Establish Melatonin-enriched Goats In Mammary Gland And Study The Inflammation Regulated By Autophagy Of High Level Melatonin Goats

Melatonin(N-acetyl-5-methoxytryptamine,MT)is an important amine hormone mainly secreted by animal pineal gland.It acts on multiple organs,tissues and cells in organisms.It exhibits multiple physiological and pharmacological functions,such as regulating biological rhythm,improving sleep quality,adjusting the immune system,enhancing immunity,reproductive modulation,antioxidation,anti-aging,anti-tumor,free radical detoxification and other effects.Aralkylamine N-acetyltransferase(AANAT)is considered as the rate-limiting enzyme for melatonin biosynthesis in vertebrates.We transfer the AANAT gene into the goat's genome directly using transgenic technology.On the one hand,to produce AANAT overexpression goats that provide the endogenously high level melatonin animal models for study its reproductive and immune functions;On the other hand,to achieve AANAT specific expression in the mammary gland of goats and to produce high level melatonin function milk.In this study,on the one hand,we produced AANAT overexpression Laso-shan dairy goats using somatic cell nuclear transfer transgenic technology.First,the pIRES2-EGFP-AANAT and CAG-AANAT expression vectors were successfully constructed.We collected two 35days fetues of Laso-shan dairy goat and we acquired the fetal fibroblast cell(FFC)lines from Laso-shan dairy goat by the attaching tissue explants cultured method.After electrotransfection FFCs and G418 selected,cell monoclones were obtained based on GFP expression,3 of which were suitable for nuclear transplantation after identification in DNA,mRNA and protein levels.We collected 472 Mll oocytes from 33 goat donors by multiple ovulation and achieved 371 fusion embryos which were divided into two groups:cultured with or without melatonin(10-7 M).The pregnancy rate and lanbing rate were significantly increased in the 10-7 M melatonin supplemented group.Seven cloned goats were survived.PCR-Sequencing and Southern blot analysis demonstrated all of them being AANAT transgenic offspring.QRT-PCR and Western blot analysis revealed that the AANAT expression was significantly elevated in the ovary,uterus and pineal gland in transgenic goats compared to the controls.Detecting the transgenic cloned goats' serum melatonin by radioimmunoassay showed that it significantly rised compared to the controls(388.12±75.18 vs.167.40±31.58 pg/ml?P<0.05).On the other hand,the mammary gland-specific expressive Laso-shan dairy goats were produced by pronuclear embryo microinjection combined with CRISPR/Cas9 gene editing technology.We successfully constructed casein promoter-driven pBC1-AANAT mammary gland-specific expression vectors.169 Laoshan Dairy Goats were used as donors for superovulation.CIDR+ 4 days 7-pin reduced FSH +PMSG+LH injection was used.The total number of collecting embryos was 2278,and the average embryo was 13.48.Transplanted to 433 recipients after pronuclear embryo microinjection of pBC]-AANAT.The pregnancy rate was 19.86%(86/433),and the average lambing rate was 21.02%(91/433).The positive rate of transgenic goats was 15.38%(14/91).The melatonin level in the transgenic goat milk was significantly increased by radioimmunoassay.In addition.using high level melatonin transgenic goat models,we studied the anti-inflammatory capacity about them using the peripheral blood mononuclear cells(PBMCs).First,semi-quantitative analysis revealed that PBMCs can express AANAT,HIOMT,and MTNR1B(MT2).After LPS stimulation of PBMCs isolated and cultured in vitro,PBMCs secreted high levels of melatonin,which can bind to MT2 receptors and then activated the p38 MAPK signaling pathway to promote the cellular autophagy,thus,to suppress the excessive inflammatory response.However,the anti-inflammatory responses were significantly differences in the intact animals.After injection of LPS in vivo,high levels of melatonin in transgenic goats inhibited ERK MAPK signaling pathway and increased the capacity of antioxidant by increasing the expression of antioxidant genes,reduced the autophagy activity by reducing the expression of autophagy-related genes,increased serum inflammatory cytokines,and enhanced the body's inflammatory response.The different responses in the cellular and intact animal levels to the LPS challenge reflected the influences of the microenvironments.Herein we provided a novel melatonin-enriching animal model to study the potential effects of endogenously produced melatonin on individual reproduction,inflammation and other functions.