| Egg production trait is one of the important economic traits in chicken and egg laying performance mainly depends on recruitment number,dominant selection,grading establishment and maturation of chicken ovarian follicles.There are many factors that affect follicle development,for example: Follicle Stimulating Hormone(FSH),Luternazing Hormone(LH)and other hormones which are secreted by Hypothalamic Pituitary Gonadal axis(HPG axis),Transforming Growth Factor beta(TGF-?)superfamily,Epidermal Growth Factor(EGF)family and some signaling pathway with its members.Our previous studies have shown that MST1,MST2,SAV1,MOB2,LATS1,LATS2 and YAP1 constitute the core members of the chicken Hippo/MST signaling pathway and that Hippo/MST signaling pathway can inhibit the development of chicken prehierarchical follicles.The results have also revealed the function and molecular mechanism of the members of Hippo/MST signaling pathway in selecting dominant follicles and follicular grading.The aim of this study is to provide a theoretical basis for further study of follicular development and analysis of regulation principle of dominant follicle selection and follicular grading,and to deeply explore the key gene with regulation egg production traits in chicken.It is of great scientific significance and application prospect to lay the molecular basis for genetic improvement and heterosis utilization of local chicken breeds in order to improve egg production traits and feed conversion rate.However,lats2 is homologous protein of lats1,there is no report about the effect and molecular regulation of LATS2 gene,which is one of the core members of Hippo/MST signaling pathway in the ovarian prehierarchical follicle development in chicken.In the current study,150 day old Hyline Brown hens were used as experimental materials and molecular genetic methods,including the relative quantitative RT-PCR,fluorescence quantitative RT-PCR,gene overexpression,siRNA interference,western blotting and EDU assay,were employed to further study,respectively from the perspectives of mRNA level and cell level,the effects of LATS2 gene expression changes on mRNA expression of the upstream genes(MST1,MST2 and SAV1)and the downstream genes(TEAD1,TEAD3 and YAP1)of Hippo/MST signaling pathway,and potential target genes of effector YAP1 moleculars_--prehierarchical follicular granulose cell proliferation marker candidate genes FSHR,STaR,CYP11A1,ESR1 and ESR2.On this basis,their regulatory relationship with LATS2 gene and their biological functions of prehierarchical follicular granulosa cell proliferation were further analyzed.The effect and functional study of chicken LATS2 gene in ovarian prehierarchical follicle development regulation were thus revealed.The results of the current study are as follows:1.The mRNA expression characteristics of LATS2 and Hippo/MST signaling pathway upstream and downstream gene in folliclesRelative quantitative RT-PCR method was used to detect m RNA expressive levels of MST1,MST2,LATS2,YAP1,TEAD1 and TEAD3 genes in ovarian stroma and different diameter follicles of 150-day Hyline brown hens,and then grey level was analyzed.The results showed that mRNA of MST1,MST2,LATS2,YAP1,TEAD1 and TEAD3 genes was expressed in different diameter follicles and that mRNA relative expression quantity of prehierarchal follicles was significantly higher than that of the preovulatory follicles(P?0.05),indicating that MST1,MST2,LATS2,YAP1,TEAD1 and TEAD3 genes were closely related to the development of chicken follicles,but the specific regulatory mechanisms need to be tested in the following experiments.2.Effects of LAST2 gene overexpression on mRNA expression of Hippo/MST signaling pathway upstream genes(MST1,MST2 and SAV1),downstream genes(YAP1,TEAD1 and TEAD3)and biomarker candidate genes(FSHR,STAR,CYP11A1,ESR1 and ESR2)of follicular granulose cell proliferation and its regulation effect on prehierarchical follicular granulose cell proliferationThe methods of vector construction and transfection,relative quantitative RT-PCR,fluorescent quantitative RT-PCR and western blotting and EDU kit were used for detection in this experiment.The results showed that overexpression of LATS2 gene caused significant up-regulation of mRNA expression quantity of MST1,MST,TEAD1 and TEAD3 genes(P?0.05),and significant down-regulation of mRNA expression quantity of SAV1 and YAP1 genes(P?0.05);It laso caused significant down-regulation of mRNA expression quantity of biomarker candidate genes(FSHR,STAR,CYP11A1,ESR1 and ESR2)(P?0.05);Proliferation of granulose cells was significantly decreased(P?0.05).The results indicated that overexpression LATS2 gene could promote mRNA expression of MST1,MST2,TEAD1 and TEAD3 genes and inhibit mRNA expression of SAV1 and YAP1 genes,and inhibit proliferation of prehierarchal follicular granulose cells,indicating that LATS2 gene has a regulatory effect on the development of prehierarchal follicles.In order to verify the reliability of the results,we need to carry next experiment.3.Effects of LAST2 gene siRNA interference on mRNA expression of Hippo/MST signaling pathway upstream genes(MST1,MST2 and SAV1),downstream genes(YAP1,TEAD1 and TEAD3)and biomarker candidate genes(FSHR,STAR,CYP11A1,ESR1 and ESR2)and its regulation effect on prehierarchical follicular granulose cell proliferationFor detection,siRNA-LATS2 interference fragments were transfected into prehierarchal follicles,and the methods of relative quantitative RT-PCR,fluorescent quantitative PCR and western blotting and EDU kit were used in this experiment.The results showed that siRNA interference of LATS2 gene caused significant down-regulation of mRNA expression quantity of MST1,MST2,TEAD1 and TEAD3 genes(P?0.05),significant up-regulation of mRNA expression quantity of SAV1 and YAP1 genes(P?0.05)and significant up-regulation of mRNA expression quantity of biomarker candidate genes(FSHR,STAR,CYP11A1,ESR1 and ESR2)(P?0.05),and proliferation of granulose cells was significantly increased(P?0.05),which are just the opposite of the results of LATS2 overexpression experiment.siRNA interference of LATS2 gene could inhibit mRNA expression of MST1,MST2,TEAD1 and TEAD3 genes,promote mRNA expression of SAV1 and YAP1 genes and promote proliferation of prehierarchal follicular granulose cells.The results of experiments 2 and 3 synthetically indicated that LATS2 gene can promote MST1,MST2,TEAD1 and TEAD3,inhibit SAV1 and YAP1,and inhibit the development of prehierarchical follicular granulose cell in chicken.4.Detection of SNPs of ESR1 and ESR2 genes and analysis of their correlation with egg production traitsIn the second and third experiments,results show the changes of ESR1 and ESR2 mRNA expression affect the proliferation and differentiation of prehierarchical follicular granulose cell in chicken,at the same time,the Dagu hens is an excellent local breed of egg and meat production.This experiment is deeply explore the key gene with regulation of egg production traits in chicken,and lays the foundation for genetic improvement and heterosis utilization of local chicken breeds to improve egg production traits and feed conversion rate.So,PCR-SSCP and DNA sequence analysis technique were used in this experiment to screen SNP loci of ESR1 and ESR2 genes correlated with egg production traits of the Dagu hens.The results showed transformation of T1101 C occurred on exon 4 of ESR1 gene and transformation of G1755 A occurred on exon 8 of ESR2 gene.Egg production of 30,43,57 and 66 –week hens of TT and GG homozygous genotypes of ESR1 and ESR2 genes was significantly higher than that of CT and GA heterozygous genotypes(P?0.05),while egg weight of 30-week hens of CT and GA heterozygous genotypes was significantly higher than that of TT and GG homozygous genotypes(P?0.05).Based on the two SNPs,4 kinds of combined genotypes could be obtained,among which egg production of 30,43,57 and 66 –week hens of TTGG combined genotype was significantly higher than that of TTGA,CTGG and CTGA combined genotypes(P?0.05).Weight of 30 and 43 –week hens of CTGA combined genotype was significantly higher than that of TTGG combined genotype(P?0.05)and egg weight of 30 and 43 –week hens of CTGA combined genotype was significantly higher that of TTGG and TTGA combined genotypes(P?0.05).Greater proportion of additive variance was explained by these two SNPs for most egg production traits were correlated with them(?1%).This study clarified that mRNA of MST1,MST2,LATS2,YAP1,TEAD1 and TEAD3 genes was expressed in ovarian stroma and different diameter follicles,the highest mRNA expression quantity of each gene was found in the prehierarchical follicles and it was significantly higher than that of preovulatory follicles(P?0.05).It revealed that LATS2 has a role in promoting MST1,MST2,TEAD1 and TEAD3 genes and has an inhibitory effect on SAV1 and YAP1,and LATS2 gene can inhibit the development of prehierarchical follicles in chicken.At the same time,the study clarified that SNP sites T1101 C and G1755 A of ESR1 and ESR2 are significantly correlated with laying traits of Dagu chicken(P?0.05).This study revealed the role of LATS2 gene can regulate the development of prehierarchical follicles,and further improve the regulatory network of follicular development in chicken.It will lay foundation for further research on mechanism of Hippo signaling pathway in regulation of prehierarchical follicles and provide theoretical basis for genetic mechanism of dominant selection and grading establishment of follicles in chicken.It will lay molecular foundation for genetic improvement and heterosis utilization of local chicken breed resources,for the purpose of improving egg production traits. |
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