Identification of bovine seminal plasma proteins with an activity to normalize endometrial epidermal growth factor concentrations in repeat breeder cows

Abstract

Epidermal growth factor (EGF) concentrations in the bovine uterus show two peaks on Days 2-4 and 13-14 during the estrous cycle in normal cows; however, these peaks were not found in about 70% of Holstein repeat breeder cows. We have demonstrated the effect of seminal plasma (SP) to normalize the endometrial EGF concentrations on Day 3 and restore fertility in repeat breeder cows. The objective of this study was to identify SP protein(s) with the activity of normalizing the endometrial EGF concentrations. Semen was collected from 5 Holstein bulls and pooled SP obtained from 30 ejaculates were used for this study. The SP protein were separated by gel filtration and 2-dimensional electrophoresis. SP fractions with molecular weight of 16-29 kDa and pI5.8-7.0 showed an activity to normalize the endometrial EGF concentrations on Day 3. Then, protein spots in these area on electrophoresis gels were extracted and subjected to liquid chromatography with tandem mass spectrometry analysis. Twelve protein spots that include four spots of osteopontin (OPN) with different molecular weights and isoelectric points were identified. Protein extracts of one of these OPN spots normalized the endometrial EGF concentrations on Day 3 in 41.9% of repeat breeder cows. The present results indicated that OPN may be the molecule responsible for the activity normalizing the EGF concentrations on Day 3 in the endometrium of repeat breeder cows