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 bitches treated with various steroid regimens showed induction of cP5 and 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 designed (a) to identify and characterize proteins synthesized de novo in explant culture from cyclic (dioestrus days 10-16) and early pregnant (dioestrus days 10-26) dogs, (b) to examine distribution of proteins by endometrium preimplantation (PI), and between (BI) and at (I) implantation 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 embryonal membranes. Proteins synthesized by endometrium were examined by incorporation of [3H]leucine or [35S]methionine on two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by fluorography or autoradiography. Nine proteins, characterized by electrophoresis and previously found between dioestrus days 3-10, appeared to be 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 sites, RBP decreased in number of isoelectric variants as gestation progressed, while the major M(r) form (23,000) was reduced to 21,500. There was no change in M(r) and isoelectric variants of RBP from cyclic dogs, suggesting differences in RBP gene regulation. Proteins synthesized by embryonal membranes appeared to be serum proteins. Immunolocalization 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 specific epithelium stained. As pregnancy progressed to day 24 of dioestrus, staining was localized to specific epithelial cells of the deep spongy zone and yolk sac. In ovariectomized bitches, CUPED was confirmed to be oestrogen dependent with staining in both luminal and glandular epithelia.(ABSTRACT TRUNCATED AT 400 WORDS)