Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment

The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [(3)H]leucine or [(35)S]methionine. The rate of incorporation of [(3)H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (Mr 54,686) and cP6 (Mr 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cp1 (Mr 87,600), synthesized after corn oil and P4 treatment, was suppressed with estradiol (E2). Proteins cP2 (MR 40,000 and Mr 42,000), present with all treatments, were intensified with P4. A high-Mr basic protein complex (cP3) and acidic protein cP4 were expressed with E2 and maintained with P4 treatment. Proteins cP5 and cP6, while not induced by E2 or P4 alone, required E2 priming for P4 induction. Protein cP5 was down-regulated while cP6 was up-regulated with P4 for 2 wk. Proteins induced by estrogen followed by 1 or 2 wk of P4 treatments were similar to those released by endometrial explants collected from pregnant and cyclic bitches on Days 3, 7, and 10 of spontaneous diestrus. Results of this study indicate that canine endometrium synthesizes de novo and secretes at least ten protein complexes, two of which are major proteins that are differentially expressed. Overall, protein expression appears different by day but not by status, suggesting that the preimplantation embryo does not have an effect on uterine endometrial protein synthesis and secretion. Expression of specific proteins during early diestrus, however, appears to be in response to specific regimens of estrogen and P4 that mimic spontaneous early diestrus.