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The effect of milk accumulation on gene expression in bovine mammary gland

ABSTRACT: We hypothesized that accumulation of milk would influence gene expression in the mammary gland of lactating dairy cows. To test this hypothesis, we enrolled 4 multiparous Holstein cows (150 ± 10 DIM) in a half-udder milk stasis experiment. On day 1 of the experiment, cows were milked at 0430 h and at 1430 h, only right udder halves were milked. Mammary biopsies were obtained from both udder halves on day 2 of the experiment at 0500 h, immediately after right udder halves were milked and 12 h since left udder halves had last been milked. Using Affymetrix GeneChip® Bovine Genome Arrays, we identified 32 genes that were differentially expressed between left (full) and right (empty) udder halves (fold change > 1.5; P < 0.05). Four of the genes were downregulated in response to milk stasis, whereas 28 were upregulated. Differentially expressed genes were associated with extracellular matrix remodeling, tight junction formation, regulation of blood flow, and apoptosis. In addition, four of the differentially expressed genes had been previously identified as candidates for local regulation of milk production in dairy cows. Expression of two of these candidates, early growth response-1 (EGR-1) and thrombospondin-1 (THBS-1), was validated using real-time quantitative RT-PCR. Consistent with microarray results, both genes were upregulated in response to 24-h of milk stasis (P < 0.03). Immunofluorescence was used to localize expression of EGR-1 protein, which was restricted to epithelia and was uniformly distributed. We conclude that accumulation of milk alters gene expression in the bovine mammary gland. In particular, EGR-1 and THBS-1 have emerged as strong candidates for local regulation of milk production in dairy cows.

ORGANISM(S): Bos taurus

PROVIDER: GSE27840 | GEO | 2014/01/01

SECONDARY ACCESSION(S): PRJNA138037

REPOSITORIES: GEO

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Project description:BACKGROUND: The lactating mammary gland responds to changes in milking frequency by modulating milk production. This response is locally regulated and, in dairy cows, the udder is particularly sensitive during early lactation. Relative to cows milked twice-daily throughout lactation, those milked four-times-daily for just the first 3 weeks of lactation produce more milk throughout that lactation. We hypothesized that the milk yield response would be associated with increased mammary cell turnover and changes in gene expression during frequent milking and persisting thereafter. Cows were assigned to unilateral frequent milking (UFM; left udder halves milked twice-daily; right udder halves milked four-times daily) on days 1 to 21 of lactation, followed by twice-daily milking for the remainder of lactation. Relative to udder halves milked twice-daily, those milked four-times produced more milk during UFM; the difference in milk yield declined acutely upon cessation of UFM after day 21, but remained significantly elevated thereafter. We obtained mammary biopsies from both udder halves on days 21, 23, and 40 of lactation. RESULTS: Mammary cell proliferation and apoptosis were not affected by milking frequency. We identified 75 genes that were differentially expressed between paired udder halves on day 21 but exhibited a reversal of differential expression on day 23. Among those genes, we identified four clusters characterized by similar temporal patterns of differential expression. Two clusters (11 genes) were positively correlated with changes in milk yield and were differentially expressed on day 21 of lactation only, indicating involvement in the initial milk yield response. Two other clusters (64 genes) were negatively correlated with changes in milk yield. Twenty-nine of the 75 genes were also differentially expressed on day 40 of lactation. CONCLUSIONS: Changes in milking frequency during early lactation did not alter mammary cell population dynamics, but were associated with coordinated changes in mammary expression of at least 75 genes. Twenty-nine of those genes were differentially expressed 19 days after cessation of treatment, implicating them in the persistent milk yield response. We conclude that we have identified a novel transcriptional signature that may mediate the adaptive response to changes in milking frequency.

Project description:Four-times daily milking during the first 3 weeks of lactation elicits an increase in milk yield, which persists through late lactation even after twice-daily milking is imposed. We hypothesized that this milk yield response would be associated with changes in mammary proliferation, apoptosis, and gene expression, which would persist throughout lactation. Six multiparous cows were assigned to unilateral frequent milking (UFM; twice daily milking of the left udder half (2X), four-times daily milking of the right udder half (4X)) on days 1 to 21 of lactation, followed by 2X thereafter. Udder halves initially milked 4X produced more milk than those milked 2X during, and after UFM treatment, through 180 days in milk (DIM). To determine the mechanisms involved in the persistent milk yield response, we obtained mammary biopsies from both udder halves at 21, 23, and 40 DIM. Rates of [3H]-thymidine incorporation into DNA in vitro and mammary cell apoptosis were not affected by UFM or DIM. Using Affymetrix GeneChipM-BM-. Bovine Genome Arrays, we determined that the differential expression (4X vs. 2X) of 18 genes was significantly affected by DIM. Within the group of 18 differentially expressed genes, we identified a cluster of 15 genes with a similar temporal pattern of differential expression. Nine of the genes in the cluster remained differentially expressed at 40 DIM, indicating that they may be involved in the persistent milk yield response. Among the genes in the cluster were chitinase 3-like (CHI3L)-1, clusterin, early growth response (EGR)-1, and sex determining region Y-box (SOX)-4. These genes have been associated with mammary development, differentiation and remodeling; all of which may be functionally related to the increase in milk yield. We conclude that frequent milking during early lactation does not alter mammary growth but is associated with changes in mammary expression of 18 genes. Future experiments will determine the function of these genes in the mammary gland, and will clarify their role in the autocrine regulation of milk production and long-term alteration of mammary function. 36 samples from 6 cows; 3 timepoints

Project description:Once daily milking reduces milk yield and alters mammary transcriptome resulting in a decrease in milk protein synthesis as well as an induction of the apoptotic signaling networks. A local regulation due to milk stasis in the tissue could contribute to this effect but such mechanisms have not yet been described. To challenge this hypothesis, cows were milked unilaterally, once daily on one udder half and twice daily on the other one, and variations in gene expression were studied in biopsies as well as in mammary epithelial cells (MEC) shed into milk during the lactation process (milk MEC). This study therefore also contributes to decipher if transcript variations in milk purified MEC can reflect that of the mammary tissue. We compared the mammary transcript profiles in biopsies collected from unilaterally once versus twice-daily milked udder halves, 504 transcripts were differentially expressed: 193 and 232 transcripts were up- and down-regulated, respectively. A first category of transcripts, which accumulation levels are mostly up-regulated, relates to mechanisms involved in cell renewal, such as cell cycle, cellular growth and proliferation, cell death and cellular development. A second family, mostly down-regulated, is involved in small molecule biochemistry, amino acid, lipid and carbohydrate metabolisms as well as molecular transport. A third category, mostly up-regulated, includes transcripts expressed in non-epithelial mammary cells such as adipocytes, endothelial and immune cells and cells from the connective tissue. These results are consistent with previous data showing a decrease in mammary synthesis activity and an activation of cell death during once daily milking. They further suggest that during once daily milking the local milk accumulation has a major effect on mammary remodeling. Interestingly, some transcripts belonged to a third family described as M-BM-+ molecular transports M-BM-;. The expression of the 21 mRNA was then analyzed by RT-par in MEC (Table 4). Seven transcripts (NUCB2, RNASE4, ABCG2, RNASE1, SLC34A2, Cap1, FABP3, LALBA, SCD) were significantly down-regulated in milk purified MEC. Six were also significantly down-regulated in mammary biopsies. These transcripts are mostly involved in milk synthesis. We therefore conclude that milk purified MEC cannot be used as general markers of variations occurring in the mammary tissue but that variations in some transcripts listed above can be useful indicators. In this experimental design, udder halves were unilaterally milked once daily and the contralateral udder halves twice daily. Each mammary biopsy RNA sample from one udder half was compared to the one of the contralateral udder half in 5 dye-swaps corresponding to 5 animal replications, minimizing the animal variability. One dye swap corresponded to one comparison meaning two slides.

Project description:Once daily milking reduces milk yield and alters mammary transcriptome resulting in a decrease in milk protein synthesis as well as an induction of the apoptotic signaling networks. A local regulation due to milk stasis in the tissue could contribute to this effect but such mechanisms have not yet been described. To challenge this hypothesis, cows were milked unilaterally, once daily on one udder half and twice daily on the other one, and variations in gene expression were studied in biopsies as well as in mammary epithelial cells (MEC) shed into milk during the lactation process (milk MEC). This study therefore also contributes to decipher if transcript variations in milk purified MEC can reflect that of the mammary tissue. We compared the mammary transcript profiles in biopsies collected from unilaterally once versus twice-daily milked udder halves, 504 transcripts were differentially expressed: 193 and 232 transcripts were up- and down-regulated, respectively. A first category of transcripts, which accumulation levels are mostly up-regulated, relates to mechanisms involved in cell renewal, such as cell cycle, cellular growth and proliferation, cell death and cellular development. A second family, mostly down-regulated, is involved in small molecule biochemistry, amino acid, lipid and carbohydrate metabolisms as well as molecular transport. A third category, mostly up-regulated, includes transcripts expressed in non-epithelial mammary cells such as adipocytes, endothelial and immune cells and cells from the connective tissue. These results are consistent with previous data showing a decrease in mammary synthesis activity and an activation of cell death during once daily milking. They further suggest that during once daily milking the local milk accumulation has a major effect on mammary remodeling. Interestingly, some transcripts belonged to a third family described as « molecular transports ». The expression of the 21 mRNA was then analyzed by RT-par in MEC (Table 4). Seven transcripts (NUCB2, RNASE4, ABCG2, RNASE1, SLC34A2, Cap1, FABP3, LALBA, SCD) were significantly down-regulated in milk purified MEC. Six were also significantly down-regulated in mammary biopsies. These transcripts are mostly involved in milk synthesis. We therefore conclude that milk purified MEC cannot be used as general markers of variations occurring in the mammary tissue but that variations in some transcripts listed above can be useful indicators.

Project description:Bovine mastitis, the infection of the mammary gland which leads to great health and economic challenges for dairy farmers is accompanied by dramatic changes in the milk proteome. In this study of naturally occurring mastitis not only have the changes in the milk proteome been quantified in subclinical and clinical mastitis but simultaneous changes in the serum proteome have also been characterised and quantified. Milk and serum samples from healthy dairy cows (n=12) were compared to those of cows with subclinical (n=10) and clinical mastitis (n=112) using TMT label-based proteomic approach. The study included the milk and serum samples taken from thirty-two dairy cows ( kept on private farms located in Croatia. All cows were checked by physical examination. Somatic cells count (SCC) and mastitis test in milk samples were performed. According to the results, cows were assigned into three groups: Group I (control, n=10) consisted of healthy cows with SCC below 400,000 cells/ml on the monthly check-up and a negative mastitis test and without any clinical sign of mastitis. Group II (subclinical mastitis, n=12) comprised cows without clinical signs of mastitis but with SCC above 400,000 cells/ml on the monthly basis and a positive mastitis test at the time of sampling. Group III (clinical mastitis, n=10) consisted of cows with clinical signs of mastitis which include changes in milk appearance (flakes and clots in milk), different stages of udder inflammation (hyperemia, edema, pain, udder enlargement and elevated udder temperature) and disturbance of general health (depression, relaxed cold ears, dehydration, elevated body temperature, increased heart and respiratory rate, decreased ruminal contraction and decreased appetite). Blood samples were taken from v. coccygea and centrifuged at 3000 g for 15 min after clotting for two hours at room temperature. Serum samples were stored at -80°C until analysis. Milk samples were taken aseptically before the morning milking. First few streams were discarded. Teat ends were disinfected with cotton swabs soaked with 70% ethanol. Samples were taken into sterile tubes and transported to laboratory on ice within a few hours.

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The effect of milk accumulation on gene expression in bovine mammary gland

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