Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle

Abstract

Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom 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 signaling and communications. Interestingly, the changes in UF-EV proteome during the bovine estrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics-based mass spectrometry was employed to compare UF-EV proteomes at day 0, 7, and 16 of the estrous cycle to understand the estrous cycle-dependent dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. One hundred fifty-nine 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). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the estrous cycle. Interestingly, comparison of hormone-synchronized EV proteomes to natural cycle UF-EVs indicated shift of signaling. Finally, UF-EVs can be used to improve embryo production in vitro.

Keywords: bovine uterine fluid; embryo culture; extracellular vesicles; proteome.

Graphical abstract

Fig. 1

Experimental design. 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 estrous 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 estrous 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). The figure was created with BioRender.com.

Fig. 2

Characterization of uterine fluid extracellular vesicles. The characteristic cup-shaped structures were identified with transmission electron microscopy from UF-EVs samples at day 0 (A), day 7 (B), and day 16 (C). The particle size profile showed particles in the range of 40 to 375 nm (D). The SD is depicted in the figure with the color gray. Protein enrichment after UF-EVs isolation was seen in exosome-related proteins, for example (E): cluster determinant (CD) 63 and 9, epithelial cell adhesion molecule (EPCAM), heat shock protein (HSP) 90AA1 and A5, integrin subunit alpha 6 (ITAG6), lysosomal-associated membrane protein 2 (LAMP2), and tumor susceptibility gene 101 (TSG101). EV, extracellular vesicle; UF, uterine fluid.

Fig. 3

The UF-EV protein profile dynamics between day 0, day 7, and day 16 of the bovineestrous cycle. The principal component analysis (PCA) showed separation in overall protein enrichment patterns between D0, D7, and D16 of the estrous cycle (A). Heatmap showed dynamic changes of significantly different protein enrichment from D0 to D16 of the estrous cycle (B). Between D0 versus D7, there was one protein enriched and five depleted (C). At D7 versus D16 of the estrous cycle, five proteins were enriched and 24 depleted (D). The highest difference was between D0 and D16, where 49 proteins were enriched and 105 depleted between the timepoints (E). EV, extracellular vesicle; UF, uterine fluid.

Fig. 4

Pathways and protein interactions of uterine fluid extracellular vesicles involved in early embryo development. Differential enrichment of selected gene ontology (GO) pathways of biological processes, molecular function, and cellular component between day 0 (D0) and day 7 (D0) (A), D0 and day 16 (D16) (B), and D7 and D16 (C). The interactions between detected proteins in the cell morphology (blue) and cell cycle (red) pathways (D). Detected protein interactions in the carbohydrate metabolic (blue), glycolysis (red), and pentose phosphate pathway (green) (E). Pathways and their proteins interactions of telomere maintenance (F), cholesterol metabolic pathway (G), antioxidant activity (H), and oocyte meiosis (I).

Fig. 5

The dynamic changes of significantly enriched receptivity-related proteins between day 0 and 16 of theestrous cycle. The changes of protein alkaline phosphatase tissue-nonspecific isoenzyme (ALPL), Annexin A1 (ANXA1), Beta-2-microglobulin (B2M), Olfactomedin 4 (OLFM4), and peptidyl-prolyl cis-trans isomerase A (PPIA) log 2 intensities of different cows on day 0, 7, and 16 of the estrous cycle are represented in different colors.

Fig. 6

Uterine fluid extracellular vesicles concentration andestrous cycle phase influence on embryo development. Embryo culture was supplemented with different concentrations of UF-EVs, where the highest rate of blastocysts was achieved using UF-EVs in the concentration of 10⁸ particles/ml. The survival probability of cleaved embryos to blastocysts in UF-EVs coculture group with 10⁸ particles/ml was significantly higher between control group (p = 0.03) and UF-EVs coculture group with 10⁷ particles/ml (p = 0.04) (A). Embryo cultures were also supplemented with UF-EVs acquired from luteal and follicular phases of the estrous cycle, which increased the blastocyst rates compared to control. However, the probability of embryos surviving to blastocysts were significantly higher only in luteal phase UF-EV coculture group (p = 0.02) than the control group (B). EV, extracellular vesicle; UF, uterine fluid. ∗ p ≤ 0.05.