ENZYME-IMMUNOASSAY FOR TESTOSTERONE AND ANDROSTENEDIONE IN CULTURE-MEDIUM FROM RABBIT OOCYTES MATURED IN-VITRO

Two enzyme immunoassays (EIAs) were validated to determine testosteron e and androstenedione levels in culture medium (Brackett's medium with or without the addition of IGF-I, hormone and serum-free), without pr evious extraction, from rabbit oocytes matured in vitro. Polyclonal te stosterone (C917), and androstenedione (C9111) antibodies were raised in rabbits using testosterone 3-carboxymethyloxime:BSA and androstened ione 3-carboxymethyloxime:BSA. Horseradish peroxidase was used as labe l, conjugated to testosterone 3 -carboxymethyloxime, and to androstene dione 6-hemisuccinate. Standard dose response curves covered a range b etween 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. Int ra- and inter-assay coefficient of variation percentages were < 6.4 an d < 7.1 for testosterone, and < 5.1 and < 6.3 for androstenedione, res pectively (n= 10). The recovery rate of known testosterone or androste nedione concentrations added to pools of culture maturation medium sam ples averaged 97.58 +/- 2.11%, and 95.73 +/- 1.59%, respectively. Comp ared 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 concentrat ions (ng/ml) were 1.80 +/- 0.09 and 0.52 +/- 0.01 for testosterone, an d 1.70 +/- 0.04 and 0.24 +/- 0.01 for androstenedione in both the oocy tes with and without cumulus cells, respectively. We concluded that ou r EIA is a highly sensitive and specific assay that provides a rapid, simple, inexpensive and nonradiometric alternative to RIA for determin ing testosterone and androstenedione concentrations in oocyte maturati on culture medium. (C) 1997 by Elsevier Science Inc.