Onset of lactation in the bovine mammary gland: gene expression profiling indicates a strong inhibition of gene expression in cell proliferation.

The mammary gland undergoes dramatic functional and metabolic changes during the transition from late pregnancy to lactation. To better understand the molecular events underlying these changes, we analyzed expression profiles of approximately 23,000 gene transcripts in bovine mammary tissue about day 5 before parturition and day 10 after parturition. At the cutoff criteria of the signed fold change >or=2 or

Effect of conjugated linoleic acid on bovine mammary cell growth, apoptosis and stearoyl Co-A desaturase gene expression.

The effects of the primary biologically active conjugated linoleic acid (CLA) isomers (cis-9, trans-11 and trans-10, cis-12; 15-150microM) on growth and survival of the bovine mammary cell-line, Mac-T, were evaluated using cell enumeration and TUNEL assay. Previous studies have shown that high concentrations of CLA induced severe milk fat depression and have had negative effects on milk yield and composition whereas the impact of lower doses has been a modest depression in milk fat percent. In this study, we show that increasing concentrations of both CLA isomers had negative impacts on cell growth, including reduced cell number at concentrations of 35microM and above (P<0.05) and a two-fold increase in induction of apoptosis in the mammary cells. Changes in cell morphology occurred with large vacuole-like structures in the cytoplasm, nuclear shrinkage and changes of nuclear shape to kidney shape. Insulin did not significantly affect apoptosis in CLA-treated cells. In addition, the effect of increased doses of CLA and the interaction of CLA and insulin on the bovine stearoyl Co-A desaturase (Scd) gene promoter was also analyzed. While a significant difference in the Scd promoter transcriptional activity was not observed in cells treated with different concentrations of CLA, insulin significantly enhanced Scd promoter activity in CLA-treated cells. Our in vitro data support the hypothesis that high levels of CLA may induce in vivo apoptosis in the mammary gland.

Cloning and expression of bovine glucose transporter GLUT12.

GLUT12 is a new member of facilitative glucose transporters. It was originally cloned from a human breast cancer cell line and its expression has been detected in rat mammary gland. Glucose transport across the plasma membrane of mammary epithelial cells is a rate-limiting factor in milk production. To examine GLUT12's expression and facilitate the study of GLUT12's potential role in supporting milk synthesis in lactating bovine mammary gland, we cloned bovine GLUT12 and examined its distribution of mRNA expression in bovine tissues. The full-length mRNA of bGLUT12 is 2,423 base pairs long and is predicted to encode a protein of 621 amino acids with a molecular weight of approximately 67 kDa. The deduced amino acid sequence of bovine GLUT12 is 87% and 82% identical to the sequences of human and mouse GLUT12. The sequence of bGLUT12 contains several characteristically conserved sugar transporter family signatures. Analysis of current bovine genomic data indicates that bovine GLUT12 gene consists of five exons. The major in vitro transcription and translation product of bovine GLUT12 cDNA migrated at an apparent molecular weight of 41 kDa. In the presence of canine microsomal membranes, the translation product increased to 43 kDa, suggesting glycosylation. GLUT12 mRNA was found in all bovine tissues examined, but most abundant in bovine spleen and skeletal muscle, at intermediate levels in bovine kidney, testes, and mammary gland, and at lower levels in bovine liver, lung and intestine. Immunofluorescence staining showed that, in the presence of insulin, bGLUT12 is mainly distributed in the cytoplasm of the transiently transfected MAC-T bovine mammary epithelial cells.