Deciphering the oviductal extracellular vesicles content across the estrous cycle: implications for the gametes-oviduct interactions and the environment of the potential embryo

Background The success of early reproductive events depends on an appropriate communication between gametes/embryos and the oviduct. Extracellular vesicles (EVs) contained in oviductal secretions have been suggested as new players in mediating this crucial cross-talk by transferring their cargo (proteins, mRNA and small ncRNA) from cell to cell. However, little is known about the oviductal EVs (oEVS) composition and their implications in the reproductive success. The aim of the study was to determine the oEVs content at protein, mRNA and small RNA level and to examine whether the oEVs content is under the hormonal influence of the estrous cycle. Results We identified the presence of oEVs, exosomes and microvesicles, in the bovine oviductal fluid at different stages of the estrous cycle (postovulatory-stage, early luteal phase, late luteal phase and pre-ovulatory stage) and demonstrated that their composition is under hormonal regulation. RNA-sequencing identified 903 differentially expressed transcripts (FDR < 0.001) in oEVs across the estrous cycle. Moreover, small RNA-Seq identified the presence of different types of ncRNAs (miRNAs, rRNA fragments, tRNA fragments, snRNA, snoRNA, and other ncRNAs), which were partially also under hormonal influence. Major differences were found between post-ovulatory and the rest of the stages analyzed for mRNAs. Interesting miRNAs identified in oEVs and showing differential abundance among stages, miR-34c and miR-449a, have been associated with defective cilia in the oviduct and infertility. Furthermore, functional annotation of the differentially abundant mRNAs identified functions related to exosome/vesicles, cilia expression, embryo development and many transcripts encoding ribosomal proteins. Moreover, the analysis of oEVs protein content also revealed changes across the estrous cycle. Mass spectrometry identified 336 clusters of proteins in oEVs, of which 170 were differentially abundant across the estrous cycle (p-value< 0.05, ratio < 0.5 or ratio > 2). Our data revealed proteins related to early embryo development and gamete-oviduct interactions as well as numerous ribosomal proteins. Conclusions Our study provides with the first molecular signature of oEVs across the bovine estrous cycle, revealing marked differences between post- and pre-ovulatory stages. Our findings contribute to a better understanding of the potential role of oEVs as modulators of gamete/embryo-maternal interactions and their implications for the reproductive success. Electronic supplementary material The online version of this article (10.1186/s12864-018-4982-5) contains supplementary material, which is available to authorized users.

Hierarchical cluster analysis showed a clear separation between S3 and S1 (A) and between S4 and S1 (B) for all small ncRNA.  Dendrograms representing results of unsupervised hierarchical clustering (HCL). Rows indicate single ncRNA, while columns represent different stages of the estrous cycle compared.
Hierarchical cluster analysis showed a clear separation between S3 and S1 (A) and between S4 and S1 (B) for rRNA. Figure S6. Comparisons of differential miRNA among stages (cut-off FDR < 0.05).
Dendrograms representing results of unsupervised hierarchical clustering (HCL). Rows indicate single ncRNA, while columns represent different stages of the estrous cycle compared.
Hierarchical cluster analysis showed a clear separation between S3 and S1 (A) and between S4 and S1 (B) for miRNA.
Dendrograms representing results of unsupervised hierarchical clustering (HCL). Rows indicate single ncRNA, while columns represent different stages of the estrous cycle compared.
Hierarchical cluster analysis showed a clear separation between S3 and S1 (A) and between S4 and S1 (B) for tRNA. Figure S8. Comparisons of differential snRNA among stages (cut-off FDR < 0.05).

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Color Key single ncRNA, while columns represent different stages of the estrous cycle compared.
Hierarchical cluster analysis showed a clear separation between S3 and S1 (A) and between S4 and S1 (B) for snRNA. Figure S9. Comparisons of differential other ncRNA among stages (cut-off FDR < 0.05).
Dendrograms representing results of unsupervised hierarchical clustering (HCL). Rows indicate single ncRNA, while columns represent different stages of the estrous cycle compared.
Hierarchical cluster analysis showed a clear separation between S3 and S1 (A) and between S4 and S1 (B) for snRNA.

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Color Key -Color Key -2 2 other ncRNA S3 vs S1 other ncRNA S4 vs S1 Figure S10. Oviductal EVs RNA isolated by the use of miRNeasy kit. RNA isolated with the miRNeasy kit (Qiagen) was analyzed using capillary electrophoresis with the Agilent RNA 6000 Pico chip (A) and the small RNA chip (B) on an Agilent 2100 Bioanalyzer ®. The y-axis of the electropherograms represents fluorescence units (FU) and the x-axis represents the nucleotide length of the RNA (nt). Peaks at 25 nt (Pico) and 4 nt (small RNA) represent the internal standard, respectively. Data shown represents the 20 samples analyzed for small ncRNA experiment. Results from the small RNA assay (for miRNeasy) showed small RNA profiles in the range of less than 20 to 200 nucleotides, that could correspond to miRNAs (» 22 nt), tRNAs (» 60-90 nt), (snoRNA (» 70-90 nt), vault RNA (» 80-110 nt), Y_RNA (84-113 nt), snRNAs (» 63-190 nt), and small ribosomal RNAs (5S and 5.8 S). B A Figure S11.
Oviductal EVs RNA isolated by the use of RNeasy micro kit. RNA was analyzed using capillary electrophoresis with the Agilent RNA 6000 Pico chip on an Agilent 2100 Bioanalyzer ®. The yaxis of the electropherograms represents fluorescence units (FU) and the x-axis represents the nucleotide length of the RNA (nt). Peak at 25 nt represents the internal standard. Data shown represents the 20 samples analyzed for mRNA experiment. Results from Pico chip showed the high integrity of RNA, with prominent 18S and 28S rRNA peaks (represented by spikes around 2000 and 4000 nt).