Development of 24 h in vitro matured bovine oocytes following parthenogenetic activation by ethanol and cycloheximide treatment

Abstract

Accurate measurement of sex steroids is essential to evaluate mouse models for human reproductive development and disorders. The recent advent of liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays that match the sensitivity of steroid immunoassay could overcome problems arising from the limited specificity of steroid immunoassay. In this current study we validate a LC–MS/MS assay for the measurement of key sex steroids from murine serum and reproductive tissues. The assay gave excellent dilutional linearity (r2 ≥ 0.98) and reproducibility (CV ≤ 10% of replicate samples) in serum and reproductive tissues with sensitive quantitation limits; testosterone (T; 2 pg), dihydrotestosterone (DHT; 10 pg), 5α-androstane-3α,17β-diol (3αDiol; 40 pg), 5α-androstane-3β,17β-diol (3βDiol; 40 pg), estradiol (E2; 0.5 pg) and estrone (E1; 0.3 pg). Using 0.1 mL sample, T was the only consistently detectable steroid (detection limit 20 pg/ml) in both male and female mouse serum. In the testis, T and DHT were quantifiable as were both diols at relatively high levels. Prostatic T levels were low and DHT was determined to be the most abundant androgen in this tissue. Uterine and ovarian levels of E2, E1 and T were measurable, with levels varying according to estrous cycle stage. Hence, we demonstrate that this LC–MS/MS method has the sensitivity, specificity and multi-analyte capability to offer accurate steroid profiling in mouse serum and reproductive tissues.