IDENTIFICATION AND IMMUNOLOCALIZATION OF PROTEINS SYNTHESIZED BY DOG ENDOMETRIUM AND MEMBRANES

Previous studies on cyclic and pregnant bitches on dioestrus days 3-10 indicated synthesis de novo of at least ten protein complexes, two of which are major protein groups (cP5, M(r) 54 600; cP6, M(r) 23 000). Protein expression differed by day but not by status, suggesting that the embryo does not affect uterine protein synthesis. Ovariectomized b itches treated with various steroid regimens showed induction of cP5 a nd cP6 only after oestrogen priming followed by progesterone. Recently , N-terminal amino acid microsequencing has identified cP6 as a member of the retinol-binding protein (RBP) family. The present study was de signed (a) to identify and characterize proteins synthesized de novo i n explant culture from cyclic (dioestrus days 10-16) and early pregnan t (dioestrus days 10-26) dogs, (b) to examine distribution of proteins by endometrium preimplantation (PI), and between (BI) and at (I) impl antation sites; (c) to characterize proteins synthesized by embryonal membranes and (d) to localize RBP and CUPED (a cat oestrogen-dependent uterine glycoprotein) immunocytochemically in the uterus and embryona l membranes. Proteins synthesized by endometrium were examined by inco rporation of [H-3]leucine or [S-35]methionine on two-dimensional sodiu m dodecyl sulphate polyacrylamide gel electrophoresis followed by fluo rography or autoradiography. Nine proteins, characterized by electroph oresis and previously found between dioestrus days 3-10, appeared to b e expressed through day 16 of cyclic and day 26 of pregnant dioestrus, although many decreased and cP5 was lost after day 13. In BI and I si tes, RBP decreased in number of isoelectric variants as gestation prog ressed, while the major M(r) form (23 000) was reduced to 21 500. Ther e was no change in M(r) and isoelectric variants of RBP from cyclic do gs, suggesting differences in RBP gene regulation. Proteins synthesize d by embryonal membranes appeared to be serum proteins. Immunolocaliza tion of RBP confirmed that oestrogen priming followed by progesterone was required for induction. In pregnant bitches, staining was present in all luminal epithelia on dioestrus day 10, but by day 17 only speci fic epithelium stained. As pregnancy progressed to day 24 of dioestrus , staining was localized to specific epithelial cells of the deep spon gy zone and yolk sac. In ovariectomized bitches, CUPED was confirmed t o be oestrogen dependent with staining in both luminal and glandular e pithelia. In PI and BI sites, CUPED was present on dioestrus days 10 a nd 13 but absent by days 15 and 17. Endometrial RBP, CUPED and other s ecretory proteins are believed to be part of the protein milieu requir ed by the conceptus before and during implantation.