cDNA Microarray Analysis Reveals that Antioxidant and Immune Genes Are Upregulated During Involution of the Bovine Mammary Gland

Abstract 

We have used cDNA microarray analysis to identify genes that play a role in bovine mammary involution. Involution was induced by termination of milking, and alveolar tissue was collected from 48 nonpregnant Friesian cows in mid lactation sacrificed at 0, 6, 12, 18, 24, 36, 72, and 192h (n=6/group) postmilking. The most highly upregulated genes were those associated with oxidative stress. Quantitative real-time reverse-transcription PCR analysis confirmed that mRNA expression of spermidine/spermine N¹-acetyltransferase was increased by 24h, superoxide dismutase 2 and metallothionein 1A by 36h, and glutathione peroxidase by 72h postmilking. The mRNA expression of the host defense proteins lactoferrin and lingual antimicrobial peptide were increased by 192h postmilking. A dramatic increase in the protein expression of lactoferrin by 192h postmilking was also detected by Western analysis. Decreased mRNA expression of the milk protein genes α_S1-, β-, and κ-casein, and α-lactalbumin were early events in the process of involution occurring within 24 to 36h postmilking, whereas β-lactoglobulin mRNA was decreased by 192h postmilking. Decreases in α-lactalbumin and β-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192h postmilking, respectively, and the cell survival factors β1-integrin and focal adhesion kinase were decreased by 72 and 192h postmilking, respectively. The results demonstrate that in the bovine mammary gland, decreased milk protein gene expression and cell survival signaling are associated with multiple protective responses to oxidative stress that occur before the induction of immune responses and mammary epithelial cell apoptosis during involution.

Key words: dairy cow, mammary involution, apoptosis, cDNA microarray