Identification And Characterization Of Genes And MiRNA Involved In Chicken Follicle Development

Animal breeding practice reveal that sexual precocity traits are inheritable, and it is likely that sexual maturity is controlled by some major genes. Therefore, by using an indigenous precocious chicken breeds, we aim to identify genes underlying early sexual maturity. To identification of these genes will be helpful for improving egg roduction as well as reproduction capability in chicken, and provide reference for the control and treatment of sexual precocity in human. Sexually maturity in animal is a complex trait, which is controlled by polygenes. Studies have been conducted in mammals, but relatively few was reported in birds.In poultry, there are strictly hierarchical properties in the mature process of follicles. Once selected, the follicle will develop by hierarchy, until ovulation, and rarely atresia. To increase the follicular quantity entering preovulatory follicle hierarchy is important for improving egg-laying property. At present, the mechanisms of follicular hierarchy selection and hierarchy maintenance are largely unknown. Studies in mammals have shown that several growth factors are involved in the regulation of follicle development acting in autocrine/ paracrine pathway. MicroRNA (miRNA) is a large class of endogenous non-coding short RNAs that plays important roles in regulating gene expression. The expression of miRNA in mammalian ovaries have been reported, but studies concerning its function and expressions in chicken ovaries are unknown. In this study, genes and miRNAs associated with the chicken ovary follicle development were identified and their function was investigated.PartⅠ: The polymorphism and functional analysis of mutations at -237 and -868 site of chicken FSHR promoter region.Follicle-stimulating hormone (FSH) is one of the important hormones regulating animal breeding activities, it is essential for animal ovarian follicle growth, development, differentiation, maturation and ovulation. The biological function of FSH is mediated by the follicle-stimulating hormone receptor (FSHR). To understand the relationship between FSHR 5′promoter region mutation and laying performance, identify DNA markers associated with breeding, is prerequisite for the poultry molecular breeding.In this study, two chicken breeds (Wenchang and Xinyang), totally 943 individuals were used to analyze the genotype frequency, hapolytypes and dipolytypes and the associations with egg production traits. By using real-time quantitative RT-PCR technology, we analyzed FSHR mRNA expression of the three different genotypes (TTG+G+, TTG+G-,TTG-G) in mutation site, construted four haplotype vector, transfected chicken follicular granulosa cell in vitro, and compared expression efficiency of the haplotype.The results revealed that, 200 bp deletion (G-) at position -868 upstream of the translation initiation codon is a predominant allele in Chinese indigenous chicken breeds, the frequency of which was up to 90%. Single site geneotypes were not significantly associated with laying egg number at 39 w and AFE in Wenchang breed, however, their diplotypes were significantly associated with AFE: TTG+G- with the low AFE(123 d), TTG+G+ with the high AFE(134 d), the difference was significant (P=0.016). In Xinyang chicken, all individuals were genotype TT at -237 site upstream of translation initiation codon. At -868 site, genotypes were significantly associated with egg number at 37 w (P<0.05): allele G- was associated with early AFE, while G+ with high laying-egg number at later period.Real-time quantitative RT-PCR results also showed that chicken FSHR mRNA expression levels of G+G+ was the highest (P<0.05) in the follicles of 4 mm. Luciferase reporter assays, using pGL3 vector constructs, showed that haplotype AG- has significantly higher promoter activity than other three haplotypes (P=0.015). We speculated that G- may affect sexual maturity, which is consistent with the extremely higher frequency in indigenous chicken breeds, whereas G+ affect high laying-egg number, and there were some synergy between the two sites, the mechanism of which needs to be further investigated.PartⅡ: TNRC6A (also known as GW182), a protein involved in RNAi and miRNA pathway, is a component of cytoplasmic compartments referred to as processing bodies (P-bodies or GW bodies) in which mRNAs, Argonaute proteins were found and involved in post-transcriptional regulation. In this study, we analyzed TNRC6A expression pattern at different periods in the developing process of hypothalamus, oviduct, liver and ovarian follicle. Real-time quantitative RT-PCR results indicated that the TNRC6A mRNA expression level was higher in ovary of 17 w single-comb white leghorn hens than 23 w, but the expression in hypothalamus, liver and oviduct were up-regulated. Among these follicles, the highest mRNA level was found in white follicles of 4 mm in diameter and the lowest in yellow follicles of 6-8 mm in diameter. After this stage, it increases following follicle growth, showing a trend towards rising, however, decreased to its lowest level in the F1 follicle of 33-34 mm in diameter. The expression level of TNRC6A mRNA has no significant difference from yellow to F1 follicles.PartⅢ: cDNA-AFLP was a nwe differential display technique with the advantage of high reproducibility, specificity and sensitivity. It was a reliable method for monitoring differential expression gene. Jining Bairi chicken comes from Shandong Province of China and is well-known for early sexual maturity. In the present study, cDNA-AFLP differential display was used to isolate genes differentially expressed in ovary of chicken with low AFE compared with high AFE. In cDNA-AFLP analysis, 403 differentially expressed fragments were identified by using 54 different combinations of EcoRI and MseI primers. After isolation, cloning and sequencing of the re-amplified bands and BLAST in GenBank we found 27 differentially expressed genes.Thirteen clones of them were confirmed by real-time qRT-PCR. The results showed that there were five genes which were significantly different in mRNA level. In which four genes were up-regulated in laying ovary. The CCT6A have a coordinating role with progesterone and ERCC8 protein was modulated by miRNA. ZNF183 zinc finger protein is one of the important transcription factors with zinc finger domain that regulates gene expression. It was found first in Xenopus oocytes and most of the zinc finger protein function are remain unknown. The poly(A) polymeraseⅡplays an important role in oocyte maturation. The Loc418883 was a down-regulated fragments in laying ovary whose function similar to vacuolar protein sorting 36 (yeast).The function of these differentially expressed genes will be studied further.PartⅣ: Solexa was a new high-throughput sequencing technology. The differentially expressed mRNA in the ovaries of 42 d and 23 w chickens were screened by this approach. The ovary tissue of three individuals were pooled and used for Solexa sequencing. The results showed 2088 mRNAs were down-regulated and 1560 mRNAs were up-regulated in laying ovary.PartⅤ: The differentially expressed miRNA in ovary of 42 d and 23 w chicken were screened by using Solexa sequencing method. The ovary tissue of three individuals were pooled and used. By this approach we obtained 72 known chicken miRNA in which 34 miRNAs were up-regulated and 38 miRNAs were down-regulated. Our study is a basis for clarification the relationship between miRNA and follicle development.In conclusion, the two polymorphisms in the promoter region of chicken FSHR gene and their relationship with age at first egg and egg production as well as their effect on gene expression were investigated. The expression profile of tissues and dynamics in a range of follicles was quantitatively characterized. Four up-regulated genes and one dow-regulated gene were identified using cDNA-AFLP approach. By Solexa sequencing, differentially expressed genes or miRNAs were revealed. These data may provide a basis for the identification of critical gene(s) underlying follicle growth in chicken.