74 THE BOVINE EMBRYO INFLUENCES THE PROTEOME OF THE OVIDUCTAL FLUID
Authors: Maillo V. , Acuña O. S. , Aviles M. , Lonergan P. , Rizos D. 
Published: December 2016

It has been shown that the equine embryo is able to modulate the proteome of the oviduct, increasing the presence of certain proteins involved in the embryo-maternal communication (Smits et al. 2016 Reprod. Fertil. Dev. doi: 10.1071/RD15481). In cattle, the presence of a single embryo did not affect the transcriptome of the oviduct, whereas multiple embryos induced changes (Maillo et al. 2015 Biol. Reprod. 92, 144). The aim of the present study was to examine the effect of the presence of an embryo on the oviduct fluid proteome. Cross-bred beef heifers were synchronized, and those in standing oestrus were randomly allocated to cyclic, not bred (n = 7), or pregnant, artificially inseminated (n = 11), groups. All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut, separating ampulla and isthmus. Each portion was flushed with 500 μL of PBS free of protein to confirm the presence of an oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (pregnant group) were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Flushings from 4 cyclic and 4 pregnant heifers (8-cell embryos) were used for proteomic analysis, including blank controls of PBS. Comparison of ipsilateral ampulla and isthmus in pregnant and cyclic heifers did not reveal any differences. Therefore, samples from ipsilateral oviducts were compared between cyclic and pregnant heifers. Total protein (150 μg) from cyclic and pregnant samples was labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3–10, 17 cm, Protean® IEF cell system, Bio Rad, Hercules, CA, USA). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). The images were obtained with the Typhoon 9410 scanner and analysed with the Progenesis SameSpots software v. 4.0. The results of image analysis showed 42 different spots between the 2 groups (ANOVA P < 0.01 and fold difference >1.5), the majority (38) of which were more abundant in pregnant heifers. Gels were stained with Coomassie blue to detect the spots. The differential spots were digested with trypsin and analysed with Agilent Ion-Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray interface. Twenty spots were highly abundant in pregnant heifers. From these, 3 different proteins were identified with score >5.0 and % SPI >76.0 by mass spectrometry, corresponding to SERPINA1, serum albumin, and serum transferrin. These proteins are abundant in plasma, suggesting that the embryo may be able to activate pathways to increase the permeability of the vascular endothelium in the oviduct. The same proteins were also identified in the equine oviducal proteome in the presence of an embryo, suggesting a possible conserved mechanism between species. In conclusion, presence of an embryo in the bovine oviduct increases the abundance of some proteins that may be related with its early development.
This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL-2012–37510 and AGL-2015–70140-R) and Science Foundation Ireland (13/IA/1983).