| Although precocious puberty is a pathologic status for humans,it is an economic trait in poultry breeding and production.Chinese indigenous chicken breeds are endowed with unique characters of precocious puberty.To study the regulatory mechanisms that underlie puberty onset in the chicken will contribute to breeding of high-production laying hens and high meat quality broilers.Meanwhile,as an important model organism,the study on precocious puberty in chicken can supply valuable cues for human's relevant researches.Puberty onset is a complicated physiological process that comprises many genes and regulatory factors.Now,it is clear that an increase in pulsatile gonadotrophin releasing hormone(GnRH)release from the hypothalamus is the determined event that causes puberty to occur.Recent studies revealed that the transcriptional suppress of some genes may play important roles in puberty onset,which prevents the puberty from earlier initiating.The observations in model organisms and mammals including humans have shown a potential link between miRNAs and puberty onset.The circulating miRNAs carried by exosomes can travel into the blood circulation and hold stably.The level changes of circulating miRNAs have been shown to be new micro-monitors of physiological and pathologic status in body.This study uses the Wenchang chicken,a famous Chinese indigenous breed for its early maturity characters,as the materials and mainly focuses on the following explored items:(1)To measure the pubertal stages and especially the timing of puberty onset in Wenchang chicken females by measuring the comb size,gonad tissues as well as LH concentrations of serum during different phases.So as to supply a basis for constructing Solexa sequencing libraries that represent before and after puberty onset states respectively.(2)To analyze the hypothalalmic expression changes of crucial genes comprised in c-Myc/LIN28/let-7and POK/PcG/TRG regulating pathways by qRT-PCR methods.(3)To determine whether miRNAs play roles in puberty onset of the chicken by Solexa deep sequencing the profiles of miRNA expression in the hypothalamus of hens from two different pubertal stages,before puberty onset(BPO)and after puberty onset(APO).(4)To set up unique and non-invasive puberty onset circulating miRNA markers by Solexa deep sequencing the profiles of miRNA expression in the serum/plasma of hens from BPO and APO and testing candidate miRNAs by qRT-PCR methods.Based on the results,the conclusions were as following:(1)Measurement of chicken puberty onset The results showed that the ovary and oviduct developed slowly and the follicles kept primitive in early period of development.This period would last about 12 weeks.Then,the gonad tissues developed explosively,the ovary weight and oviduct length of 13 weeks increased 125%and 120%respectively compared to that of 12 weeks,accompanied by emergence of pre-hierarchical follicles.Circulating Luteinizing Hormone(LH)changes were correlated with the rapid growth of gonad tissues,it increased significantly at 13weeks and reached its peak during 14?15 weeks,but decreased to lower levels again at the age of first laying.Based on these,we determined the crucial "timing" for transition from juvenile to puberty onset for Wenchang hens is about 13 weeks.It supplied an essential precondition for constructing Solexa sequencing libraries that represented before and after puberty onset states respectively.(2)Expression patterns of genes involved in transcriptional regulation pathways qRT-PCR test revealed the relative expression levels of c-Myc and LIN28B genes in hypothalamus decreased significantly(P<0.05,P<0.01)at the transition timing of puberty onset in Wenchang chicken,that is 12 to 13 weeks.The lower level of c-Myc could last to the age of first laying,but that of LIN28B recovered to higher level before the age of first laying.Accompanied with these changes were the increases of let-7a?let-7b and let-7g.The relative expression levels of BCL6 and ZBTB7A were lower before puberty onset,and BCL6 level increased significantly(P<0.01)after puberty onset,but the significant changes for ZBTB7A occurred at 15 week,later than BCL6.The relative expression levels of YY1and EED were higher before puberty onset,and decreased significantly(P<0.01)after puberty onset.However,the decrease only kept 2-3 weeks,and then recovered to higher levels from 15 weeks.The relative expression levels of Oct-1 were lower before puberty onset,and increased significantly(P<0.01)after puberty onset.The higher level of Oct-1could last to the age of first laying.But the relative expression levels of TTF1decreased significantly(P<0.01)after puberty onset,which was contrary to previous reports.It suggested the presence of other pathways.Overall,the results supported the hierarchical transcription gene networks,which were suggested as c-Myc(?)?LIN28(?)-| let-7(?)and POK(?)-| PcG(?)-| TRG(?).The results provide new insights into functions of the transcriptional regulators in chicken puberty onset.In the view of evolution,the two regulating pathways might be conserved in vertebrates and mammals.(3)Hypothalamic miRNA profiles before and after puberty onset374 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 BPO to APO.Five differentially expressed miRNAs were validated by real-time quantitative RT-PCR(qRT-PCR)method.2013 putative genes were predicted as the targets of' the 15 most differentially expressed miRNAs(| log2(fold-change)I>2.0,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 APO than in BPO.Furthermore,functional analysis suggested that these 15 miRNAs play important roles in transcriptional regulation and signal transduction during puberty onset.The results provide new insights into miRNAs functions in chicken puberty onset.Considering the characteristics of miRNA functional conservation,the results will contribute to the researches in other animals and humans.(4)Circulating miRNA profiles and unique markers for measurement of puberty onset192 conserved and 19 novel miRNAs were identified as co-expressed miRNAs in the chicken serum and plasma.Among them,4 were serum-special and 1 was plasma-special.With respect to one common miRNA,the relative reads in serum and plasma at one time point were roughly the same.130 conserved miRNAs were showed to be differentially expressed(reads>10,P<0.05)during the transition from BPO to APO,with 68up-regulated and 62 down-regulated.4829 putative genes were predicted as the targets of the 40 most differentially expressed miRNAs(| log2(fold-change)|>1.0,P<0.01).GO enrichment and KEGG analysis suggested these miRNAs are involved in protein hydrolysis,oocyte division,MAPK signaling,basic transcription pathways and so on.qRT-PCR tests showed the level changes of let-7a,miR-29c-3p,miR-217-5p,miR-375,miR-215-5p,miR-155,miR-9-5,miR-19b-3p and miR-133a-3p were coincided with that of Solexa sequencing during puberty onset.Expression levels of the 9 candidate miRNAs were stable in early development periods,however,with respect to the transition timing of puberty onset(from 12 to 13 weeks),the levels of miR-29c-3p(p=0.000),miR-375(p=0.002),miR-215-5p(p=0.013),miR-9-5p(p=0.034),miR-19b-3p(p=0.008),miR-133a-3p(p=0.006)and let-7a(p=0.002)except for miR-217-5p(p=0.112)and miR-155(p=0.174)changed significantly.The results show that these circulating miRNAs can be served as novel biomarkers to measure Wenchang chicken puberty onset.Due to highly conserved nature of miRNAs,the findings from our study will provide cues for other animal's puberty onset measurement as well as human precocious puberty diagnose. |
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