| Muskrat(Ondatra zibethicus Linnaeus)is a kind of economic animals for seasonal multiple estrus reproduction.Themorphologyofthe glandinlower abdomenshows obvious seasonal change,that is,the development expands in the breeding period,but shrinks in the non breeding period.The muskrat musksecreted by adult male muskrat in the breeding period contains the same main components and pharmacological effects as natural musk,which has the potential to replace natural musk.Therefore,it is necessary to explore the mechanism of the development and secretion of the scent gland in order to increase the yield of muskrat musk.Programmed cell death(PCD)is a kind of suicide protective measure initiated by cells to maintain homeostasis and regulated by genes.Necroptosis and macroautophagy are the modes of PCD.Necroptosisinduces the cell necrosis-like apoptosis.Macroautophagy refers to the lysosomal mediated process of cell self degradation,in order to maintain the metabolic needs of cell itself and the renewal of some organelles.In this study,the scent glands of muskrat in different months were collected and analyzed by morphological observation,RT-qPCR and Western blotting.The morphological results showed that the scent gland developed rapidly from the end of February to the end of April,the shape of the scent gland was relatively stable from the end of May to the end of July,the epidermis of the gland was shrunk and the volume was significantly reduced at the end of August,and the scent gland continued to shrink and become smaller at the end of September.The expression of related genes of necroptosis pathway and macroautophagy pathway was detected by fluorescence quantitative PCR,and the mRNA expression of these genes in different periods was obtained.The effect of genes on the development or atrophy of muskrat scent gland was studied by Western blotting.There are 36 samples in this experiment.and three technical repeats were set for each sample.Statistical analysis the transcriptional changes of the necroptosisfunction genes MLKL anditsrelated genes rip1,rip3,PGAM5,DRP1,p65,autophagy markers LC3-? and macroautophagy related genes ulkl,FIP200,atg13,pi3kcb,Akt,mTOR_,Beclinl(atg6),JNK,DAPK,LKB1(STK11),AMPK,TSC1,p62,atg9b,atg2aandapoptosisrelated genes BCL2,Bax,caspases 3 genes.Compared with ozsg2 group,the transcription level of MLKL gene in ozsg3,ozsg4,ozsg5,ozsg7,ozsg8,ozsg9 and ozsg10 experimental group was 1.49 times,1.7 times,3.13 times,2.1 times,8.38 times,15.22 times and 13.3 times of ozsg2 group,respectively;The transcription level of LC3-? gene in the experimental group was 1.9 times,2.42 times,2.76 times,2.45 times,4.76 times,5.58 times and 5.04 times of ozsg2 group,respectively;The transcription level of BCL2 gene in experimental group was 1.21 times,1.04 times,0.57 times,0.6 times,0.47 times,0.48 times and 0.47 times of ozsg2 group.respectively.With the increase of months,the expression of mlkl gene and LC3-?gene was up-regulated,and the expression of BC.L2 gene was down regulated,andthere was significant difference in the expression between the rapid development and rapid atrophy(P<0.05).The result of Western blotting was consistent with that of qPCR,protein levels of P65 and MTOR were higher in scent glands in February and April,and lowest in September.These results suggest that BCL2 and other cell survival promoting genes regulate the survival of the scent gland cells and inhibit the death of the scent gland cells,so as to promote the development and function of the scent gland of muskratin the rapid development period of muskrat's scent gland from February to April.Autophagy was induced by autophagy genes such as LC3-?.which could induce andpromote the atrophy of scentgland.MLKL and other cell death promoting genes regulate the cell death of scentgland in muskrat. |
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