| The level of follicular development directly affects litter size in sheep,and BMP/SMAD signaling pathway which involved in the oocyte maturation and granulosa cell proliferation is closely related to the follicular development.In order to study the effects of BMP/SMAD signaling pathway on litter size of Small Tail Han(STH)sheep,quantitative real-time PCR(qRT-PCR)were performed to investigate the expression of related genes(BMP4,BMPR1,BMPR1B,BMPR2,SMAD4,SMAD5 and SMAD6)in 14tissues(heart,liver,spleen,lung,kidney,brain,cerebellum,hypothalamus,ovary,pituitary,uterus,oviduct,thyroid,and adrenal gland)from STH sheep with different physiological periods(Follicular and Luteal phase)and different fecundity(monotocous and polytocous ewes).Seven sheep breeds(Small Tail Han sheep,Suffolk sheep,Tan sheep,Sunite sheep,Prairie Tibetan sheep,Tan sheep,Cele black sheep)were selected and and Sequenom MassARRAY~?SNP assay was applied to genotype single nucleotide polymorphism sites(SNPs)of above genes.Then the association was analyzed between above genes and litter size in Small Tail Han sheep.The main results include:1.Expression profile of genes related to BMP/SMAD signaling pathway in 14 tissues of STH sheep in follicular phaseThe results showed that BMP4,BMPR1A,BMPR2 and SMAD4 genes expressed in all 14 tissues(heart,liver,spleen,lung,kidney,thyroid,adrenal gland,brain,cerebellum,hypothalamus,pituitary,ovary,uterus and oviduct);the BMPR1B gene is mainly expressed in the hypothalamus,ovary,uterus and oviduct tissues;the SMAD5gene is mainly expressed in the uterus and oviduct;the SMAD6 gene is mainly expressed in lung,kidney and ovary tissues.It is indicated that the different transcription levels of the above genes may be related to the breeding activities of STH sheep2.Expression profile of genes related to BMP/SMAD signaling pathway in gonadal axis of STH sheep with different physiological periods and different fecundityThe results in follicular phase showed that the expression of BMP4 and SMAD4genes in hypothalamus,pituitary and ovary of polytocous ewes were extremely significantly higher than that of monotocous ewes(P<0.01);the expression of BMPR1B gene in the hypothalamus of polytocous ewes was extremely significantly higher than that of monotocous ewes(P<0.01);the expression of SMAD5 gene in the pituitary of monotocous ewes was extremely significantly lower than that of the polytocous ewes(P<0.01);the expression of SMAD6 gene in the hypothalamus of polytocous ewes was significantly higher than that of the monotocous ewes(P<0.05).The results in luteal phases showed that the expression of BMPR1B gene in the hypothalamus of polytocous ewes was significantly higher than that of monotocous ewes(P<0.05);the expression of SMAD5 gene in the hypothalamus and ovary of monotocous ewes was significantly lower than that of the polytocous ewes(P<0.05),but the expression in pituitary of the monotocous ewes higher than that of polytocous ewes;the expression of SMAD6 gene in the hypothalamus of monotocous ewes was significantly higher than that of the polytocous ewes(P<0.05).Therefore,the differential expression of the above genes in the gonadal axis may play an important role in the regulation of follicular development and atresia,oocyte maturation and ovulation,and formation of the corpus luteum in STH sheep,which is one of the reasons for the high number of litter size in STH sheep.3.Association analysis between polymorphism of genes related to BMP/SMAD signaling pathway with litter size in STH sheepIt was found that the gene genotype and allele frequencies of g.63454744 T>G locus of BMP4 gene,g.41128335A>T,g.41127600C>T,g.41127598A>G locus of BMPR1A gene,g.29380950G>A,g.29380965A>G,g.29401381C>T locus of BMPR1B gene,g.203707935A>G locus of BMPR2,g.44857543T>C,g.44831116G>A locus of SMAD5 and g.13320337A>C locus of SMAD6 in the monotocous and polytocous groups were significantly different(P<0.05),preliminary indications that these locus are related to the litter size in STH sheep.Correlation analysis between different genotypes and litter size of STH sheep showed that g.63454744T>G locus of BMP4 gene might have significant positive effects on litter size of STH sheep,g.29380965A>G locus of BMPR1B gene might have significant negative effects on litter size of STH sheep,while,a mutation in each additional copy of the and g.203708352A>G locus of BMPR2 gene can reduce the litter size 0.6 of STH sheep.Correlation analysis between the combined genotypes of three locus and FecB of litter size in STH sheep showed that the g.63454744T>G locus mutation may be similar to FecB gene effect,g.29380965A>G and g.203708352A>G locus may attenuate the downstream signal intensity of BMP/SMAD signaling pathway,thereby weakening the effect of FecB gene in the ovary of STH sheep,reducing the number of ovulation and decreasing the litter size,which indicated they may be the key locus that affect the litter size of STH sheep and it can be used for the breeding of litter size trait selection in sheep. |
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