Kge014

Venous blood samples were collected from the median cubital vein in the antecubital fossa of each participant using a standard venipuncture technique. In each high intensity interval exercise trial, approximately 10 ml of whole blood was collected from each participant at each time point (pre-exercise, 5 min, 1h, 24h, and 48h after exercise) for a total of 10 blood draws per participant. All blood samples sat for 30 min at room temperature before being centrifuged at 3000xg and 4°C for 15 min in a benchtop centrifuge (Allegra ZIR centrifuge, Beckman Coulter, USA). Serum was then aliquoted into microcentrifuge tubes and stored at -80°C until analysis. Plasma was also used to measure hematocrit.
Serum levels of sclerostin, CTXI, PINP, and baseline estradiol were measured in duplicate using commercially available immunoassay (ELISA) kits (SCL, cat.# DSST00, R&D Systems, Inc., CTXI, cat.# E-EL-H0835, Elabscience, and PINP, cat. # E-EL-H0185, Elabscience) following the manual instructions. Our in-house inter- and intra-assay coefficients of variation (CV) for sclerostin, CTXI, and PINP were 8.2% and 3.7%, 10% and 11.6%, and 7.2% and 9.9%, respectively. Baseline (i.e., resting) serum estradiol was measured prior to each trial using one ELISA kit (Estradiol, cat.# KGE014, R&D Systems, Inc., intra-assay CV: 8.2%).

Culture supernatants from GCs or TCs monolayers were taken from individual wells to detect hormone levels 48 h after siRNA transfection with additional 12-h LH co-culture. After centrifugation at 2500 g at 4°C for 15 min, the supernatant was stored at −80°C for hormone measurement. The concentrations of Estradiol (E₂; KGE014, R&D Systems), testosterone (T, RnD, KGE010), progesterone (P₄, EA, Merck-Millipore, STTHMAG-21K-02) and androstenedione (AND, LSBio, ELISAKit-LS-F39181) were determined by ELISA using kits according to the manufacturer's protocols, respectively. A reported physiological concentration (2.5 ng/mL) of luteinizing hormone (LH) was added for 12 h to induce and maintain hormone secretion. Intra- and inter-assay precisions as described by coefficients of variations were T, ≤3.1% and ≤6.3%; AND, <8% and 10%. The detection limits of E₂, T, P₄ and AND were 12.3–3000 pg/mL, 0–10 ng/mL, 0.156–10 ng/mL, and 0.156–10 ng/mL, respectively.

The tissue was placed in 250 μL of T-Per tissue protein extraction reagent containing Halt Protease Inhibitor and ground (Thermo Scientific, Rockford, IL, USA). Ground samples were frozen and thawed, followed by centrifugation and decanting of the supernatant. The quantitation of protein in the tissue samples was performed in duplicate for Nrnx3, Nnat, Ephx2, Ppm1e, Kcnip1, Scn5a, Kcng1, Vdr, and Nptxr using ELISA (MyBioSource, San Diego, CA, USA). Total protein was determined in each sample using a BCA protein assay (Thermo Scientific, Waltham, WA, USA). Values represent the pg of protein measured by ELISA per mg of total protein. Blood was also collected after the animals were euthanized and the estradiol levels were measured in the plasma to confirm the estrous cycle stage as determined from the vaginal smears. To measure estradiol levels, the estradiol ELISA kit was used according to the manufacturer’s directions and samples were analyzed in duplicate (catalog # KGE014, R & D systems, Minneapolis, MN, USA).

Circulating FSH (CSB-E06871m, CUSABIO, MD, USA), corticosterone (KGE009, R&D Systems, MN, USA) and 17β-estradiol (KGE014, R&D Systems, MN, USA) were determined with commercial ELISA kits, according to the manufacturer’s instructions. Serum free fatty acid (FFA) and triglyceride (TG) levels were measured via a nonesterified free fatty acids assay kit (A042-2-1) and triglyceride assay kit (A110-1-1) from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Mice were euthanized by an overdose of pentobarbital sodium administered i.p. on days 3, 7 11, and 14. The uterus was harvested according to the OECD guidelines [28 ] and its wet weight was measured using a precision balance (d = 0.001 g) on days 3–14. In the additional three OVX mice, the uterus was harvested, and the effects of estrogen administration were examined in the in topical EB wound treatment, subcutaneous E2 pellet, and topical E2 skin application groups. Plasma was prepared from each mouse’s blood, isolated through cardiac puncture, and frozen until the time of assay. Enzyme immunoassay analysis (EIA) was performed to determine the concentration of E2 in the plasma on day 14, according to manufacture’s instruction (KGE014, R&D systems Inc., Tokyo, Japan).