Altered Type Ⅰ Collagen Level In Ovary Of Myostatin Homozygous Mutant Pig

The follicle is the functional unit of ovary,which develops in the ovarian matrix composed of extracellular matrix(ECM)molecules,smooth muscle cells,fibroblasts and other substances.Collagen,as a main component of ovarian extracellular matrix,plays an important role in ovarian growth and follicular development.Myostatin(MSTN),a member of transforming growth factorβ(TGF-β)superfamily,is a highly conserved negative regulator of skeletal muscle.Studies have shown that MSTN gene involves in regulation of collagen accumulation in addition to its regulatory role in development of skeletal muscle fiber.However,the effect of MSTN gene on ovarian collagen content has not been explored in existing literatures.Therefore,the following experiments will be conducted in this study to clarify the changes of collagen content in the ovary of MSNT gene deficient pigs,providing new insights for mammalian ovarian system biology.In experiment 1,the levels of total collagen in ovaries of MSTN gene deficient sows were detected by hydroxyproline,Sirius red staining method was used to detect the levels of total collagen in ovaries of wild-type pigs and MSTN gene deficient pigs of the same age,and the expressions of collagen-related genes and proteins in ovaries were further detected by molecular biological methods.Immunofluorescence staining was performed for COL1A1 protein in ovarian sections of wild-type pigs and MSTN gene deletion pigs to observe the expression of COL1A1 in ovarian cortex and follicles.Cumulus cells of wild-type pigs and MSTN gene deletion pigs were collected to detect the expression of collagen-related genes and protein levels.In experiment 2,small interfering RNAs(siRNA)targeting MSTN genes were constructed.After inhibiting the expression of MSTN with siRNA in cumulus cells,molecular biological techniques such as QRT-PCR and Western blotting were used to detect the expression of collagenrelated genes and proteins.The main results are as follows:1.The total collagen content in ovary of MSTN gene deletion pigs was significantly lower than that of wild-type pigs(200.3±1.5μg/g vs 161.93±3.1μg/g,P<0.05),and the number of large follicles in MSTN-/-groups was significantly decreased,while the number of middle and small follicles was significantly increased.2.The expression levels of collagen-related genes COL1A1 and COL3A1 in ovaries of MSTN deficient pigs were significantly lower than those of wild-type pigs(P<0.01),the expression of collagen-related protein COL1A1 was down-regulated(P<0.01),and there was no difference in COL3A1 protein expression(P>0.05).Compared with wild-type pigs,the immunofluorescence intensity of COL1A1 protein in ovarian cortex and follicle was significantly decreased in MSTN gene deletion pigs.3.The expression levels of collagen-related genes COL1A1 and COL3A1 in cumulus cells of MSTN deficient pigs were significantly lower than those of wild-type pigs(P<0.01),the expression of collagen-related protein COL1A1 was down-regulated(P<0.01),and there was no difference in COL3A1 protein expression(P>0.05).4.The interference sequence of porcine MSTN was designed and tested to validate whether the interference fragment could reduce the expression level of MSTN in porcine cumulus cells.Subsequently,interference of MSTN expression with siRNA significantly reduced COL1A1 gene and protein expression(P<0.01),weakened cell viability and promoted apoptosis,and BAX expression was significantly increased(P<0.05).In conclusion,in the current study,the effect of MSTN on ovarian collagen level was investigated using MSTN deficient pig through various experimental methods including qRT-PCR,Western blotting,and immunofluorescence staining.In the current study,the followings were found.In MSTN deficient pigs,the expression of type I collagen-related genes and proteins in cumulus cells was significantly lower than that in wild-type pigs of the same age.Meanwhile,interfering MSTN expression with siRNA in cultured cumulus cells significantly reduce the expression of type I collagen-related genes and proteins.This study provides a new understanding of the effect of MSTN on ovarian collagen level and gives new insights into mammalian ovarian system biology.