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* The University of Arizona, Tucson 85721-0038; and
and
Michigan State University, East Lansing 48824
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: bovine • gene expression • heat stress • mammary
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). When the stress is environmental, the strain is measured as a change in body temperature, metabolic rate, productivity, or heat conservation and/or dissipation mechanisms. At the cellular level, acute environmental change initiates the "heat shock" or cellular stress response. Changes in gene expression associated with a reaction to an environmental stressor involves acute responses at the cellular level (in most if not all cells) as well as changes in gene expression across a variety of organs and tissues associated with the acclimation response.
Early work by Guerriero and Raynes (1990) demonstrated elevated heat-shock proteins in response to thermal stress (TS) in bovine blood leukocytes. Subsequently, Hansen and coworkers evaluated the heat-shock response in the early bovine embryo and demonstrated that moderate heat shock (41° C) causes increased heat-shock protein synthesis, decreased protein synthesis, mitochondrial swelling, and movement of organelles away from the plasma membrane associated with cytoskeletal reorganization (Edwards and Hansen, 1997; Edwards et al., 1997; Rivera and Hansen, 2001; Rivera et al., 2003). However, no one has evaluated global changes in gene expression during the heat-shock response in bovine cells, nor has anyone considered the direct effects of TS on bovine mammary epithelial cells (BMEC). Therefore, it was our objective to profile gene expression during heat shock using primary BMEC cultured in collagen gels.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Collagen was isolated from rat tail tendons by dissolving in 0.017 M acetic acid at 4 g/L. The tendon-acetic acid solution was mildly agitated by stirring for 48 h at 4° C. Simple purification of the collagen solution was obtained by high-speed centrifugation (10,000 x g) at 4° C for 1 h in a fixed-angle rotor (JA-20; Beckman, Coulter Inc., Fullerton, CA) using a Beckman Coulter high-speed centrifuge (J2 HS).
Tissue Dissociation and Isolation of Epithelial Cells
Primary BMEC were isolated from mammary gland tissue samples of a multiparous, pregnant (6 to 7 mo of gestation), nonlactating Holstein dairy cow immediately after slaughter. The udder was removed from the cow at the abbatoir and taken to the laboratory. The hair was removed from the outer skin of the udder and the skin surface was sterilized with betadine solution and 70% ethanol. A scalpel was used to make an incision deep into the parenchyma of the gland, and 100 g of tissue was aseptically removed and placed in storage medium consisting of Medium 199 (M199, 11150-059, Gibco, Grand Island, NY), 100 µg/mL of penicillin/streptomycin (15140-148, Gibco), and 20 mg/mL of gentamycin sulfate (G1264, Sigma, St. Louis, MO). Samples (10 g) were finely minced using standard razor blades and were frequently moistened with storage medium during mincing.
Subsequent tissue dissociation and epithelial cell isolation closely followed the protocol described by McGrath (1987). Each 10-g sample of minced tissue was dissociated in a 500-mL trypsinization flask using 100 mL of dissociation medium consisting of 0.15% collagenase type II (4174; Worthington Biochemical Corp., Lakewood, NJ), 0.075% hyaluronidase (H-3506, Sigma), and 5% fetal bovine serum (16000-044, Gibco), brought to volume with M199, and continuously shaken at 225 rpm and 37° C using a gyratory bench-top shaker. To separate the desired smaller cell aggregates from the larger pieces containing connective tissue, the dissociated tissue was filtered using Nitex mesh filter paper (150-µm pores). Fragments unable to pass the filter were resuspended in fresh dissociation medium and returned to the shaker, followed by a second round of filtration. All filtrates were spun, and cells were resuspended in M199 plus 0.1% DNase I (DN-25, Sigma). All cell isolates were combined, filtered again, and re-suspended in M199 + 10% fetal bovine serum.
The epithelial fraction was then isolated by carefully layering 1 mL of the cell suspension (approximately 7.5 x 107 cells) onto preformed 40% Percoll (17-0891-01, Amersham Biosciences, Piscataway, NJ) gradients (10.8 mL of Percoll:16 mL of M199:1.2 mL of 10x Way-mouth’s medium per tube). Gradients were formed several hours before use by centrifugation at 20,000 x g for 1 h at 20° C, and stored at 4° C until use. After cell suspensions were layered, the gradients were spun at 121 x g for 10 min (brake off). The epithelial cell fraction sedimented to the 1.065 to 1.07 g/mL-density region. Epithelial cells were removed by pipette, diluted 1:1 with M199, pelleted, and resuspended in M199. Cell density was checked to determine the final resuspension volume, and cells were resuspended in freezing solution (M199 + 10% fetal bovine serum + 10% di-methyl sulfoxide). Cells were evenly aliquotted into 1.5-mL cryovials at approximately 1 x 107 cells/mL. After an overnight incubation at –80° C, the cryogenically stored cells were submerged in liquid nitrogen for long-term storage.
Cell Cultures and Sampling
Cells were thawed, transferred to a single 15-mL tube, diluted 1:1 with Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium (F-12, 11039-021, Gibco), spun for 4 to 5 min at 121 x g, and resuspended in 500 µL of DMEM/F-12. The cell suspension was then ready to be added to the neutralized collagen solution. Stock collagen was neutralized using a mixture of 10x Hanks’ balanced salt solution and 0.34 N NaOH (10:9) at a ratio of 2 mL per 8 mL of collagen. Before adding cells, a base layer of 300 µL of collagen per well was added to the 12-well culture plate(s) at room temperature. Resuspended cells (suspended in 500 µL of DMEM/F-12) were added to the remaining neutralized collagen solution at this time, and kept on ice until the base layer gelled (2 to 4 min); 500 µL/well of the collagen-cell suspension was then pipetted slowly over the top of the base layer and allowed to gel for 20 to 30 min at room temperature or in an incubator at 37° C and 5% CO2. The appropriate medium was then added directly to the culture at 500 µL/well.
Experimental Design
For growth, primary BMEC were cultured in serum-free medium consisting of DMEM/F-12, 0.1% BSA, penicillin/streptomycin (15070, Invitrogen Corp., Carlsbad, CA), bovine IGF-I (Monsanto Company, St. Louis, MO, 75 ng/mL), and r-human epidermal growth factor (25 ng/mL, 13247-051, Gibco). Medium was exchanged every 48 h. On d 7 of culture, 1 of the culture plates was placed in an incubator at 42° C (thermal stress). Samples were collected 24 h before, immediately before (0 h), 15 and 30 min, and 1, 2, 4, 8, 16, and 24 h after initiating thermal stress.
For DNA quantification, 4 gels/sample were collected at –24, 0, 8, 16, and 24 h relative to TS initiation. Gels were frozen at –20° C, and DNA was estimated by thawing the samples, dissolving the collagen gels with 0.1 to 1.0% collagenase in a 37° C water bath (15 to 30 min), and proceeding with the PicoGreen Fluorometric DNA Quantification assay as outlined by the manufacturer (Molecular Probes, Inc., Invitrogen Corp.). For RNA, 2 gels/sample were collected at all time points except –24 h. TRIzol reagent (Invitrogen) was immediately added (1.5 mL per gel) after sampling and the gels were allowed to dissolve in the TRIzol at room temperature (about 10 min), after which the samples were stored at –80° C until processing. Total RNA was used for all real-time PCR; heat-shock protein 70 (HSP-70) gene expression was quantified at all time points, whereas all other genes were quantified only at 1, 2, and 4 h of heat shock. Amplified RNA generated from the samples representing the same 3 time points (i.e., 1, 2, and 4 h) was used for the microarray analysis (Figure 1). The RNA manipulation methodologies are described in detail below.
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Total RNA was isolated from cell culture samples using TRIzol reagent (Invitrogen Corp.) following the manufacturer’s protocol with the following modifications. For precipitation, only half of the recommended volume of isopropanol was used, with the other half being replaced with salt solution (0.8 M sodium citrate, 1.2 M NaCl). The concentration, purity, and integrity of the RNA were confirmed on the Agilent 2100 BioAnalyzer (Agilent Technologies Inc., Palo Alto, CA, as well as spectrophotometrically (SmartSpec 3000; BioRad, Hercules, CA).
Approximately 2 µg of total RNA (pooled) was amplified using the MessageAmp II aRNA Kit (1750, Ambion Inc., Austin, TX) and the amplified product evaluated on the BioAnalyzer. Reverse transcription (RT) reactions were performed using the EndoFree RT Kit (1740, Ambion) with a random hexamer primer mix (Integrated DNA Technologies Inc., Coralville, IA) in the presence of 2 mM amino allyl dUTP. Purified cDNA was labeled either green or red with Alexa Fluor 555 or 647 (Molecular Probes), respectively, by incubating in the dark for 1 h at room temperature.
Hybridizations and Scanning
Immediately after labeling, the 2 samples to be hybridized on the same slide were combined and purified using QIAquick purification columns (Qiagen, Valencia, CA). An equal volume of 2x hybridization buffer [8x saline sodium citrate (SSC), 60% formamide, and 0.2% SDS] was added along with 10 g of Cot-1 DNA and 10 g of poly-dA to a final volume of 122 µL. The combined sample was hybridized to a bovine cDNA microarray containing approximately 18,000 sequences developed at Michigan State University as part of the National Bovine Functional Genomics Consortium (Suchyta et al., 2003).
Hybridization was conducted at 47° C, and the slides were subsequently washed with 2 wash solutions (1x SSC, 0.1% SDS; and 0.1x SSC, 0.01% SDS) using a GeneTac Hybridization Station (Genomic Solutions Inc., Ann Arbor, MI). Slides were rinsed in 0.1x SSC, and dried. To minimize photobleaching and enhance slide preservation, DyeSaver2 (Genisphere Inc., Hatfield, PA) was applied to each array, and allowed to dry in the dark for no less than 1 h. Slides were scanned using the arrayWoRxe CCD scanner (Applied Precision Inc., Issaquah, WA), and raw intensity values determined using softWoRx Tracker v2.2 spot finding analysis software (MolecularWare Inc., Irvine, CA).
Data Analysis and Clustering
The microarray data analysis approach we implemented was based on fitting spot intensity values to a linear ANOVA model (Greer et al., 2006). A linear ANOVA model was implemented with partitioned error to accommodate replications in printed spots. All data were transformed using the Linlog variance stabilization method, which utilizes a linear transformation at low intensities, in which additive error is dominant, and a log transformation at high intensities, in which multiplicative error is dominant. Data were then normalized for intensity and array position (column and row) by a lowess regression before fitting to the linear ANOVA model. Genes were clustered across treatments by standard hierarchical clustering using SAS (SAS Inst., Inc., Cary, NC), with Ward’s minimum variance as the metric.
Real-Time PCR
One microgram of total RNA was DNase-treated at room temperature for 15 min in 10-µL reactions containing 0.5 U of DNase I (amplification grade, Invitrogen). Then, EDTA was added to a final concentration of 2.5 mM and the DNase was inactivated at 65° C for 15 min. The resulting RNA was used for cDNA synthesis (20-µL reactions) using iScript cDNA Synthesis Kit (1708891, BioRad). Analysis was conducted using the iCycler IQ Real-Time PCR Detection System (Bi-oRad). Several genes were considered for use as the internal control gene, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (S18), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and ß-actin; after standard curve analyses of all candidates across several samples, HPRT1 was chosen. The resulting gene expression data were calculated using the 2–
CT method (Livak and Schmittgen, 2001). All gene-specific forward and reverse primer sequences are available upon request.
Fixation and Immunofluorescence
For structural analysis by fluorescence microscopy, an entire gel was fixed within the culture plate well by adding 2 mL of 2% paraformaldehyde and allowing it to set for 1.5 h at room temperature. Two washes were then applied using 50 mM glycine for 20 to 25 min per wash at room temp. The fixed gel was submerged in 1 x PBS and stored at 4° C. Fixed samples were labeled according to the published protocol (Larson, 1988). Briefly, samples were permeabilized by incubation in PBS containing 0.1% Triton X-100 for 10 min, then the samples were incubated in PBS containing Texas Red-X phalloidoin (17471, Invitrogen Corp.). The cells were subsequently visualized and captured at 100 x total magnification using an Olympus IX-70 inverted epifluorescence microscope.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Inducible HSP-70 gene expression was dramatically stimulated between 1 and 2 h and appeared to peak within 4 h of TS. This was followed by a down-regulation resulting in transcript levels close to baseline values after 8 h (Figure 1
). Immunofluorescent and confocal microscopy of collagen gel whole mounts stained with phalloidin indicated a dramatic reduction of the prominent, complex networks of ductal structures seen in TN cultures (Figure 3). After 24 h of TS, ductal extensions were no longer visible. Light microscopy evaluation of the cultures suggested that the process of morphological regression began within 8 h of heat shock because the TS cultures were no longer undergoing branching morphogenesis, and an increasing disparity in ductal growth was observed between the TS and TN cultures during this time.
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, samples corresponding to 1, 2, and 4 h from both TS and TN cultures were analyzed. Analysis of the microarray data was conducted using CARMA (Computational Analysis of Replicated Measures for Arrays; Greer et al., 2006). Briefly, data were transformed using the Linlog variance stabilization method and then normalized for intensity and array position by a lowess regression. Data is fitted to a linear ANOVA model, generating a significance value used to determine differential expression. Initially, 1,174 genes were considered differentially expressed using a P-value cutoff of 0.05. This number was narrowed somewhat by removing genes that were both near the P-value cutoff and low in terms of spot intensity. Significance estimates were also obtained for every sample comparison. This permitted subsequent filtering of the gene list to 1) separate out gene-by-time effects, and 2) determine when the expression patterns were significantly changing (1, 2, and/or 4 h). Over 700 genes demonstrated significant changes between TN samples (i.e., due to time) and were subsequently eliminated, ultimately resulting in a list of nearly 340 TS-responsive genes (Figure 5, Tables 1, 2, and 3). A large number of genes were regulated in both the control and TS cultures; however, this is not surprising because these were growing cells. The Venn diagrams in Figure 5a and b display the pattern of up- and downregulation across the 3 time points. Figure 5 also demonstrates that more genes are being downregulated (Table 2) than upregulated (Table 1), as might be expected during a stress response. A subset of genes (n = 31) demonstrated both significant up- and downregulation, depending on the time point (Table 3).
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, Table 1) during the first 1 to 2 h after initiation of stress were associated with stress response, DNA repair, and protein repair. Genes downregulated (Figure 6, Table 2) during this period were associated with the cell cycle, metabolism, and structural proteins. Collectively, these changes in gene expression are consistent with a complete shutdown of cell growth and ductal branching, and upregulation of the cell survival mechanisms. By 8 h after stress initiation, the BMEC were losing thermotolerance as HSP-70 gene expression declined to basal levels and expression of genes associated with apoptosis increased.
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). Fifteen genes were measured, with some analyzed at multiple time points (e.g., 2 and 4 h), leading to 19 gene x treatment comparisons between microarray and reverse transcription-PCR. Seventeen of the 19 (89%) demonstrated a consistent trend in expression pattern between the 2 technologies. These results provided some confirmation of the expression profiles revealed by the microarray hybridizations. |
DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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, these changes include 1) inhibition of DNA synthesis, transcription, RNA processing, and translation; 2) inhibition of progression through the cell cycle; 3) denaturation and misaggregation of proteins; 4) increased degradation of proteins through proteasomal and lysosomal pathways; 5) disruption of cytoskeletal components; 6) alterations to metabolism that lead to a net reduction in cellular ATP; and 7) changes in membrane permeability that lead to an increase in intracellular Na+, H+, and Ca2+ concentrations. In our studies, TS induced the changes in gene expression noted above along with rapid regression of BMEC ductal structures. Transcriptional activity indicated a downregulation of a number of genes associated with branching morphogenesis and microtubule activity, thereby suggesting a repression of the genomic signals responsible for promoting ductal growth and networking. Overall, the transcriptome profile indicated downregulation of genes involved in cell structure, metabolism, biosynthesis, and intracellular transport. Of the upregulated genes, the majority were involved in cellular repair, protein repair, and degradation, and apoptosis after loss of thermotolerance when HSP-70 gene expression fell to basal levels. These data indicate that morphogenic activity in the mammary epithelium might depend upon the expression profile of a core set of genes, and that structural assembly is under a positive mode of regulation (i.e., morphogenesis is "on" by default). In turn, the transition from structural assembly to disassembly might be controlled at the genomic level by simply shutting down cellular biosynthesis and core morphogenic genes. In contrast, transcription of genes encoding repair enzymes and apoptotic proteins is kept off until the cell requires them and removes the inhibition, which suggests a negative mode of regulation. An additional group of genes dominated by patterns of downregulation were those involved in BMEC differentiation and milk synthesis. This suggests that 1) even during growth and morphogenesis, BMEC express detectable mRNA levels of some lactogenic genes; and 2) heat-induced BMEC regression includes transcriptional repression of genes involved in milk synthesis. This strongly implies that milk yield losses in lactating dairy cows exposed to TS are due in part to direct repression of genes associated with milk synthesis.
Thermotolerance in BMEC appeared to be lost after 8 h of exposure to TS when HSP-70 gene expression returned to basal levels, which was associated with increased expression of genes in the apoptotic pathways, indicating these cells were in the process of undergoing apoptosis. These studies were carried out using BMEC from nonadapted and nonacclimated cattle. Future studies will examine the response of BMEC from Bos indicus cattle and in BMEC cultured at slightly higher than normal temperatures to induce cellular acclimation. Results to date indicate that a portion of the loss in milk yield during acute thermal stress is associated with direct effects of thermal stress on BMEC.
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IMPLICATIONS |
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Footnotes |
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2 Corresponding author: rcollier{at}ag.arizona.edu
Received for publication August 11, 2005. Accepted for publication December 23, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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