Milking and Feed Restriction Regulate Transcripts of Mammary Epithelial Cells Purified from Milk

Abstract

Feed restriction and once-daily milking (ODM) reduce milk yield in dairy cows and the amount of glucose taken up by the mammary gland. The modulation of mammary glucose uptake may be the consequence of modifications to glucose transport, capacity for lactose synthesis, and cell death in mammary epithelial cells (MEC). The aim was to demonstrate the usefulness of a new method to purify MEC from milk somatic cells and to examine the effects of feed restriction and ODM on mammary transcripts. Five Holstein cows were subjected to a 2 x 2 factorial arrangement of 2 milking frequencies and 2 feeding levels, during which the cows were milked once or twice daily while fed a diet providing either 98 or 70% of requirements. The cows were equipped to study net mammary balance of glucose. On d 7 of each experimental week, milk and lactose yields and mammary glucose uptake were measured. Cells were isolated from fresh milk by centrifugation to generate total milk cell samples. Mammary epithelial cells were separated from total milk cells by using magnetic beads associated with anticytokeratin 8 antibodies. Total RNA was extracted from both total milk cells and purified MEC samples. Real-time reverse transcription PCR was performed to determine mRNA levels in purified MEC under feed restriction and under ODM. Purified MEC samples revealed higher total RNA quality (RNA integrity number = 8) and were better suited to the measurement of mammary transcripts than total milk cell samples (RNA integrity number = 4). Significant correlations were obtained between mRNA levels and net glucose balance data (0.465 < r < 0.680), demonstrating the validity of results obtained by using purified MEC. Feed restriction induced a significant reduction (by half) in type 1 glucose transporter mRNA levels without any effect on alpha-lactalbumin (alpha-LA), galactosyltransferase, kappa-casein, bcl2, or bax mRNA levels. When compared with twice daily milking, ODM reduced kappa-casein (-86%) and alphaLA (-73%) mRNA levels and up-regulated bax and bcl2 mRNA levels (7- and 9-fold). The results suggest that the regulation of glucose uptake and milk yield is dependent on the transcription of glucose transporters under feed restriction and on the transcription of alphaLA under ODM. Further studies are required to con-firm the suggested onset of cell death after 7 d of ODM.

Use of milk epithelial cells to study regulation of cell activity and apoptosis during once-daily milking in goats

“…These techniques are invasive and are not adapted for timeline studies. A new non-invasive method using purified milk MEC developed by Boutinaud et al (2008) was adapted to goats and consisted in the use of milk-purified MEC. The purification of MEC from goat milk permitted the recovery of a sufficient quantity of MEC for extracting total RNA and for carrying out RT-PCR analyses.…”

“…Milk samples were collected at the morning milking after 1 and 5 weeks of each period (Figure 1) for MEC purification as described by Boutinaud et al (2008). Briefly, 1.4 kg of fresh milk was centrifuged at 1500 g for 15 min at 48C.…”

“…PCR amplifications of cDNA samples were carried out using the primers already described for cyclophilin, a-LA, k-CN (k-casein), GT, GLUT1, Bax and cytokeratin-8 (Boutinaud et al, 2008) and for Bcl-2, r18S, caspase-3, b-actin and RPLP0 ).…”

“…The apoptotic state of a tissue can be evaluated by quantifying certain proteins involved in this death process. Previous studies carried out in mammary tissue or MEC used mRNA quantification of the pro-apoptotic proteins caspase-3 and Bax, together with the anti-apoptotic protein Bcl-2, as indicators of cell death (Wareski et al, 2001;Sorensen et al, 2006;Boutinaud et al, 2008).…”

“…The collection of MEC from milk is a non-invasive alternative, which was previously used to study mammary gene expression in cows (Hayashi et al, 2004;Murrieta et al, 2006) and in goats ). An improvement in the method was developed in cows, consisting of the specific purification of MEC from somatic milk cells using magnetic beads (Boutinaud et al, 2008). The use of milk MEC enables repeated sampling without carryover effects.…”