Spatiotemporal Expression Of VDAC1、VDAC2 And MOSPD1 In Ovarian Prehierarchical Follicles And Association With Egg Production In Hens

The egg production traits, such as age at the first egg, egg production and egg weight, play an important role in high-quality hens breeding. However, the degree of prehierarchical follicles development, the number of dominant follicles that be raised and selected from hens follicles at the same time, are the key factors on determining hens laying ability. As the important members of VDAC protein families that existed in mitochondrial outer membrane extensively, VDAC1 gene and VDAC2 gene are important factor in cell energy conversion and apoptosis. MOSPD1 gene is the number of four major sperm protein family numbers, which located in X chromosome of mouse and coded sperm protein domain and two main C end transmembrane region[1]. MOSPD1 gene plays an important role in some mesenchymal cells and epithelial cells, such as osteoblasts, myoblast and lipoma cells, has a higher expression level in dense cells quantity, and participate in the process of ovarian cancer. But, the study about the expression of three genes in hens follicle development especially the degree of prehierarchical follicles development hasn’t been reported.Therefore, our study is to detect the genes location and expression in prehierarchical follicles by means of Semi-quantitative RT-PCR and in-situ hybridization technologies, then analyse the correlation with hens egg production traits to find new molecular markers.Experiment one, by semi-quantitative RT-PCR and RNA in-situ hybridization, select 150 days Hy-Line Brown laying hens with a regular cycle, we detect MOSPD1、VDAC1 and VDAC2 gene location and expression in prehierarchical follicles. The results of Semi-quantitative RT-PCR test show that, VDAC1 and VDAC2 m RNA keep highly expression in Hy-Line Brown laying hens prehierarchical follicles, hierarchical follicles and Ovarian stroma, and the differences are not significant（P>0.05）. Where in, the expression level of VDAC1 m RNA in 6-7mm and F3 is higher than others, and the expression level in F1 is the lowest; the expression level of VDAC2 m RNA in prehierarchical follicles（diameter less than 8mm） is higher than that in hierarchical follicles（F6, F5, F4, F3, F2 and F1） before ovulation. Among them, the expression level in 4-5mm, 6-7mm and 7-8mm follicles are higher than that in others, and the expression level in F2 and F1 are the lowest. The result of RNA in-situ hybridization showed that VDAC1 and VDAC2 genes located in 150 days Hy-Line Brown hens hierarchical follicular oocytes, granulosa cells and cells, which is consistent with the test results of semi-quantitative RT-PCR. Which, VDAC1 m RNA expression level of the prehierarchical follicular granulosa cells, film cells and oocytes was no significant difference, and the level of VDAC2 m RNA expression in cells of the granular layer and the film was significantly lower than that in oocytes. MOSPD1 m RNA has a significant expression level in 120 tested tissue samples, which has a relative higher significant expression in 1-4mm follicles（1.068705±0.260） and F1（1.013716±0.465, P<0.05） and lower significant expression in F3（0.085935±0.172） and F4（0.069848±0.142）. On expression abundance, MOSPD1 gene expression is under VDAC1 and VDAC2 gene, has dose dependent on regulating in different periods of follicular. Under the condition of this research, due to the limitation of the in situ hybridization detection technology and the lower expression of MOSPD1 gene m RNA in prehierarchical follicular, we haven’t get the positive expression results.By inference, VDAC1、VDAC2 and MOSPD1 gene likely play an important role in 150-day-old Hy-Line Brown hens prehierarchical follicules development, but the specific regulatory mechanisms have yet to be explored.Experiment two, by PCR-SSCP and DNA sequencing, we selected Dagu hens which from jilin tianze, to detect the SNP loci of VDAC1（NC₀06100） gene and VDAC2（NC₀06093） gene, then analysis the assotiation Dagu hens Egg production traits. The polymorphism test results showed that, the genotypes of VDAC1 genes detection by SSCP were AA and AB, in this population we has not detected genotype BB, which the Haploid genotype AA genotype frequency was higher than AB genotype, and that of allele A is higher than the frequency of allele B.By Sequence alignment, we found one mutation site in the sixth exon, which named G15362448 A. By SSCP genotyping, we found that VDAC2 gene was detected two genotypes which named GG and GT, and the haplotype frequency of GG was higher than the GT genotype haplotype, the allele frequency of G was greater than allele T. The sequence alignment results showed one mutation site in 3’ control area which named G14420258 A conversion. The association analysis of laying traits showed that, in the number of eggs and egg weight of 30 weeks, the haplotype AB of VDAC1 gene was significantly higher than the AA genotype（P <0.05）, and the haplotype VDAC2 GT gene Type was significantly higher than GG genotype（P <0.05）. VDAC1 gene AB genotype had the highest egg number of 30weeks（22.20 ± 2.16） and egg weight of 33weeks（56.86 ± 3.40）. The 43-week, 57 week and 66 week egg number of VDAC2 GT gene haplotype was significantly higher than GG genotype（P <0.05）. GT genotype had the highest 30 w, 57 w and 66 w egg number, about 18.83 ± 1.62,78.00 ± 3.93 and 122.80 ± 2.26. Therefore, we can have an inference that, the haplotype AB of VDAC1 gene and GT of VDAC2 gene were predominant genotype. Both of them provide the theory basis for marker-assisted selection of quality advantage traits of laying hens egg production, used as potential molecular markers on laying early selection of hens laying production.