104 ESTROUS CYCLE-DEPENDENT CHANGES IN THE BOVINE ENDOMETRIUM PROTEOME

Abstract

Among the reproductive tissues, the endometrium plays a central role in the context of embryo-maternal communication and pregnancy recognition. During the estrous cycle, characteristic morphological and functional changes occur in the bovine endometrium, being crucial for uterine receptivity. These changes are mainly regulated by the hormones progesterone, estradiol, and oxytocin. The bovine estrous cycle, with a length of 21 days, can be divided into 4 stages: i) estrus (considered as Day 0, low progesterone level and time of ovulation); ii) metestrus (Days 1 to 5, corpus luteum formation, rising progesterone level); iii) diestrus (Days 6 to 17, high progesterone); and iv) proestrus (Days 18 to 20, corpus luteum degeneration; declining progesterone). The principles of hormonal regulation during the estrous cycle are well understood; however, in-depth knowledge of the detailed molecular mechanisms is still incomplete. To elucidate the underlying biochemical processes, the proteomes of bovine endometrial samples of all 4 stages (cycle Day 0, Day 3.5, Day 12, and Day 18) were compared in a quantitative manner. To maximize the accuracy of protein quantification, sophisticated 2-dimensional (2D) fluorescence difference gel electrophoresis (2D-DIGE) experiments were performed, using internal pooled standards for inter-gel normalization. To enhance the resolution of 2D-polyacrylamide gel electrophoresis separation, the proteins were analyzed by 2 overlapping pH gradients. In total, 28 individual DIGE experiments (14 2D gels × 2 pH gradients) were performed, corresponding to 84 gel images. With a refined statistical analysis of spot intensities, we were able to identify a total of 91 spots altered by at least a factor of ±2 (P < 0.05) in intensity between at least 2 of the 4 stages. Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) identification of these spots showed that they originated from 66 different proteins. Moreover, for 14 of these proteins, several polymorphic variants could be identified. Gene ontology analysis of the protein IDs revealed a broad diversity of biological and biochemical functions as well as cellular localizations of these proteins. Several proteins detected (e.g. FK506 and 20 alpha-HSD) are crucial components for uterine receptivity and represent interesting targets for further functional studies. This study was supported by the Deutsche Forschungsgemeinschaft (FOR 478).