36 Analysis of miRNA content of oviduct and uterine extracellular vesicles across the bovine estrous cycle
Authors: Hamdi M. , Mazzarella R. , Cañon-Beltrán K. , Cajas Y. N. , Leal C. L. V. , Gutiérrez-Adán A. , González M. E. , da Silveira J. C. , Rizos D. 
Published: January 2021

With the aim of investigating the possible hormonal regulatory effect of the oestrous cycle on miRNA content in the extracellular vesicles (EVs) of bovine oviducal and uterine fluids (OF, UF), we performed a bioinformatic analysis of these miRNAs, their target genes, and their biological pathways. Reproductive tracts were collected from slaughtered heifers and selected according to their corpus luteum morphology, corresponding to the 4 stages of the oestrous cycle (n=5 per stage; S1: days 1 to 4, S2: days 5–10, S3: days 11–17, S4: days 18–20) and transported to the laboratory on ice. EVs were obtained by size exclusion chromatography (PURE-EVs-Hansa Biomed) from the flushing of 1.2mL and 2.5mL of OF and UF, respectively. To concentrate the EVs, they were ultracentrifuged and suspended in 100µL of PBS. Total RNA extraction was obtained from 70µL of the previous pellet, using miRNeasy Mini Kit (Qiagen). Then, 100 to 200ng of the obtained RNA was reverse transcribed using miScript II RT Kit (Qiagen). MicroRNA (miRNA) expression profiling was done by primer-based real-time quantitative PCR of 383 mature miRNA sequences. Possible miRNA target genes and their biological pathways were predicted using the miRWalk database. Among EV miRNAs in OF, bta-miR-130a, bta-miR-382, and bta-miR-1291 were the most abundant at all stages of the oestrous cycle, displaying a significantly progressive increase from stages 1 to 4 (P&lt;0.05). In UF, bta-miR-17-5p, bta-miR-206, bta-miR-22-5p, bta-miR-502a, and bta-miR-503-3p were the most abundant at all stages of the cycle, showing greater differences between S1 and S3 (P&lt;0.05). Other miRNAs were exclusively present in a specific stage of the oestrous cycle in OF: bta-miR-21-5p (S1), bta-miR-146a (S2), bta-miR-128 (S3), and bta-miR-147 (S4). In UF, bta-miR-218 (S1), bta-miR-208b (S2), bta-miR-340 (S3), and bta-miR-335 (S4) were found. Table 1 presents some of these miRNAs, their predicted target genes, and functional pathways. In conclusion, this study highlights the effect of the oestrous cycle on miRNAs contained in the EVs of OF and UF. These miRNAs are related to relevant biological pathways implicated in oviduct and uterus modulation across the cycle, but they may also prepare those organs for embryo/conceptus presence and development.

Table 1.
Micro (mi)RNAs of oviductal (OF) and uterine fluid (UF) extracellular vesicles (EVs), their target genes, and biological pathways

Reproductive fluid
miRNAs
Target genes
Target pathways

OF
bta-miR-130a
BMPR2, SMAD5, SMAD4
BMP signalling

bta-miR-1291
SLC2A1
Glucagon signalling

bta-miR-21–5p
LIF
Pluripotency stem cells regulation

UF
bta-miR-17-5p
STAT3
Prolactin signalling

bta-miR-206
ESR1
Oestrogen signalling

bta-miR-340
HRAS
Ras/MAPK/ERK signalling (embryo implantation)

This research was funded by MINECO-Spain AGL2015-70140-R, PID2019-111641RB-I00, RTI2018-093548-B-I00; SENESCYT-Ecuador (YNC); FAPESP-Brazil 2017/20339-3 (CLVL), 2014/22887-0 (JCS), 2019/04981-2 (RM); CNPq-Brazil 304276/2018-9, 420152/2018-0 (CLVL).