| With the development of social economy,people’s demand for rabbit meat is increasing.The supply of rabbit meat can not meet people’s demand.Therefore,it is urgent to improve the reproductive performance of rabbits.The reproductive performance of animals is affected by many factors.In addition to nutrition,the reproductive capacity of rabbits is mainly determined by the reproductive characteristics of the female animals.The development of ovarian follicles greatly affects the reproductive functions of the female animals,such as estrus,ovulation and pregnancy.Granulosa cells(GCS)play a very important role in the development of follicles.The proliferation of GCS and the secretion of progesterone and estradiol affect the development and maturation of follicles,and then affect the reproductive performance of rabbits.Cathepsin S(CTSS)and cathepsin B(CTSB)are members of cysteine cathepsin family.Although CTSS/CTSB plays an important role in animal reproduction,human reproduction,tumor growth and other physiological and pathological processes,the function of CTSS/CTSB in the development of rabbit granulosa cells(GCS)is still unknown.In this study,we isolated and identified rabbit granulosa cells,and used recombinant adenovirus and small interfering RNA technology to overexpress and interfere with CTSS/CTSB gene in rabbit granulosa cells.By detecting the secretion of progesterone and estradiol and the proliferation and apoptosis of granulosa cells,we explored the regulatory mechanism of CTSB/CTSS gene on rabbit granulosa cells.1.Clone the full length of the CDS region of the CTSSICTSB gene by RT-PCR.The length of the CDS region of the CTSS gene is 1020 bp,and the length of the CDS region of the CTSB gene is 1023 bp.Through tissue expression profile analysis,CTSB gene expression is highest in abdominal fat and liver,followed by muscle and liver,and uterine tissue and spleen are the lowest.CTSS has the highest expression in the spleen,followed by the heart and liver,and the uterus is the lowest.2.In order to explore the functional mechanism of CTSS/CTSB gene in rabbit granule cells,we isolated and cultured rabbit granule cells in vitro,and identified the rabbit granule cells by HE staining and FSHR immunofluorescence methods.The cells were all spindle-shaped,Oval or multi-projection,with large and round nuclei,obvious nucleoli,abundant and uniform cytoplasmic granules,and close connections between cells with pseudopods.FSHR fluorescent granule cells accounted for 99%,the separation purity of rabbit granule cells reached 99%,and the separation of rabbit granule cells was successful.3.Construction of CTSS/CTSB gene adenovirus overexpression vector and small interfering RNA screening,p Ad Easy-CTSS and p Ad Easy-CTSB overexpression vectors were obtained by constructing shuttle vector and homologous recombination,transfected,packaged and expanded,The best MOI is 40 for rabbit granule cell gradient inoculation.RT-qPCR results showed that:48 hours after p Ad Easy-CTSS and p Ad Easy-CTSB recombinant adenoviruses infect rabbit granular cells,the CTSS gene was up-regulated by more than 20,000 times;the CTSB gene was up-regulated by more than 200 times.Design three pairs of small interfering RNAs,by screening Si-CTSB-118/Si-CTSS-127 fragments,the interference effect is the most significant,and the interference efficiency reaches 80%.4.In order to explore the function of cathepsin B/S in rabbit granule cells(GCS).Using siRNA interference and adenovirus overexpression systems to overexpress and interfere with CTSB/CTSS genes in granular cells,studies have found that CTSB promotes the proliferation of GCS by increasing the expression of anti-apoptosis(BCL2)and proliferation(PCNA)related genes,and at the same time through Decrease the expression of pro-apoptosis to inhibit the expression of GCS apoptosis(BAX)related genes and reduce the apoptosis of granulosa cells.In addition,we found that CTSB can also promote the secretion of progesterone and estradiol in granulosa cells by regulating the expression of CTP11A1 and CYP19A1.Similar to the CTSB gene,the overexpression of CTSS can significantly promote the secretion of progesterone(P4)and estrogen(E2)by increasing the expression of STAR and CYP19A1(P<0.05).At the same time,the overexpression of CTSS increases the proliferation of GCS by up-regulating the expression of proliferation-related genes(PCNA)and anti-apoptotic genes(BCL2).CTSS activation can significantly reduce cell apoptosis(P<0.05).And CTSS gene knockdown significantly reduced the secretion of P4 and E2 and the proliferation of rabbit GCS,and at the same time increased the apoptosis of rabbit GCS.In summary,this study successfully cloned the CTSB/CTSS gene CDS region,and constructed an adenovirus overexpression system,successfully isolated and identified rabbit granulosa cells.The study found that the CTSS/CTSB gene regulates rabbit GCS hormone secretion,cell proliferation and apoptosis.An important role in death.These results can provide a basis for a better understanding of the molecular mechanism of rabbit reproduction. |
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