miRNA expression profile in chicken ovarian follicles throughout development and miRNA-mediated MMP expression

Accumulating evidence has demonstrated the role of microRNAs (miRs) in the avian ovary. In this study, high-throughput transcriptome analyses were employed to study the differential miR expression profiles in the chicken ovary, aiming to reveal miR-targeting matrix metalloproteinase (MMP) expression during follicular growth, maturation, and atresia. Using tissues of chicken ovarian follicles at key steps of development (slow growing - white, the most recently recruited - small yellow, and preovulatory - F2) and regression (the third postovulatory), 14 small RNA (sRNA) libraries were constructed. The 25 most highly expressed known miRs were identified along with eight significantly differentially expressed (DE) miRs (gga-miR-let-7d, gga-miR-31-3p, gga-miR-138-1-3p, gga-miR-1552-5p, gga-miR-92-3p, gga-miR-31-5p, gga-miR-202-3p, and gga-miR-6648-3p) which were further examined by quantitative real time-PCR (qRT-PCR) in white, yellowish, small yellow, and atretic follicles as well as in the granulosa and theca layer of yellow preovulatory F3-F1 follicles (n = 6 hens). These miRs were mainly associated with four pathways: inhibition of MMPs, axonal guidance signaling, HIF1α signaling, and GP6 signaling. Four predicted target genes (i.e. MMP-16, ADAM10, COL4A2, and COL4A5) were examined by qRT-PCR and negatively correlated with DE miRs. The identified candidate miR:mRNA target pairs include gga-miR-31-5p or gga-miR-92-3p:MMP-16, gga-miR-31-5p or gga-miR-92-3p:ADAM10, let-7d:COL4A2, and gga-miR-138-1-3p:COL4A5 are potentially associated with MMP modulation in the hen ovary, mostly in the granulosa and theca cells of the largest preovulatory follicles. These results provide a novel insight to the role of miRs in follicle development by identifying a miR target network that is putatively engaged in remodeling of the extracellular matrix during ovarian follicle development in chickens.