Studies relating to protein expression in the uterus of the cow

Abstract

Embryo loss is a major cause of reproductive wastage in the cow. The majority of embryo loss occurs in the first 16 days after fertilisation when the embryo is critically dependent on the maternal uterine environment for survival. Despite the central role of uterine fluid in the normal growth and development of the embryo, there is limited information on the protein composition of these fluids. The main objectives of the studies in this thesis were to examine the protein composition of the bovine uterus during the oestrous cycle and to examine the relationship between the concentration of systemic progesterone and uterine protein expression in the cow. In the first study, the concentration of retinol-binding protein (RBP) in the bovine uterus was found to vary across the cycle and was 5-15-fold higher (P<0.001) on Day 15 than Days 3, 7 and 11. Additionally, the concentration of uterine RBP seems to be regulated in a local manner as the concentration in the uterine horn ipsilateral to the corpus luteum (CL) was more than 2-fold higher (P<0.001) than the contralateral horn on Day 15. This indicates that RBP is possibly regulated by a local increase in the concentration of progesterone. Uterine and plasma concentrations of RBP were similar on days 3 to 11, however, uterine RBP concentrations were 6-15 fold higher than blood plasma RBP concentrations on day 15. This shows evidence of active transport and/or synthesis of proteins into the uterus which previously were thought to be due to transduation of plasma. There was no relationship between the concentration of systemic progesterone and concentration of RBP on day 7 (P>0.05) of the cycle, which was surprising given that previous studies have indicated that uterine RBP gene expression was positively associated with the concentration of systemic progesterone. In the second study, IGF binding protein 2 (IGFBP-2), IGFBP-3, IGFBP-4 and IGFBP-5 were identified in uterine fluid on days 3, 7, 11 and 15 of the oestrous cycle. There was a local effect on the concentration of IGFBPs where the concentration was greater on the ipsilateral side than that on the contralateral side for IGFBP-2 (P<0.05), 3 (P<0.01) and 5 (P<0.01) on day 15. This difference is a further indication of a local controlling mechanism regulating proteins between the uterine horns. Similar to RBP expression this study could find no significant relationship between the concentration of systemic progesterone and IGFBP concentrations on Day 7 of the oestrous cycle. In the third study, changes in the global pattern of uterine proteins between Days 3 and Day 15 of the oestrous cycle were examined using two-dimensional electrophoresis (2-DE). Six proteins were found to be upregulated on Day 15 compared to Day 3. Three proteins of these were identified as aldose reductase, plakoglobin and heat shock protein 27 while the other three proteins were identified as bovine serum albumin. Aldose reductase, an enzyme directly involved in the production of sorbitol and indirectly of fructose, was 10-fold higher (P<0.0001) on Day 15 compared to Day 3. Plakoglobin (Pg) was upregulated 2.3-fold (P<0.0001) on Day 15 compared to Day 3. Pg is a component of cellular junctions and its up-regulation may have a role in the uterine glandular epithelium. Heat shock protein 27 (Hsp27) was higher on Day 15 than on Day 3 (P<0.01) and Hsp27 was 1.4-fold higher in the ipsilateral compared to the contralateral uterine horn (P<0.01). Hsp27 may be secreted in response to potential stresses in the uterus or act as a molecular chaperone. On Day 7 there was no difference (P<0.05) in the pattern of proteins secreted between cows with low (2.7±0.10ng/ml) and high (4.8±0.13 ng/ml) concentrations of systemic progesterone on Day 7. The results of these studies have shown that dramatic changes occur in protein expression across the bovine oestrous cycle. Additionally, it emphasises the need for gene studies to be followed with protein studies as an adjunct or complementary tool. Proteins have a wide range of essential roles in the uterus and together these studies provide novel information on protein expression in the uterus of the cow.