Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence.
Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.
Department of Animal Breeding and Husbandry, University of Bonn, Bonn, Germany.
Md Mahmodul Hasan
MM
Sohel
Michael
M
Hoelker
Sina Seifi
SS
Noferesti
Dessie
D
Salilew-Wondim
Ernst
E
Tholen
Christian
C
Looft
Franca
F
Rings
Muhammad Jasim
MJ
Uddin
Thomas E
TE
Spencer
Karl
K
Schellander
Dawit
D
Tesfaye
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002450:Cell Communication; D002458:Cell Fractionation; D054885:Cumulus Cells / Q000166:cytology / Q000378:metabolism; D055354:Exosomes / Q000378:metabolism*; D005260:Female; D015571:Follicular Fluid / Q000378:metabolism*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D058977:Molecular Sequence Annotation; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D009866:Oogenesis / Q000235:genetics*; D010078:Oxazines; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013799:Theca Cells / Q000166:cytology / Q000378:metabolism; D014461:Ultracentrifugation
D035683:MicroRNAs; D010078:Oxazines; D012333:RNA, Messenger; C006797:Brilliant Cresyl Blue