For in vitro experiments, a fresh stock solution of metformin (1,1-dimethylbiguanide hydrochloride; Sigma-Aldrich, Oakville Ontario) was prepared (0.167 mg/ml) in serum-free media (SFM), containing 10X Dulbecco’s modified Eagle’s medium/F12, 30% glucose, 7.5% NaHCO3, 1 M Hepes, l-glutamine, hormone mix, and penicillin and streptomycin. Dilutions were made in SFM to reach the desired end concentration. For in vivo experiments, metformin was prepared fresh daily in sterile 1X phosphate-buffered saline (PBS). For experiments in naïve early postnatal preweaning animals, mice received subcutaneous injections of metformin (20 mg/kg) once daily from P9 to P12. In juveniles and young adults, mice received intraperitoneal injections of metformin (200 mg/kg) once daily from P21 to P27 and from P50 to P56, respectively. When investigating the ideal dose of metformin in males, mice received 20 or 200 mg/kg of metformin via intraperitoneal injections for 4 or 7 days. For experiments investigating the effects of hormones, estradiol (5.6 mg/kg, Sigma-Aldrich, Missouri, USA) or vehicle (sesame oil, Sigma-Aldrich, Missouri, USA) was administered subcutaneously from P17 to P27, along with concurrent intraperitoneal metformin (200 mg/kg) or PBS injections from P21 to P27, as mentioned above. For testosterone administration, testosterone propionate (100 μg, Toronto Research Chemicals, Ontario, Canada) or vehicle (sesame oil) was injected subcutaneously from P9 to P12, along with concurrent intraperitoneal injections of metformin (20 mg/kg) or PBS. To investigate the effect of post–H-I metformin treatment, mice received daily subcutaneous injections (20 mg/kg) from P9 to P27, followed by drug delivery through an implanted subcutaneous osmotic pump (20 mg/kg per day; Durect Corporation, California, USA) from P28 to P41.

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