title: Enzyme immunoassay for testosterone and androstenedione in culture medium from rabbit oocytes matured in vitro.
abstract: Two enzyme immunoassays (EIAs) were validated to determine testosterone and androstenedione levels in culture medium (Brackett's medium with or without the addition of IGF-I, hormone and serum-free), without previous extraction, from rabbit oocytes matured in vitro. Polyclonal testosterone (C917), and androstenedione (C9111) antibodies were raised in rabbits using testosterone 3-carboxymethyloxime:BSA, and androstenedione 3-carboxymethyloxime:BSA. Horseradish peroxidase was used as label, conjugated to testosterone 3-carboxymethyloxime, and to androstenedione 6-hemisuccinate. Standard dose response curves covered a range between 0 and 1 ng/well. The low detection limits of the technique were 11.43 pg/ml for testosterone, and 2.32 pg/ml for androstenedione. Intra- and inter-assay coefficient of variation percentages were < 6.4 and < 7.1 for testosterone, and < 5.1 and < 6.3 for androstenedione, respectively (n= 10). The recovery rate of known testosterone or androstenedione concentrations added to pools of culture maturation medium samples averaged 97.58 +/- 2.11%, and 95.73 +/- 1.59%, respectively. Compared with RIA, EIA values were in close agreement for testosterone (n= 15, r= 0.96, P< 0.001), and androstenedione (n= 15, r= 0.94, P< 0.001). Culture medium samples were obtained at the end of oocyte in vitro maturation (14-16 h). Mean +/- SE culture maturation medium concentrations (ng/ml) were 1.80 +/- 0.09 and 0.52 +/- 0.01 for testosterone, and 1.70 +/- 0.04 and 0.24 +/- 0.01 for androstenedione in both the oocytes with and without cumulus cells, respectively. We concluded that our EIA is a highly sensitive and specific assay that provides a rapid, simple, inexpensive and nonradiometric alternative to RIA for determining testosterone and androstenedione concentrations in oocyte maturation culture medium.
authors.affiliation: Departamento de Fisiología Animal. Facultad de Veterinaria UCM. 28040 Madrid, Spain.
authors.forename: J C
authors.initials: JC
authors.lastname: Illera
authors.forename: P L
authors.initials: PL
authors.lastname: Lorenzo
authors.forename: G
authors.initials: G
authors.lastname: Silván
authors.forename: C J
authors.initials: CJ
authors.lastname: Munro
authors.forename: M J
authors.initials: MJ
authors.lastname: Illera
authors.forename: M
authors.initials: M
authors.lastname: Illera