Characterization of proteins produced in vitro by bovine endometrial explants

Endometrial tissues were obtained from 17 pregnant (P, estrus/mating = Day 0; Day 16, n = 4; Day 19, n = 6; Day 22, n = 3; Day 24, n = 4) and six nonpregnant (NP; Day 16, n = 4; Day 19, n = 2) cattle, as well as from one cyclic (nonbred) cow (Day 4), one later-pregnant cow (Day 69), and both ligated and pregnant uterine horns of three unilaterally pregnant cattle (UP; Day 270). Tissues (approximately equal to 500 mg wet tissue/explant) were cultured for 24 or 48 h in modified minimal essential medium (MEM), in the presence of radioactive amino acid and/or amino sugar substrate(s) (L-[3H] leucine, L-[14C] leucine, and D-[3H] glucosamine), in order to characterize substrate uptake and de novo synthesis and release of proteins and polypeptides in vitro. Endometrial explants from all cattle produced proteins de novo from radiolabeled substrates. Chromatographic (gel filtration, cation, and anion exchange) and two-dimensional polyacrylamide gel electrophoretic analyses revealed complex patterns of primarily acidic radiolabeled polypeptides in dialyzed MEM, which were absent from endometrial tissue homogenates. No qualitative differences were noted in the array of proteins released into MEM associated with either pregnancy status (P vs. NP, UP) or stage of gestation (Days 16, 19, 22, 24, 69, and 270). Medium from all endometrial explants was enriched in polypeptides in four Mr (X 10(3)/pH classes (I, approximately equal to 14/greater than 7.2; II, 19-24/5.4-6.3; III, 28-31/6.9-7.3; and IV, greater than or equal to 150/less than or equal to 5.1).