Study On The Mechanism And Polymorphism Of ADAMTS1 Gene In Mongolian Sheep Twins

Mongolia sheep is a low reproductive capacity due to seasonal oestrus,inadequate nutrition carried by the sheep themselves,the harsh natural environment and inadequate care by the herders.Numerous studies have shown that a disintegrin and metalloproteinase with thrombospondin motifs 1(ADAMTS1)is closely associated with granulosa cell proliferation and development,but the mechanism by which ADAMTS1 regulates granulosa cell proliferation and apoptosis,and the mechanism remains unclear by which it produces the doublet trait in Mongolian sheep.The PI3K/AKT pathway plays an irreplaceable role in follicle development.During ovarian granulosa cell development,it is crucial to investigate whether the PI3K/AKT pathway-mediated regulation of granulosa cell development by ADAMTS1 affects twin birth traits in Mongolian sheep.In this thesis,we propose to investigate the changes in ADAMTS1 expression on ovaries of Mongolian sheep producing twin lambs and Mongolian sheep producing singleton lambs,and the mechanisms of PI3K/AKT-mediated ADAMTS1 regulation on granulosa cells,using Mongolian sheep ovaries and granulosa cells,as follows:1.Twin and single lambing Mongolian sheep were studied by natural estrus and divided into twin estrus group,twin interestrus group,single estrus group and single interestrus group.The relative expression levels of key genes on ADAMTS1 and PI3K/AKT pathway on the ovaries were examined to screen for genes that differed between twin and singleton Mongolian sheep,The relative expression of key genes and proteins of ADAMTS1 and PI3K/AKT pathway on the ovaries was examined.The results showed that the expression of ADAMTS1,PI3 K and AKT were significantly higher in the twin estrus and twin interestrus groups than in the single estrus and single interestrus groups(P<0.05),both in terms of genes and proteins.2.Mongolian ovarian granulosa cells were extracted and divided into control and experimental groups,the control group was untreated and the experimental group was supplemented with PI3 K inhibitor LY294002.cell numbers were measured at 0,24,48 and72 h.The cell status was observed after 48 h and the relative expression of ADAMTS1 and PI3K/AKT key genes and proteins were measured.The results showed that compared with the control group,the granulosa cells in the test group grew slowly,decreased in number and showed abnormal cell morphology with wrinkling and distension;the expression of ADAMTS1,PI3 K and AKT genes and proteins were significantly lower(P<0.05),indicating that the ADAMTS1 and PI3K/AKT signal path regulates the proliferation and development of granulosa cells and affects the performance of Mongolian sheep in producing twin litters.3.PCR amplification and sequencing were performed on whole blood from Mongolian sheep producing twins and singleton litters.The results revealed a base mutation at position210 in exon 5 of ADAMTS1 with no change in amino acid sequence,and the Mongolian sheep double-lambing population was in Hardy-Weinberg equilibrium(P>0.05)with moderate polymorphism in PIC,indicating that the Mongolian sheep samples were randomly mated without human intervention,and that the locus could be stably inherited,and could be this locus was used as a genetic marker for Mongolian sheep.