The Dynamic Change Of Adipocytes And Its Mechanism In Dairy Goat Mammary Gland Remodeling

Adipose tissue is an important part of the mammary gland(MG),adipocytes can secrete special adipokines that affect the state of epithelial cells and MG function,have important physiological significance for the healthy development of MG and lactation process.Basing on the level of physiology and tissue origin,the function of the MG in ruminants is more similar for that of the human body,providing an ideal model for studying the development and disease of MG.The essence of the periodic remodeling is the overlapping of involution and regeneration of MG tissue.The MG of ruminants have a special regenerative degeneration phenomenon,that is,the superposition of pregnancy and dry period.The cells turnover rapidly while the MG degenerating,and the alveolar structures are maintained during this period.In addition,the remodeling process is usually completed within 2 months.During MG remodeling,the adipocytes change accordingly,but the changing law and mechanism remain to be elucidated.In this study,the dairy goat was used as the research model,and the changes of MG tissue in remodeling were characterized through histological studies.It would be of great significance to analyze the biological connotation of MG development and ensure MG health.(1)Characterization of changes in MG tissue during MG remodeling.Three dairy goats were selected for in vivo sampling at 5 time points during the MG remodeling(8 weeks before birth,4 weeks before birth,1 week before birth,1 week after birth,and 4 weeks after birth)to obtain dairy goat MG samples;The MG alveolar and adipocytes were characterized by HE(Hematoxylin-Eosin)staining,immunofluorescence,transmission electron microscopy(TEM)experiment,and lipid droplet staining.HE staining showed that during MG remodeling,the alveolar shrank and collapsed first,and the areas of the alveolars were smallest at 4 weeks before parturition(4WBP),then gradually increased after 4WBP.Immunofluorescence detection showed that in 4WBP,the number of adipocytes increased significantly;after 4WBP,the proportion of adipocytes gradually decreased.The results of TEM showed that a certain number of cell types with adipocyte characteristics existed between the alveolars,but the lipid droplet content was less,and other organelle structures were more prosperous.Bodipy staining showed that the inter-alveolar cells were almost unstained,while the mammary epithelial cells that constituted the alveolar showed a lot of fluorescent staining,and the fluorescence was defined as the milk fat component in milk.These results show that: in the process of mammary gland remodeling in dairy goats,the acinus first atrophies and collapses and then regenerates;most of the adipocytes are in an incomplete mature state,and the proportion of cells first increases and then decreases,and the proportion of cells reaches the highest in 4WBP.(2)Study on the mechanism of changes in MG adipocytesAccording to the breeding time and expected date of delivery,4 dairy goats were selected,and MG tissues were collected by biopsy method at 4WBP and 1 week after parturition(1WAP)respectively.The complex digestive enzyme system was used to isolate the adipocytes and then performed transcriptome sequencing to explore the biological process that regulating adipocyte changes in the MG remodeling of dairy goats.Data analysis showed that we obtained high-quality transcriptomic data at 4WBP and1 WAP,with a total of 21,280 genes in MG adipocytes.Compared with 1WAP,4WBP had 2008 differential expressed genes,of which 1095 were significantly up-regulated and913 were down-regulated,the filter condition was | log2(Fold Change)| >1,P<0.05.The results of GO enrichment analysis showed that the up-regulated biological function pathways of 4WBP were mainly related to transcriptional expression and cell division.The results of KEGG enrichment analysis showed that significantly different signaling pathways in adipocytes included cell cycle,MAPK,PI3K-AKT,TNF signaling pathway and so on.It suggested that 4WBP adipocytes underwent extensive self-renewal,including proliferation,apoptosis,and differentiation.MAPK and PI3K-AKT signaling pathways(P<0.01)were activated in 4WBP adipocytes,maintaining the proliferation and differentiation of adipocytes.(3)Effects of changes in adipocytes on MG developmentBased on human telomerase lentiviral system,goat immortalized preadipocyte cell line was screened and obtained;SB203580 and PD169316 were added to specifically inhibit MAPK pathway inhibitors,and MK2206 was added to specifically inhibit PI3 KAKT pathway,to explore the effect on adipocyte differentiation;the medium of early(D0),mid-differentiation(D10),and late-differentiation(D15)adipocytes was collected for culturing goat mammary epithelial cells(GMEC);cell scratching and cell cycle(PI staining)assay were used to detect its effect on the proliferation of GMEC.The results showed that the immortalized goat preadipocyte line had a good ability to differentiate maturely.The results of oil red O staining of adipocytes showed that the differentiation rate of immortalized preadipocytes was significantly reduced after the MAPK(P<0.01)and PI3K-AKT(P<0.01)pathways were inhibited.Compared with the control group,D15 medium after 48 h culturing significantly reduced the proliferation of epithelial cells,which were showed in the scratch area(P<0.01).The cell cycle of mammary epithelial was detected by flow cytometry,and the results showed that the proportion of epithelial cells was higher in G1 phase and lower in S phase.It was suggested that after adipocyte differentiation medium culturing,GMEC was probably blocked in G1 phase,which reduced the proportion of S phase,and finally leads to the inhibition of GMEC proliferation.The above results suggested that MAPK and PI3K-AKT signaling pathway could significantly promote the maturation and differentiation of adipocytes,and mature adipocytes had an inhibitory effect on the proliferation of GMEC.In conclusion,during the MG remodeling,the dairy goat mammary adipocytes showed the change rule of first increased and then decreased,and the adipocytes proportion in 4WBP increased significantly which meant 4WBP was a critical time point for MG remodeling.The changes of adipocytes were regulated by MAPK and PI3K-AKT signaling pathways.The regular changes of adipocytes in MG remodeling had a significant impact on the proliferation of epithelial cells,and the adipocytes could participate in the process of regulating MG remodeling.This study characterized the changes of mammary adipocytes and provided basic data for analyzing the biological mechanism of MG remodeling.