The Number And Distribution Of Mouse Mammary Stem Cells At Different Developmental And Lactation Stages

The development of mammary gland is driven by mammary stem cells.Therefore,it is very important to regulate the proliferation and differentiation potential of mammary stem cells to improve the performance and mammary gland health of mammals.However,it is based on an understanding of the characteristics and dynamics of mammary stem cells during development and lactation.Up to now,the number and distribution patterns of mammary stem cells at different stages of development and lactation have not been well understood.Mammary tissues of mice at 1 day after birth,3 weeks of age,6 weeks of age,14.5 days of gestation,14.5 days of lactation,and 10 days of dry milk were collected in this experiment.The distribution pattern and number of mammary stem cells in different developmental and lactation stages were studied by whole-mount,hanging bulb culture,fluorescence immunohistochemistry and flow separation.The development of ducts and acinus in different stages of development and lactation was observed by Whole-mount.The results showed that mammary ducts branched slowlyfrom birth to 3 weeks of age.Up to 6 weeks of age,ducts branched extensively,approximately 11 times as many as at 3 weeks of age,and the number of terminal terminal buds(TEB)increased,approximately 2.6 times as much as at 3 weeks of age.The duct growth rate was approximately twice as fast as that of the whole breast.From 3 to 9 weeks of age,duct length increased linearly.Finally,ducts cover the entire fat pad of the breast and small acinus buds appear;During pregnancy,the mammary gland acinus expands massively to form complex duct epithelial trees,which continue to grow throughout lactation.At involuting phase,acinar lumen and ductal lumen atrophy and collapse,and mammary epithelial cells apoptosis.The existence of dipotent stem cells in adult mammary gland was confirmed by hanging bulb culture and immunofluorescence.Immunohistochemistry was used to locate mammary stem cells at different stages of development and lactation.Cytokeratin 8(K8)was used to label luminal cell line cells,keratin 14(K14)was used to label basal lineage cells,and K8 and K14 double labeled mammary dipotent stem cells.The results showed that at the time of birth,the mammary stem cells of newborn mice were located in the mammary bud with enriched epithelial cells and the extension of the surrounding ducts.At 3 weeks,the mammary stem cells were mainly distributed on the lateral side of the luminal epithelial cell layer.At 6 weeks of age and adulthood,a small amount of mammary stem cells were distributed at the junction of luminal cells and myoepithelial cells,and the rest were distributed in the stroma near ducts.During pregnancy and lactation,the mammary stem cells were mainly located on the lateral layer of the acinar cavity.In involuting phase,mammary stem cells were partially located at the junction of atrophy ductal myoepithelial cell layer and luminal epithelial cell layer and lateral to acinar epithelial cell layer,while the rest were distributed in stroma.Mammary epithelial cells are composed of luminal cells in the inner layer and basal cells in the outer layer.In this study,PCR technology was used to analyze the expression of characteristic genes of different cell groups(Lgr5 and K5 were characteristic genes of basal lineage cells,and Elf5,Sox10 and K18 were characteristic genes of lumen lineage cells).The results showed that the expression of Lgr5 was the highest during pregnancy and the lowest at3 weeks.The expression of K5 was the highest at 6 weeks,and the lowest at birth and involuting phase.The expression level of Elf5 was the highest during pregnancy and involuting phase.Sox10 expression was the highest during pregnancy.The expression of K18 was highest at 9 weeks,pregnancy and lactation.It suggests that basal lineage cells are more active during the rapid extension of mammary ducts.During lactation,luminal lineage cells were more active.During pregnancy,the rapid expansion of acinus requires both basal and luminal lineage cells.Flow cytometry was used to classify and screen the cells and observe the changes of the number of different cell groups in different periods.The results showed that the number of stem cells was higher at birth and decreased at 3 weeks.From 6 weeks to gestation,the number of stem cells increased and decreased during lactation and involuting phase.The number of luminal cell populations increased from birth to 6 weeks,decreased briefly at 9weeks of age,increased sharply during gestation and lactation,and decreased at involuting phase.These results suggest that the number and distribution of stem cells and the expression of genes of different mammary epithelial lineages are changed with the development of mammary glands and the different stages of lactation.The number of stem cells increase mainly during puberty and pregnancy,and the number of stem cells is the highest during pregnancy.