High-Throughput Sequencing Reveals Hypothalamic MicroRNAs as Novel Partners Involved in Timing the Rapid Development of Chicken (Gallus gallus) Gonads

Onset of the rapid gonad growth is a milestone in sexual development that comprises many genes and regulatory factors. The observations in model organisms and mammals including humans have shown a potential link between miRNAs and development timing. To determine whether miRNAs play roles in this process in the chicken (Gallus gallus), the Solexa deep sequencing was performed to analyze the profiles of miRNA expression in the hypothalamus of hens from two different pubertal stages, before onset of the rapid gonad development (BO) and after onset of the rapid gonad development (AO). 374 conserved and 46 novel miRNAs were identified as hypothalamus-expressed miRNAs in the chicken. 144 conserved miRNAs were showed to be differentially expressed (reads > 10, P < 0.05) during the transition from BO to AO. Five differentially expressed miRNAs were validated by realtime quantitative RT-PCR (qRT-PCR) method. 2013 putative genes were predicted as the targets of the 15 most differentially expressed miRNAs (fold-change > 4.0, P < 0.01). Of these genes, 7 putative circadian clock genes, Per2, Bmal1/2, Clock, Cry1/2, and Star were found to be targeted multiple times by the miRNAs. qRT-PCR revealed the basic transcription levels of these clock genes were much higher (P < 0.01) in AO than in BO. Further functional analysis suggested that these 15 miRNAs play important roles in transcriptional regulation and signal transduction pathways. The results provide new insights into miRNAs functions in timing the rapid development of chicken gonads. Considering the characteristics of miRNA functional conservation, the results will contribute to the research on puberty onset in humans.