A SENSITIVE EIA FOR 17-BETA-ESTRADIOL AND PROGESTERONE IN CULTURE-MEDIUM FOR OOCYTE IN-VITRO MATURATION PROCEDURES

A sensitive heterologous enzyme immunoassay (EIA) was validated to det ermine 17 beta-estradiol (E2) and progesterone levels, without previou s extraction, in culture medium from rabbit oocytes matured in vitro w ith and without the addition of IGF-I. Polyclonal E2 (C902), and proge sterone (C914) antibodies were raised in rabbits using 6-keto-17 beta- estradiol 6-carboxymethyloxime:BSA, and 11 alpha-hydroxyprogesterone 1 1 alpha-hemisuccinate:BSA. Horseradish peroxidase was used as label, c onjugated to 17 beta-estradiol 3-hemisuccinate, and to progesterone 3- carboxymethyloxime. Standard dose response curves covered a range betw een 0 and 1 ng/well (100 mu l). The low detection limits of the techni que were 1.99 pg/well for E2, and 13.21 pg/well for progesterone. Intr a-and interassay coefficient of variation percentages (% CV) were < 6. 3 and < 7.8 for E2 and progesterone, respectively (n = 10). The recove ry rate of known E2 or progesterone concentrations added to a pool of culture maturation medium averaged 96.39 %, and 98.65 %, respectively. Compared with RIA, EIA values were in close agreement for E2 (n = 15, R = 0.96, P<0.001), and progesterone (n = 15, R = 0.99, P < 0.001). M edium samples were obtained after oocyte maturation in vitro for 16 h. Use of IGF-I significantly elevated steroids production in the oocyte surrounded cumulus cells. The EIA described here is highly sensitive and specific assay, and provides a rapid, simple, inexpensive, and non -radiometric alternative to RIA for determining E2 and progesterone le vels in oocyte culture medium.