Quantification of ovarian steroidogenesis in the domestic fowl by incubation of intact large follicles.

The output of progesterone, androstenedione, and estradiol-17 beta from the five largest preovulatory follicles (F1 to F5 in descending order) of 30 57-wk-old Single Comb White Leghorn hens killed 3 to 3.5 h prior to the next midsequence ovulation was measured by radioimmunoassay of incubation media. Follicles were incubated for 3 h at 39 C with different levels of bovine luteinizing hormone (bLH) (0, .125, .25, .5, 1.0, and 2.0 micrograms per follicle) in Medium 199 and HEPES (N-2-hydroxyethyl piperazine-N'-2-ethane sulfonic acid) (pH = 7.2). The amount of incubation media used was calculated in proportion to follicular size. In the absence of exogenous bLH, the average amount of progesterone produced in vitro by the F1 preovulatory follicle was 113 ng per follicle. In contrast, the average amount of progesterone produced by the F2 to F5 follicles combined was 3.75 ng. The output of androstenedione was highest (P less than .05) in F2, when compared with any of the other follicles in the hierarchy, including F1. The low secretion (less than 2.5 ng per follicle) of estradiol-17 beta from F1 to F5 follicles supports a previous conclusion that large follicles are probably not the major source of estradiol-17 beta in laying hens. The five largest follicles removed from hens killed shortly (less than 4 h) after an in vivo surge of LH were refractory to exogenous bLH stimulation. The amount of progesterone, androstenedione, and estradiol-17 beta obtained by the incubation of intact large follicles is consistent with published results obtained by the incubation of isolated follicular cells. Incubation of intact follicles provides a relatively simple, yet reliable, procedure for the measurement of steroidogenesis of avian large follicles.