Marion Boutinaud

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

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.

Comparison of five different RNA sources to examine the lactating bovine mammary gland transcriptome using RNA-Sequencing.

The objective of this study was to examine five different sources of RNA, namely mammary gland tissue (MGT), milk somatic cells (SC), laser microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured milk mammary epithelial cells (mMEC) to analyze the bovine mammary gland transcriptome using RNA-Sequencing. Our results provide a comparison between different sampling methods (invasive and non-invasive) to define the transcriptome of mammary gland tissue and milk cells. This information will be of value to investigators in choosing the most appropriate sampling method for different research applications to study specific physiological states during lactation. One of the simplest procedures to study the transcriptome associated with milk appears to be the isolation of total RNA directly from SC or MFG released into milk during lactation. Our results indicate that the SC and MFG transcriptome are representative of MGT and LCMEC and can be used as effective and alternative samples to study mammary gland expression without the need to perform a tissue biopsy.

Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows

In most mammals, prolactin (PRL) is essential for maintaining lactation, and yet the short-term suppression of PRL during established lactation by bromocriptine has produced inconsistent effects on milk yield in cows and goats. To assess the effect of the long-term inhibition of PRL release in lactating dairy cows, 5 Holstein cows in early lactation received daily intramuscular injections of 1mg of the PRL-release inhibitor quinagolide for 9 wk. Four control cows received the vehicle (water) only. During the last week of the treatments, one udder half was milked once a day (1×) and the other twice a day (2×). Blood samples were harvested at milking in wk -1, 1, 4, and 8. The daily injections of quinagolide reduced milking-induced PRL release but not the basal PRL concentration. Quinagolide induced a faster decline in milk production, which was about 5.3 kg/d lower in the quinagolide-treated cows during the last 4 wk of treatment. During wk 9, the inhibition of milk production by quinagolide was maintained in the udder half that was milked 2× but not in the half milked 1×. Milk production was significantly correlated with the quantity of PRL released at milking. Quinagolide did not affect the release of oxytocin at milking. Serum concentration of insulin-like growth factor-1 was not affected by treatment or correlated with milk production. Serum concentrations of leptin and the calciotropic hormone stanniocalcin were not affected by the treatment. In conclusion, the chronic administration of the PRL-release inhibitor quinagolide decreases milk production in dairy cows. The effect is likely the result of the reduced release of milking-induced PRL and is modulated at the level of the gland by milking frequency.

Effects of polyunsaturated fatty acids from plant oils and algae on milk fat yield and composition are associated with mammary lipogenic and SREBF1 gene expression

The main aim of the present study was to examine the effects of long-term supplementing diets with saturated or unprotected polyunsaturated fatty acids from two different plant oils rich in either n-3 or n-6 fatty acids (FAs) plus docosahexaenoic acid (DHA)-rich algae on mammary gene expression and milk fat composition in lactating dairy cows. Gene expression was determined from mammary tissue and milk epithelial cells. Eighteen primiparous German Holstein dairy cows in mid-lactation were randomly assigned into three dietary treatments that consist of silage-based diets supplemented with rumen-stable fractionated palm fat (SAT; 3.1% of the basal diet dry matter, DM), or a mixture of linseed oil (2.7% of the basal diet DM) plus DHA-rich algae (LINA; 0.4% of the basal diet DM) or a mixture of sunflower oil (2.7% of the basal diet DM) plus DHA-rich algae (SUNA; 0.4% of the basal diet DM), for a period of 10 weeks. At the end of the experimental period, the cows were slaughtered and mammary tissues were collected to study the gene expression of lipogenic enzymes. During the last week, the milk yield and composition were determined, and milk was collected for FA measurements and the isolation of milk purified mammary epithelial cells (MECs). Supplementation with plant oils and DHA-rich algae resulted in milk fat depression (MFD; yield and percentage). The secretion of de novo FAs in the milk was reduced, whereas the secretion of trans-10,cis-12-CLA and DHA were increased. These changes in FA secretions were associated in mammary tissue with a joint down-regulation of mammary lipogenic enzyme gene expression (stearoyl-CoA desaturase, SCD1; FA synthase, FASN) and expression of the regulatory element binding transcription factor (SREBF1), whereas no effect was observed on lipoprotein lipase (LPL) and glycerol-3-phosphate acyltransferase 1, mitochondrial (GPAM). A positive relationship between mammary SCD1 and SREBF1 mRNA abundances was observed, suggesting a similar regulation for these genes. Such data on mammary gene expression in lactating cows presenting MFD contribute to strengthen the molecular mechanisms that govern milk fat synthesis in the mammary glands. In purified MEC, the dietary treatments had no effect on gene expressions. Differences between mammary tissue and milk purified MEC gene expression were attributed to the effect of lipid supplements on the number of milk purified MEC and its RNA quality, which are determinant factors for the analysis of gene expression using milk cells.

Mammary cell activity and turnover in dairy cows treated with the prolactin-release inhibitor quinagolide and milked once daily

To assess the regulation of mammary cell activity, survival, and proliferation by prolactin (PRL), 5 Holstein cows in early lactation received daily i.m. injections of 1mg of quinagolide, a suppressor of PRL release, for 9 wk, whereas 4 control cows received the vehicle (water) only. During the last week of treatment, one udder half was milked once a day (1×) and the other twice a day (2×). Mammary biopsies were harvested 1 wk before and 4 and 8 wk after the start of quinagolide treatment. The quinagolide injections reduced milk yield and resulted in lower levels of κ-casein and α-lactalbumin mRNA in the mammary biopsies at wk 4 compared with the control cows. In the mammary tissue of the quinagolide-treated cows at wk 8 of treatment, cell proliferation (as determined by proliferating cell nuclear antigen labeling) was lower and apoptosis (as determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay) was higher than in the mammary tissue of the control cows. During differential milking, mammary epithelial cells (MEC) were extracted from the milk by centrifugation and purified by immunocytochemical binding to allow variations in the levels of mammary transcripts to be observed. After 9 wk of treatment, levels of α-lactalbumin and κ-casein mRNA were lower in the MEC isolated from milk of the quinagolide-treated cows. This effect was associated with lower PRL receptor mRNA levels and a tendency toward lower viability in the milk-isolated MEC from the 2×-milked glands. The decrease from 2× milking to 1× milking also downregulated α-lactalbumin and κ-casein transcripts in the milk-isolated MEC. Viability was higher for the MEC collected from the 1×-milked udder halves compared with the 2×-milked halves. In conclusion, the reduction in milk yield after chronic administration of the PRL-release inhibitor quinagolide is associated with a reduction in mammary cell activity, survival, and proliferation in lactating dairy cows. Reduced milking frequency was also associated with a decrease in MEC activity.

New developments on the galactopoietic role of prolactin in dairy ruminants.

In most mammals, prolactin (PRL) is essential for maintaining lactation and its suppression strongly inhibits lactation. However, the involvement of PRL in the control of ruminant lactation is less clear because inconsistent effects on milk yield have been observed with short-term suppression of PRL by bromocriptine. By contrast, in vitro studies have provided evidence that PRL helps to maintain the differentiation state and act as a survival factor for mammary epithelial cells. Therefore, a series of experiments were conducted to assess the galactopoietic role of PRL. In a first experiment, daily injections of the PRL inhibitor quinagolide reduced milking-induced PRL release and induced a faster decline in milk production. Milk production was correlated with PRL released at milking. Quinagolide reduced mammary cell activity, survival, and proliferation. During the last week of treatments, differential milking (1× vs 2×) was applied. The inhibition of milk production by quinagolide was maintained in the udder half that was milked 2× but not in the udder half milked 1×, suggesting that the response to PRL is modulated at the gland level. In a second experiment, cows were injected with quinagolide, quinagolide + injection of bovine PRL at milking time, or water. As in the first experiment, quinagolide reduced milk, protein, and lactose yields. Although PRL injections at milking time were not sufficient to restore milk yield, they tended to increase milk protein and lactose yields and increased the viability of milk-purified mammary epithelial cells. Recently, we investigated the use of quinagolide at drying off. Treating late-lactation cows with quinagolide decreased milk production within the first day of treatment and induced faster increases in somatic cells and bovine serum albumin content in mammary secretions after drying off, which indicates an acceleration of mammary gland involution. In conclusion, these data, combined with data from other studies, provide a good body of evidence indicating that PRL is galactopoietic in dairy cows. However, the response to PRL appears to be modulated at the mammary gland level.

Effects of nutrient restriction on mammary cell turnover and mammary gland remodeling in lactating dairy cows

The aim of this study was to investigate the effects of a severe nutrient restriction on mammary tissue morphology and remodeling, mammary epithelial cell (MEC) turnover and activity, and hormonal status in lactating dairy cows. We used 16 Holstein × Normande crossbred dairy cows, divided into 2 groups submitted to different feeding levels (basal and restricted) from 2 wk before calving to wk 11 postpartum. Restricted-diet cows had lower 11-wk average daily milk yield from calving to slaughter than did basal-diet cows (20.5 vs. 33.5 kg/d). Feed restriction decreased milk fat, protein, and lactose yields. Restriction also led to lower plasma insulin-like growth factor 1 and higher growth hormone concentrations. Restricted-diet cows had lighter mammary glands than did basal-diet cows. The total amount of DNA in the mammary gland and the size of the mammary acini were smaller in the restricted-diet group. Feed restriction had no significant effect on MEC proliferation at the time of slaughter but led to a higher level of apoptosis in the mammary gland. Gelatin zymography highlighted remodeling of the mammary extracellular matrix in restricted-diet cows. Udders from restricted-diet cows showed lower transcript expression of α-lactalbumin and kappa-casein. In conclusion, nutrient restriction resulted in lower milk yield in lactating dairy cows, partly due to modulation of MEC activity and a lower number of mammary cells. An association was found between feed restriction-induced changes in the growth hormone-insulin-like growth factor-1 axis and mammary epithelial cell dynamics.

Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling

Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

DNA Methylation and Transcription in a Distal Region Upstream from the Bovine AlphaS1 Casein Gene after Once or Twice Daily Milking

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1±0.2 kg/day instead of 16.2±0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

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

Generally, once-daily milking (ODM) decreases milk yield. This effect may be the consequence of a decrease in mammary epithelial cell (MEC) activity or a reduction in their number. The aim of this study was to determine the effect of ODM on the synthetic activity and rate of apoptosis of MEC using a non-invasive method. Eight Alpine goats were subjected to ODM or twice-daily milking for two 5-week periods. MECs were purified by centrifugation and immunocytochemical binding in milk after 1 and 5 weeks of each period. mRNA levels of some proteins involved in lactose and milk protein synthesis and in apoptosis were evaluated using real-time PCR. Isolation of MEC from milk was a useful method to investigate transcriptional regulation in a timeline study. ODM induced greater decreases in milk, lactose and protein yields after 1 week than after 5 weeks. This suggests an adaptation of the mammary gland to ODM, which reduces the inhibitory effect of this practice. Reductions in milk component yields were associated with lower α-lactalbumin transcripts, suggesting a transcriptional decrease of lactose synthesis during ODM. Glucose transporter GLUT1 transcripts were downregulated under ODM, suggesting that lactose precursor uptake by MEC might be involved in the regulation of lactose synthesis. κ-Casein mRNA levels tended to be lower during ODM. ODM increased levels of the pro-apoptotic transcript Bax after both 1 and 5 weeks, but no variation was observed in the Bax/Bcl-2 ratio. ODM affected cell synthetic activity through transcriptional regulation and may have induced apoptosis. The reduction of the negative effect of ODM on milk yield suggests that Alpine goats are able to adapt to ODM. Further studies are needed to investigate the effect of ODM on MEC turnover.