| Goose is a species with important economic value such as meat,down,eggs,liver,etc.Goose industry is a sunrise industry for the development of animal husbandry industry.Down,which is commonly used in people’s daily life,is developed from the skin feather follicles of ducks,geese and other waterfowls.Among them,goose down has good thermal performance and good quality,which is favored by people,but the yield of goose down can not fully meet the needs of people’s life.In recent years,with the rise of down industry,the team of goose breeding industry is also growing.Under the background of people’s increasing demand for down,people gradually began to pursue both quantity and quality.Some studies have found that melatonin can play an important role in regulating the development of skin feather follicles through its receptors.However,as a highly conserved indole hormone secreted by the pineal gland,its mechanism of action is relatively complex and it’s not fully understood.Therefore,the main purpose of this research is to study the effect of melatonin on the development of skin feather follicles in Embryo of Jilin White Goose.On one hand,we detected the m RNA expression of melatonin membrane receptor and nuclear receptor genes in skin feather follicles in Embryo of Jilin White Goose,on the other hand,the protein localization and quantitative study of Mel1a and RORαreceptors in skin feather follicles in Embryo of Jilin White Goose were carried out,therefore,the effect of exogenous addition of melatonin on the dermal fibroblasts of the embryonic back of Jilin White Goose was further explored.The results of the study are as follows:（1）The m RNA expression levels of AANAT,ASMT,Mel1a,Mel1b,Mel1c and RORαgenes in skin feather follicles of Jilin White goose in embryonic stage at different time points（E14,E18 and E28）were detected by q PCR.The results showed that those six genes were continuously expressed throughout the developmental stages of the embryonic back skin feather follicles of Jilin White Goose,and the expression levels had temporal differences.Among them,the expression of Mel1a gene in the back skin feather follicles in embryo of Jilin White Goose showed a gradually decreasing trend at three embryonic ages,and the expression of Mel1a gene at E28was significantly lower than that at E18（P<0.05）,and extremely significantly lower than that at E14（P<0.01）.Furthermore,the expression levels of Mel1b,Mel1c and RORαincreased at first and then decreased.The expression of mel1b and mel1c in E18 was the highest,which was extremely significantly higher than the expression in E14 and E28（P<0.01）.In addition,the expression of RORαin E14 was extremely significantly lower than that in E18（P<0.01）,and significantly lower than those at E28（P<0.05）.However,there was no significant difference in the expression at E18and E28.（2）The distribution of Mel1a and RORαin the skin feather follicles in embryo of Jilin White Goose was detected by immunohistochemistry.The results showed that:the positive expression sites of the two proteins in the skin feather follicles in embryo of Jilin White Goose were almost the same.At E14,the positive expression sites were concentrated in the epidermis（E）and the placode（P）.At E18,the expression of the two proteins continued to be high in the epidermis（E）,and also appeared in the outer root sheath（ORS）,inner root sheath（IRS）and feather crest（FC）.At E28,the positive expression sites began to migrate to the epidermal collar（EC）and dermal papilla（DP）.（3）The protein expression levels of MEL1A and RORαreceptors in the back skin feather follicles in Embryo of Jilin White Goose were detected by Western blot.The results showed that Mel1a and RORαproteins were continuously expressed in the back skin feather follicles in embryo of Jilin White Goose,and the expression levels tended to increase gradually.The expression levels of both receptors were the lowest at E14,which were both extremely significantly lower than those at E18 and E28（P<0.01）.However,the expression levels of both receptors were the highest at E28,which were significantly higher than those at E18（P<0.05）.（4）The dermal fibroblasts of the embryonic back of Jilin White Goose were cultured by exogenous addition of melatonin and its blocking agent,and the MTT method was used to detect that the optimal concentration of exogenous addition of melatonin was 1×10-8 mol/L,and at the 24h and 36h,the cell viability of this group was significantly higher than that of the other test groups（P<0.05）.The best concentration of exogenous addition of melatonin-blocking agent was 1×10-4 mol/L,and at 18h,24h,30h,36h and 42h,the cell activity of the MEL group was the lowest and the difference was significant（P<0.05）.Moreover,the total concentration of melatonin receptor was detected by ELISA,and the results showed that the total concentration of melatonin receptor in MEL group was the lowest,and that in MEL+LUZ group was slightly higher than that in MEL group,and the difference was not significant between the two groups（P>0.05）,but the total concentration of melatonin receptor in MEL group and MEL+LUZ group was extremely significantly lower than those in CON group and LUZ group（P<0.01）.In addition,the total concentration of melatonin receptor in CON group was the highest,which was not significantly different from that in the LUZ group（P>0.05）.Finally,the q PCR technology was used to detect the m RNA expression levels of AANAT,ASMT,Mel1a,Mel1b,Mel1c,RORα,Ki67 and PCNA in the each group of the cells.The results showed that Mel1a,Mel1b,Mel1c,RORα,Ki67 and PCNA expression levels in MEL group were extremely significantly higher than those in other groups（P<0.01）.In addition,the expression of AANAT and ASMT was not detected in dermal fibroblasts of the embryonic back of Jilin White Goose. |
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