Characterization of bovine uterine fluid extracellular vesicles proteomic profiles at follicular and luteal phases of the oestrous cycle

Abstract

Extracellular vesicles (EV) have been identified in uterine fluid (UF), however the bovine UF-EV profile during different phases of the oestrous cycle has not yet been established. Therefore, we compared the UF-EV, and their protein profile at follicular and luteal phases of the oestrous cycle. UF samples were collected from healthy uteri of six live and six slaughtered cows at follicular or luteal phases. Isolation of EV was performed using tangential flow filtration followed by size exclusion chromatography. EV were characterized by nanoparticle tracking analysis (NTA), fluorescence NTA, zeta potential, and transmission electron microscopy. Mass-spectrometry was used to evaluate EV protein profile from live cows. Particle concentrations (mean ± SD) were higher (P < 0.05) at follicular than at luteal phase in both live (1.01 × 108 ± 1.66 × 107 vs 7.56 × 107 ± 1.80 × 107, respectively) and slaughtered cows (1.17 × 108 ± 2.34 × 107 vs 9.12 × 107 ± 9.77 × 106, respectively). The proportion of fluorescently labelled EV varied significantly between follicular and luteal phases across live (28.9 ± 1.9% vs 19.3 ± 2.8%, respectively) and slaughtered cows (26.5 ± 6.3% vs 27.3 ± 2 .7%, respectively). In total, 41 EV proteins were differentially expressed between the phases. Some of the proteins were involved in reproductive processes, cell adhesion and proliferation, and cellular metabolic processes. The results indicated differences in bovine UF-EV concentration and protein profile at follicular and luteal phases, which would suggest that EV modulate uterine microenvironment across the oestrous cycle. Further research is needed to understand the effect of EV changes throughout the oestrous cycle.

Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle

“…In one of our previous studies, we have identified 2587 proteins in the UF-EVs enriched from cows with natural cycle, where 41 proteins were differentially expressed between follicular and luteal phases of the estrous cycle ( 13 ). When comparing the results of present study of UF-EV proteome acquired from synchronized cows and the previous UF-EV proteome study from naturally cyclic cows, we identified that 51.7% of proteins were overlapping in both datasets.…”

“…Thereafter, 500 μl of sample was added on top of the filter in the column. Immediately after adding the sample, 500 μl fractions were collected from which fraction 6 to 9 contained EVs ( 13 ). Fractions 6 to 9 were pooled and further concentrated up to 500 μl using Amicon Ultra-2 ml centrifugal filters (10 kDa cut-off, Merck Millipore Ltd) by centrifuging at 4 °C at 2000 g. Enriched EV samples were stored at −80 °C in protein low-binding tubes.…”

“…Depending on the cargo content, EVs influence normal physiological and pathological conditions ( 7 ). Recent studies have shown that UF-EVs modulate reproductive events such as endometrial development towards receptivity ( 12 , 13 , 14 ), endometrial-embryo crosstalk during pre-implantation period to support conceptus elongation and survival ( 15 ), regulation of maternal immune system allowing embryo attachment ( 16 ), and facilitation of embryo implantation ( 17 , 18 ). However, the exact type of EVs and EV-cargo composition which support these biological processes are yet to be discovered.…”

Exosomal miRNAs from maternal vaginal discharge as biomarkers for preterm labor: non-invasive liquid biopsy

“…Exosomes have become a focal point in biomedical research due to their crucial influence on various biological functions and their ability to modulate microenvironments 21,22 . Exosomes, classified as membrane-bound nanovesicles with sizes ranging from 30 to 200 nm 23 , are released from diverse cell types and bodily fluids, including blood 24 , saliva 25 , breast milk 26 , urine 27 , tears 28 , and vaginal discharge 29 . These small vesicles transport bioactive compounds like proteins, nucleic acids, and lipids 30,31 .…”

Elusive physiological role of prostatic acid phosphatase (PAP): generation of choline for sperm motility via auto-and paracrine cholinergic signaling

“…ENPP6 exists in two forms: a glycosylphosphatidylinositol (GDI)-anchored enzyme and, after cleavage of the GDI-anchor, a soluble enzyme. However, ENPP6 is predominantly expressed in the brain and kidney (Greiner-Tollersrud, 2014), although it has been detected in bovine uterine fluids (Piibor et al, 2023). Glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) is widely expressed in humans, including in the uterus, and can also generate choline from GPC (Gallazzini et al, 2008;Lang et al, 2008).…”