Bovine Mammary Gene And Protein Expression Profiling During The Onset Of Lactation

Lactogenesis includes two stages. Stage I begins a few weeks before parturition and ischaracterized by mammary differentiation and progressive expression of milk proteins as wellas secretion of pre-colostrum. Stage II is initiated around the time of parturition and extendsfor several days afterwards. This stage is characterized by the closure of tight junctionsbetween alveolar cells and the formation and secretion of colostrum and milk.To better understand the molecular events underlying these changes, genome-wide geneexpression profiling was conducted using digital gene expression (DGE) on bovine mammarytissue at three time points (on approximately day35before parturition (-35d), day7beforeparturition (-7d) and day3after parturition (+3d)). Approximately6.2million (M),5.8million (M) and6.1million (M)21-nt cDNA tags were sequenced in the three cDNA libraries(-35d,-7d and+3d), respectively. After aligning to the reference sequences, the three cDNAlibraries included8,662,8,363and8,359genes, respectively. With a fold change cutoffcriteria of≥2or≤-2and a false discovery rate (FDR) of≤0.001, a total of812genes weresignificantly differentially expressed at-7d compared with-35d (stage I). This differentialexpression accounted for9.7％of transcripts in the-7d cDNA library. There were234genesupregulated and578genes downregulated, accounting for28.8％and71.2％, respectively, ofdifferentially expressed genes. Gene ontology analysis showed that the upregulated geneswere mainly associated with cell cycle, lipid metabolism, and those downregulated weremainly associated with immune response and biological adhesion. A total of1,189genes weresignificantly differentially expressed at+3d compared with-7d (stage II), accounting for14.2％of the transcripts in the+3d cDNA library. Of these genes with significant expressionchanges,274(23.0%) genes were upregulated, and these genes were mainly associated withthe immune response.915(77.0%) genes were downregulated, and these genes were mainlyassociated with the cell cycle. Moreover, there were1672genes significantly differentiallyexpressed at+3d compared with-35d, with209(12.5%) genes upregulated and1463(87.5%)genes downregulated. And the significantly enriched biological processes in these differentially expressed genes were metabolic processes. The results suggest that themammary gland begins to lactate not only by a gain of function but also by a broadsuppression of function to effectively push most of the cell’s resources towards lactation.In addition, the protein expression profiling was studied using isobaric tags for relativeand absolute quantitation (iTRAQ) on bovine mammary tissue at three time points (-35d,-7d,+3d). After aligning to the reference sequences, a total of2271proteins were identified. Andproteins with relative molecular mass less than100kDa accounted for80%of all identifiedproteins. With a fold change cutoff criteria of>1.2and P-value of <0.05, a total of373proteins were significantly differentially expressed at-7d compared with-35d (stage I). Geneontology analysis showed that the differentially expressed proteins were mainly associatedwith biosynthetic process, cellular component organization or biogenesis, and nucleic acidmetabolic process. A total of273proteins were differentially expressed at+3d compared with-7d, and they were mainly associated with regulation of primary metabolic process, regulationof macromolecule metabolic process and regulation of gene expression.The association analysis of protein and transcription were done. Many differentiallexpressed proteins were also differentially expressed at transcriptional level at-7d comparedwith-35d. The correlation coefficient for them was0.6983. It suggested that the proteinexpression increased with the increasing of transcription. A total of329differentiallyexpressed proteins had no significant difference at transcriptional level. GO enrichmentanalysis showed that these genes were mainly associated with translation, glucose metabolism,and electron transport chain. The correlation coefficient for differentially expressed genesboth at transcriptional level and protein level was0.5509at+3d compared with-7d. A total of223differentially expressed proteins had no significant difference at transcriptional level. GOenrichment analysis showed that these genes were mainly associated with translation andimmune process.In a word, during stage I of lactogenesis, the mammary cells proliferated, some milkproteins and immunoglobulins were upregulated, some enzymes involved in citric acid cyclewere upregulated, genes associated with extral cellular matrix were downregulated, genesassociated with immune process were downregulated, and some enzymes involved inglycolysis were downregulated. During stage II of lactogenesis, genes associated withimmune process were upregulated, some milk proteins were upregulated, and some genesassociated with cell cycle process were downregulated. The association analysis showed thatthe relationship between differentially expressed genes and differentially expressed proteinswas positive correlation. And post-transcriptional and post-translational regulation might have a role in the regulation of these genes, which were associated with translation, glucosemetabolism and electron transfer chain.