Abstract
Simple SummaryMilk synthesis is vital for maintaining the normal growth of newborn animals. Abnormal mammary gland development leads to a decrease in female productivity and the overall productivity of animal husbandry. This study characterized the dynamic miRNA expression profile during the process of mammary gland development, and identified a novel miRNA regulating expression of β-casein—an important milk protein. The results are valuable for studying mammary gland development, increasing milk production, improving the survival rate of pups, and promoting the development of animal husbandry.Mammary gland morphology varies considerably between pregnancy and lactation status, e.g., virgin to pregnant and lactation to weaning. Throughout these critical developmental phases, the mammary glands undergo remodeling to accommodate changes in milk production capacity, which is positively correlated with milk protein expression. The purpose of this study was to investigate the microRNA (miRNA) expression profiles in female ICR mice’s mammary glands at the virgin stage (V), day 16 of pregnancy (P16d), day 12 of lactation (L12d), day 1 of forced weaning (FW1d), and day 3 of forced weaning (FW3d), and to identify the miRNAs regulating milk protein gene expression. During the five stages of testing, 852 known miRNAs and 179 novel miRNAs were identified in the mammary glands. Based on their expression patterns, the identified miRNAs were grouped into 12 clusters. The expression pattern of cluster 1 miRNAs was opposite to that of milk protein genes in mammary glands in all five different stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the predicted target genes of cluster 1 miRNAs were related to murine mammary gland development and lactation. Furthermore, fluorescence in situ hybridization (FISH) analysis revealed that the novel-mmu-miR424-5p, which belongs to the cluster 1 miRNAs, was expressed in murine mammary epithelial cells. The dual-luciferase reporter assay revealed that an important milk protein gene—β-casein (CSN2)—was regarded as one of the likely targets for the novel-mmu-miR424-5p. This study analyzed the expression patterns of miRNAs in murine mammary glands throughout five critical developmental stages, and discovered a novel miRNA involved in regulating the expression of CSN2. These findings contribute to an enhanced understanding of the developmental biology of mammary glands, providing guidelines for increasing lactation efficiency and milk quality.
Highlights
The mammary gland is an epidermal appendage that has evolved over 300 million years, and distinguishes mammals from all other animals due to its distinctive anatomical structure [1]
We investigated the expression patterns of three milk protein genes—whey acidic protein (WAP), CSN2, and CSN1S1—by Quantitative Real-Time PCR (qPCR) at all five stages (Figure S1B)
It is worth noting that the murine mammary gland has a similar small RNA expression pattern across the L12d and FW1d stages, which is due to the samples of FW1d having been collected at 24 h of forced weaning, which was performed right after L12d
Summary
The mammary gland is an epidermal appendage that has evolved over 300 million years, and distinguishes mammals from all other animals due to its distinctive anatomical structure [1]. The mammary gland is a complex and unique glandular organ that only develops fully after birth [2]. The rudimentary ductal system that has existed since the embryonic period starts to formally develop and fill the fat pad [3]. During pregnancy, the gland undergoes numerous changes, including ductal proliferation and lobuloalveolar differentiation under the combined action of progesterone and prolactin. Alveoli generated via mammary differentiation secrete milk. The lack of demand for milk at weaning triggers the process of involution, whereby the gland remodels the epithelial tree back to a simple ductal architecture [4]. The mammary gland undergoes obvious remodeling throughout these stages, including the biological processes of cell proliferation, differentiation, and apoptosis. Molecular signaling pathways, and transcription factors have been found to control these processes [5–7]
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