Luminex multiplex system

At the end of the treatments, blood samples were collected from each mouse and the serum was separated and analyzed, as previously described (21 (link)). Briefly, testosterone (T) quantification was performed through a competitive inhibition enzyme immunoassay technique (Mouse Testosterone ELISA Kit, CSB-E05101 m, China), whereas P4, estradiol (E2) and LH levels were measured using a magnetic bead immunoassay based on Luminex Multiplex System (Merck Millipore, Germany), according to the manufacturer’s instructions; respectively, P4 and E2 were analyzed by the “Steroid/Thyroid Hormone Magnetic Bead Panel” (#STTHMAG-21K) and the “Mouse Pituitary Magnetic Bead Panel” (#MPTMAG-49K) was used for LH.

After 20 consecutive days of treatments, blood samples were collected from each mouse before sacrifice. To separate serum, blood was kept at 4 °C for 1 h, followed by two consecutive centrifugations for 10 min at 15000 g at 4 °C. Serum was kept at −20 °C until hormonal assay was performed. Analysis of Testosterone (T) was performed employing a competitive inhibition enzyme immunoassay technique (Mouse Testosterone ELISA Kit, CSB-E05101m, Cusabio, Hubei Province, China; minimum detectable concentration (minDC) = 0.1 ng/ml; intra-assay and inter-assay precisions (mean of the percentages of coefficients of variation) are both <15 %). According to the manufacturer’s instructions, no significant cross-reactivity or interference between mouse testosterone and analogues was observed. Levels of Progesterone (P4), Estradiol (E2) and LH were measured using a magnetic bead immunoassay based on Luminex Multiplex System (Merck Millipore, Darmstadt, Germany), according to the manufacturer’s instructions; respectively E2 and P4 were analyzed by the “Steroid/Thyroid Hormone Magnetic Bead Panel” (# STTHMAG-21K; minDC = 0.02 ng/ml for E2 and 0.09 ng/ml for P4; intra-assay and inter-assay precisions are <10 % for both analytes), while the “Mouse Pituitary Magnetic Bead Panel” (# MPTMAG-49K; minDC = 1.34 pg/ml; intra-assay precision <15 % and inter-assay precision <20 %) was used for LH.