Uterine extracellular vesicles as multi-signal messengers during maternal recognition of pregnancy in the mare

Abstract

In contrast to other domestic mammals, the embryo-derived signal(s) leading to maternal recognition of pregnancy (MRP) are still unknow in the mare. We hypothesize that these embryonic signals could be packed into uterine extracellular vesicles (uEVs), acting as multi-signal messengers between the conceptus and the maternal tract, and contributing to MRP. To unveil these signals, the RNA and protein cargos of uEVs isolated from uterine lavages collected from pregnant mares (P; day 10, 11, 12 and 13 after ovulation) and cyclic control mares (C; day 10 and 13 after ovulation) were analyzed. Our results showed a fine-tuned regulation of the uEV cargo (RNAs and proteins), by the day of pregnancy, the estrous cycle, and even the size of the embryo. A particular RNA pattern was identified with specific increase on P12 related to immune system and hormonal response. Besides, a set of proteins as well as RNAs was highly enriched in EVs on P12 and P13. Differential abundance of miRNAs was also identified in P13-derived uEVs. Their target genes were linked to down- or upregulated genes in the embryo and the endometrium, exposing their potential origin. Our study identified for first time specific molecules packed in uEVs, which were previously associated to MRP in the mare, and thus bringing added value to the current knowledge. Further integrative and functional analyses will help to confirm the role of these molecules in uEVs during MRP in the mare.

Dynamic regulation of the transcriptome and proteome of the equine embryo during maternal recognition of pregnancy

“… 26 In contrast, at protein level, a more constant profile was found without differences regardless embryo size or pregnancy day. This is in line with our results derived from uEVs, 41 where no differences for P19 at mRNA and protein levels were found across pregnancy days. Furthermore, this is also in line with our concurrent study of the endometrium 23 ; where no differences across pregnancy days were observed in the GE, where P19 was highly expressed.…”

“…Furthermore, this is also in line with our concurrent study of the endometrium 23 ; where no differences across pregnancy days were observed in the GE, where P19 was highly expressed. Overall, the low expression of P19 in the equine embryo when compared to uEVs 41 and endometrium, 23 further supports the maternal origin of P19 protein and mRNA, and suggests the transport of P19 to the embryo via EVs.…”

“…The analysis of all identified proteins using the PANTHER classification system and DAVID analysis (Gene Ontology main category cellular component) revealed that 44% and 24% of the proteins, respectively, belonged to the extracellular region (potentially secreted). Furthermore, all proteins and DAP were compared to proteins identified in equine uterine extracellular vesicles (uEVs) found in a parallel study, 41 revealing an overlap of 9% of proteins between embryo and uEVs (Table S6 ).…”

“…Integration of the proteomic and transcriptomic datasets (2200 proteins and 14,061 RNAs identified) showed that 95% of the proteins were also identified at the mRNA level. The remaining 101 proteins which did not overlap with the mRNAs were compared to the uEVs protein cargo dataset, 41 and about 68% of these proteins were found in uEVs. Further comparison between all proteins identified in the embryo and proteins identified in corresponding uEVs showed that almost half of the embryo proteins were also found in uEVs.…”

“…Genetic studies showed an implication of miR‐499a‐5p in recurrent pregnancy loss in woman 78 . Furthermore, miR‐16‐5p has been shown to regulate placental angiogenesis and development by targeting expression of VEGF, 79 suggesting together with its relatively high expression in equine uEVs 41 a regulatory effect on the endometrium. In cultured bovine embryos, elevated miR‐30c‐5p levels were associated with decreased developmental competence 80 .…”