Abstract
This study aimed to characterize proteins and exosomal microRNAs (miRNAs) in the uterine flushings (UF) of cows associated with Day 7 (D7) pregnancy using the embryo donor cows of the embryo transfer program. Superovulated cows either were inseminated (AI cows) or remained non-inseminated (Ctrl cows). UF was collected on D7 in the presence of multiple embryos (AI cows) or without embryos (Ctrl cows) and subjected to isobaric tags for relative and absolute quantification protein analysis. A total of 336 proteins were identified, of which 260 proteins were more than 2-fold higher in AI cows than Ctrl cows. Gene ontology analysis revealed that many differentially expressed proteins were involved in “neutrophil-related” and “extracellular vesicular exosome-related” terms. In silico analysis of proteins with higher concentrations in the UF of AI identified 18 uniquely expressed proteins. Exosomes were isolated from the UF, from which RNA was subjected to miRNA-seq, identifying 37 miRNAs. Of these, three miRNAs were lower, and six miRNAs were higher in the UF of AI cows than those of Ctrl ones. The principal component analysis displayed a close association in miRNA and protein between bta-miR-29a, bta-miR-199b, SUGT1, and PPID. In addition, the receiver operating characteristic curve analysis showed that SUGT1 was the best predictor for the presence of embryos in the uterus. These findings suggest that the presence of multiple D7 embryos in the uterus can lead to significant changes in the protein composition and exosomal miRNA contents of UF, which could mediate innate immunological interactions between the pre-hatching embryo and the uterus in cows.
Highlights
Up to 50% of potential bovine pregnancies are lost in the first week following insemination (Diskin and Morris, 2008; Wiltbank et al, 2016)
The isobaric tags for relative and absolute quantification (iTRAQ) analysis revealed a significant difference in protein content and level in the Day 7 (D7) uterine flushings (UF) from AI cows with multiple embryos vs. those of Ctrl cows without embryos
A total of 336 proteins were identified, of which 260 proteins were more than 2-fold higher (Figure 2A) in AI cows compared to Ctrl cows
Summary
Up to 50% of potential bovine pregnancies are lost in the first week following insemination (Diskin and Morris, 2008; Wiltbank et al, 2016). Proper interactions between the early embryo and mother are crucial for successful pregnancy in cows. Pregnancy is established by complex interactions of the factors/molecules derived from both the developing embryo and the cow’s endometrium (Talukder et al, 2020). IFNT regulates the maternal immune response toward an antiinflammatory (Th2) action to tolerate the semi-allogenic embryo (Sponchiado et al, 2017; Talukder et al, 2017; Passaro et al, 2018; Rashid et al, 2018; Sponchiado et al, 2019). In addition to IFNT, other factors from both the pre-hatching embryo or the endometrium may modulate maternal immunity. The nature of these interactions during the pre-hatching period of pregnancy has not yet been well characterized
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