeks before the onset of behavioral tests. On the day of testing, the females (either tests or stimuli) were administered progesterone (P) (P0130, Sigma-Aldrich, USA) (500 µg/mL, s.c.) 3 hours prior to test commencement.

The in vitro hypertrophy induction protocol for the H9c2 cardiomyoblast cell model (Watkins et al. 2010 ) included treatment with the hypertrophic agonist endothelin-1 (ET-1, E7764, Sigma, Germany). For these procedures, cardiomyoblasts were cultured to optimally control the hypertrophic responses of ET-1. Ethanol (E) served as the solvent in each group. Cardiomyoblasts were washed, cultured, and treated with serum-free medium and 1% of antibiotics, in maintenance DMEM, for the duration of each experiment. Cells were then incubated for 24 h at 37 °C, and cultured in the presence of 5% CO₂. After 24 h, the cardiomyoblasts were exposed to 100, 1000, and 10,000 nM of ET-1, respectively. Cells were pretreated with 200 nM of β-E (E2758, Sigma, Schnelldorf, Germany) for 6 h before exposing to 1000 nM of ET-1.

HEC-1A cells and JAR cells (American Type Culture Collection, Manassas, VA, USA) were grown in McCoy’s 5 A and RPMI 1640 medium. RL95-2 cells were grown in DMEM/F12 (1:1) with 0.005 mg/mL insulin. The three cells lines were all supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. When the cells reached 80% confluence, they were subsequently starved of serum for 3 h before treatment with P4 (Sigma, P0130), E2 (Sigma, E2758) or E2 and P4 (concentrations can be seen in figure legends) under serum-free conditions. Cells were harvested for RNA after treatment 48 h and for protein after treatment 72 h.

Adult mice (8–12 week-old female mice or 14-16 week-old male mice) were bilaterally ovariectomized or castrated. For sex hormone replacement, a 2 cm length of silastic tubing (inner diameter: 1.58 mm; outer diameter: 3.18 mm) containing 36 μg 17β-estradiol/mL (E2758, Sigma)⁴² in sesame oil or testosterone powder^{43 (link)} (T1500, Sigma) were inserted on the dorsal aspect of the animal’s neck immediately after gonadectomy. Mice were allowed to recover for 1 week before further treatment.
For ablation experiments, 8- to 10-week-old female mice or 14- to 16-week-old male mice were intraperitoneally injected with 20 ng/g bodyweight diphtheria toxin (DT; 322326, EMD Millipore) twice with a 3-day interval between injections. For activation experiments, 2.5 mg of clozapine-N-oxide (CNO; Tocris Bioscience) in 200 ml of drinking water was administered to the mice with a high-fat diet (22% carb, 24% protein, 54% fat, E15742-347, Ssniff). For FSH administration, mice with a high-fat diet received daily 30 IU/kg i.p FSH (GONAL-f, Merck) injections. Mice were weighed and monitored daily before being euthanized as experimental mice. Mice were humanely euthanized if postsurgical complications progressed to a pre-defined humane endpoint at which the mice started to suffer.

Explant cultures were utilized to further investigate the role of both estradiol and A2m on condylar fibrocartilage degradation. Entire mandibles with condyle and condylar fibrocartilage intact were harvested from 13-week old WT female mice and cultured in a 24-well plate with 1.5 mL of FBS-free BGJb medium supplemented with 1% Penicillin-Streptomycin (ThermoFisher Scientific) overnight. Following overnight incubation, media was removed, samples were washed 2 × with sterile PBS, and fresh media with either 0 M, 10⁻⁸ M, or 10⁻⁶ M 17β-estradiol (17β-estradiol, Sigma E2758, for the estradiol study), 100 nM and 200 nM A2m (from human plasma, Sigma SRP6314), or 50 nM and 100 nM Pi15 (synthetic – based on human peptide sequence, abcam ab23016) was added and incubated for 48 hours. Estradiol concentrations were chosen based on similar studies^{21 (link)} and further substantiated by evidence of circulating estrogen levels in humans^{59 (link)}. A2m concentrations were determined based on previous evidence in a knee osteoarthritis model^{49 (link)}. After 48 hours of treatment, supernatant was collected for zymogram assays and collagen type I and II cleavage ELISAs. Messenger RNA from both left and right samples was pooled together and utilized for RTPCR. For gelatin zymography and ELISA analysis, supernatant from only the left specimen was utilized.