The progesterone-receptor complex in the nuclear fraction of the bovine endometrium

Abstract

A nuclear-exchange assay was utilized to characterize and quantitate the progesterone-receptor complex in the nuclear fraction of the bovine endometrium. Eight multiparous cows, four primiparous cows and three heifers were observed twice daily for estrus and were randomly assigned to experimental procedures at the onset of estrus. Endometrium was removed, via surgery, on day 4, 10 to 12, and 18 of the cycle and on the day of estrus. The tissue was placed in cold tris-glycerol (TG) buffer [.01 M tris with 30% glycerol (v /v ), pH 7 .4 ], blotted dry, weighed, and placed in fresh TG buffer and packed in ice. Following dicing, the tissue was homogenized in a 25 x 200 mm Pyrex tissue grinder, filte re d through one layer of organza and centrifuged at 800 x £ for 20 minutes. The supernatant was decanted and the isolated nuclear pellet was washed three times, resuspended, gently rehomogenized, and diluted to the equivalent of 25 mg of tissue per ml of TG buffer. Five hundred microliters of the nuclear preparation were allowed to equilibrate with varying levels of tritiated progesterone (^H-P) with or without a 100- fold concentration of unlabeled P. The saturation analysis was submitted to Scatchard plot analysis which resulted in an exponential curve f i t indicating at least two binders. Additional tubes containing a 100-fold concentration of cortisol were utilized to linearize the Scatchard plot.