| As one of the most important economic traits of poultry,egg laying traits(egg number and egg weight)have significant differences among breeds and strains,which were mainly due to the follicular development processes of chickens,among them,the development of prehierarchal follicles could contribute to screen the dominant follicles,and the direct impact on the laying performance of hens.Therefore,this study used the prehierarchal follicles as the experimental object,explored deeper the influencing factors of prehierarchal follicles development.More recent researchs suggested that,Spalt(Sall)family had been implicated with an important effect on embryonic development and reproductive organ development in human and mouse except for in the regulation of many organs development.But the explicit roles of chicken SALL1(csal1)and SALL3(csal3)in prehierarchical follicle(PF)development remains not so far clear.This study,we used the Hy-Line brown layers as the experimental materials.The spatial and temporal expression pattern of csal1 and csal3 genes in m RNA and protein level in the prehierarchal follicles were detected using RT-q PCR and immunofluorescence.The genetic effects of csal1 and csal3 genes on egg-production-related traits in Chinese Dagu hens were detected by PCR-SSCP.The m RNA expression levels of CCND1,Bcat,St AR,CYP11A1 and FSHR genes in granulosa cells(GCs)by RT-q PCR and proliferation of GCs by Brd U assay were detected after overexpression and si RNA of csal1 and csal3 genes,in order to explore the potential role of csal1 and csal3 genes in the proliferation,differentiation and follicular selection of GCs.The primary reaesrch results are as below:1.Expression of csal1 and csal3 genes in prehierarchical folliclesThe result of qualitative PCR showed the csal1 and csal3 gene m RNA were highly expressed in prehierarchical follicles from different developmental stages(diameter 1-3.9 mm,4-4.9 mm,5-5.9 mm,6-6.9 mm,7-8 mm)which suggest that csal1 and csal3 genes are involved in the growth and development of prehierarchical follicles.The expression level of csal1 and csal3 gene m RNA using RT-q PCR in all developmental stages of prehierarchical follicles showed that,with the proliferationand differentiation of granulosa cells from prehierarchical follicles,the m RNA expression patterns of csal1 gene increased gradually,the highest expression abundance was observed in the prehierarchical follicles with diameter 6-6.9 mm.With the development of prehierarchical follicles,the gradually increased of csal3 m RNA abundance in the examined follicles from 1-3.9 mm up to 7-8 mm in diameters,and the highest expression abundance was determined in the prehierarchical follicles with diameter 7-8 mm.These results preliminarily revealed that csal1 and csal3 genes are involved in the regulation of the growth and development of prehierarchal follicle development at the level of transcription.2.Immunolocalization of CSAL1 and CSAL3 in the chicken ovarian folliclesThe protein of CSAL1 and CSAL3 proteins were found to be predominantly expressed in the GCs and oocytes of the PFs examined as well as in the ovarian stroma and epithelium in various sized prehierarchichal follicles of the adult hen ovary by immunofluorescence,and the two proteins mainly distributed in cytoplasm.It was speculated that both CSAL1 and CSAL3 might participate in the regulation of the development and maturation of prehierarchichal follicles of different sizes by translocation to nuclear.3.Genetic effects of csal1 and csal3 genes on egg production-related traits in Chinese Dagu hensThe SNPs of csal1 and csal3 genes were screened by PCR-SSCP,and the correlation between polymorphisms and laying traits(egg weight and egg yield)from Dagu hens were studied.The results showed that the SNP(729 C>A)in exon 2 of csal1 gene and the SNP(1014 T>A)in exon 2 of csal3 gene were correlated with some laying traits(egg weight and egg yield),and the corresponding percentage of additive genetic variance were relatively higher.In addition,among the four combinations,TATT genotype was significantly correlated with egg laying performance.The results indicated that two SNPs associated with egg laying traits which were screened from csal1 and csal3 genes involved in follicular development could be used as potential molecular markers to provide molecular biological basis for breeding high-yield laying hens.4.The inhibiting effect of csal1 and csal3 genes on prehierarchal follicle developmentIn the granulosa cells of prehierarchal follicular which were successfully transfected with p Yr-adshuttle-4-csal1/csal3 and p Yr-adshuttle-4 blank vector usingthe Lipofectamine 2000 transfection reagent,the expression levels of CCND1,Bcat,St AR,CYP11A1 and FSHR genes were dramatic decline(P<0.05)which were detected using RT-q PCR and the cell proliferation rate of the GCs is dramatically decreased(P<0.05)by Brd U assay.This suggested that the overexpression of csal1 and csal3 genes inhibited GC proliferation,differentiation and follicle selection.In this process,after overexpression of csal1 gene,the expression level of csal3 gene m RNA was significantly increased(P<0.05).Similarly,after overexpression of csal3 gene,the expression level of csal1 gene m RNA was significantly increased(P<0.05).After overexpression of the two genes,the expression level of the above five genes and granulosa cells proliferation were significantly lower than other groups.It is speculated that csal1 gene might act synergistic inhibitory effect with csal3 gene in the growth and development of prehierarchical follicles.5.Further verification of synergistic inhibitory effect of csal1 and csal3 in prehierarchal follicle developmentTo further conform the exact role of csal1 and csal3 genes in the prehierarchical follicle development evaluated by overexpression of the foregoing,si RNA-mediated knockdown of csal1 and csal3 genes were performed by transfection of csal1/3-specific si RNA into the GCs.The results showed that the expression levels of the five genes were upregulated significantly(P<0.05)using RT-q PCR,and the proliferation of granulosa cell was significantly promoted(P<0.05)by Brd U assay.This demonstrated that csal1 and csal3 genes inhibited GC proliferation,differentiation and follicle selection.In this process,the expression level of csal3 gene was significantly inhibited after csal1 si RNA interference(P<0.05),similarly,the expression level of csal1 gene was significantly inhibited after csal3 si RNA interference(P<0.05).After si RNA of the two genes,the expression level of the above five genes and granulosa cells proliferation were significantly higher than other groups.These results further confirmed that csal1 gene served as a suppressor in the growth and development of prehierarchical follicles by synergistical action with csal3 gene.The current data showed that csal1 and csal3 m RNA expressed at different developmental stages of the prehierarchical follicles and their proteins were present in the granulosa cells and oocytes as well as in the ovarian stroma and epithelium.Two single nucleotide polymorphic loci associated with egg laying traits which were screened from csal1 and csal3 genes involved in follicular development could be usedas potential molecular markers to provide molecular biological basis for breeding high-yield laying hens.The experimental results of overexpression and si RNA indicated that csal1 and csal3 gene might play synergistic inhibitory effects in GC proliferation,differentiation and follicle selection during the PF development.The current data provide a basis for further investigation of molecular mechanisms of csal1 and csal3 genes in controlling the PF development and growth of the hen ovary.It also offered a theoretical foundation for a more in-depth research on the genetic mechanism of follicle recruitment,the selection of dominant follicles and establishment of follicle hierarchy.This may also contribute to a foundation for a more in-depth research on detecting the major genes for egg-laying traits and the local breed improvement and heterosis utilization which be provided with very significantly scientific significance and academic value. |
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