Uterine environment is tightly and finely regulated via various signalling pathways mediated through endocrine, exocrine, autocrine, juxtracrine and paracrine mechanisms. In utero signalling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo and even inter-kingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signalling and communications. Interestingly, the changes in UF-EV proteome during the bovine oestrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered.
Researchers from the Estonian University of Life Sciences employed shotgun quantitative proteomics-based mass-spectrometry to compare UF-EV proteomes at day 0, 7 and 16 of the oestrous cycle to understand the oestrous cycle dependant dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. 159 UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased in vitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (P ≤ 0.05). These findings highlight the importance of bovine UF-EV in uterine communications throughout the oestrous cycle. Interestingly, comparison of hormone synchronized EV-proteomes to natural cycle UF-EVs indicated shift of signalling. Finally, UF-EVs can be used to improve embryo production in vitro.
Six healthy cows were selected for the study, who were subjected to modified Double-Ovsynch for synchronizing ovulation. After which uterine fluid was collected from every cow on day 0 (n = 6), 7 (n = 6) and 16 (n = 6) of the oestrous cycle. From all uterine fluids (n = 18), the extracellular vesicles (EVs) were isolated using size exclusion chromatography-based method. Uterine fluid EVs were characterized with nanoparticle tracking analysis for EVs size profile and concentration (n = 18), transmission electron microscopy for EVs morphology (n = 1 pooled per group), and mass-spectrometry on a sample before and after EVs isolation to evaluate the protein enrichment of EV-related proteins after isolation. Moreover, change in the proteome of uterine fluid EVs during the different timepoints of the bovine oestrous cycle was evaluated using mass-spectrometry (n = 4 per group). The change of Ras homolog family member A (RHOA) protein abundance was validated using Western Blot analysis (n = 4 per group). Lastly, the uterine fluid EVs (n = 3 pooled per group) impact on embryo development was assessed. In the first experiment, influence of different concentrations (109, 108 and 107 particles/mL) of uterine fluid EVs on blastocyst rates was evaluated and compared with a control (no EVs added to the culture). In the second experiment, influence of uterine fluid EVs at luteal or follicular phase to the blastocyst rates was assessed and compared with a control (no EVs added to the culture).