J. Anim Sci. 2007. 85:395-403. doi:10.2527/jas.2006-234
© 2007 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Identification of differentially expressed transcripts in bovine rumen and abomasum using a differential display method1
S. G. Roh*,2,
M. Kuno*,
D. Hishikawa*,
Y. H. Hong*,
K. Katoh
,
Y. Obara,
H. Hidari
and
S. Sasaki*
* Department of Food Production Science, Faculty of Agriculture, Shinshu University, Nagano-ken 399-4598, Japan;
and
Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan; and
and
Field Center of Animal Science and Agriculture, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
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Abstract
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The rumen has several important physiological functions: absorption, transport, metabolic activity, and protection. To clarify the molecular basis underlying the physiological function of the rumen, reticulum, omasum, and abomasum, we used mRNA differential display to isolate and identify differentially expressed genes in these tissues. We isolated 18 transcripts that coexpressed in the rumen, reticulum, and omasum. Five genes, ribosomal protein 19 (RPS19), basic helix-loop-helix domain containing class B2 (BHLHB2), NADH dehydrogenase flavoprotein 2 (NDUFV2), exosome component 9 (EXOSC9), and ribosomal protein 23 (RPS23), were highly expressed in the rumen of adult Holstein and Japanese Black cattle. Significant differences of expression were observed in the abomasum compared with the rumen, reticulum, and omasum. To investigate the expression pattern of these genes during the neonatal growth stage, the relative levels of gene expression were analyzed in the rumen and abomasum of 3-wk-, 13-wk-, and 18- to 20-mo-old Holstein cattle. The expression level of RPS19 did not change with age in the rumen and abomasum. The levels of BHLHB2, NDUFV2, and EXOSC9 mRNA in the abomasum decreased (P < 0.05) after weaning and declined (P < 0.05) further in adults; in contrast, expression in the rumen was not altered. Interestingly, the levels of RPS23 mRNA in the rumen increased (P < 0.05) after weaning and further increased in the adult; however, the level of expression of this gene decreased (P < 0.05) in the abomasum with weaning and age. This indicates that the 4 tissues, especially the rumen and abomasum, have different developmental pathways after birth and subsequent onset of rumination.
Key Words: abomasum • cattle • differential display reverse transcriptase polymerase chain reaction • rumen
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INTRODUCTION
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In cattle, development of the rumen occurs in the neonate after the initiation of solid feed intake and the concomitant establishment of microbial fermentation and VFA production. At present, it is not known whether development of the rumen occurs in response to a direct or indirect action of VFA on the ruminal epithelium. A number of studies have investigated gene expression patterns in the rumen. Wang et al. (1996)
reported 2 cDNA clones encoding small proline-rich proteins expressed in sheep ruminal epithelium, reticulum, omasum, and skin, but not in the abomasum. The expression patterns of leptin and cholecystokinin receptor genes have been studied in calf stomachs. It was found that mRNA levels of both genes are affected by the change in physiological status brought about by weaning and VFA feeding (Yonekura et al., 2002). Overall, it is now clear that a number of genes are involved with the development of the rumen after weaning and that the expression patterns of these genes can be influenced by feed ingredients.
To date, most research on rumen physiology has concentrated on the role of microorganisms. There is considerable uncertainty on the identity of the genes involved in rumen development and in the genetic control of the developmental switch that initiates postnatal development of the rumen. It is clearly essential that these genetic processes are clarified to achieve a more complete understanding of the development and function of the rumen. To our knowledge, there are no reports comparing gene expression patterns in bovine rumen, reticulum, omasum, and abomasum.
We have exploited the differential display reverse transcription (DDRT)-PCR technique to examine changes in gene expression patterns in the gastric tissues of neonatal calves, with the ultimate aim of identifying candidate genes for the control of rumen development.
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MATERIALS AND METHODS
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Animals
All experiments were conducted in accordance with the Shinshu University Guide for the Care and Use of Experimental Animals. Rumen, reticulum, omasum, and abomasum were sampled from 12 Japanese Black cattle (18 to 24 mo of age) and 6 Holstein cattle (18 to 20 mo of age) immediately after slaughter at a local abattoir. Six of the 12 Japanese Black cattle were used for DDRT-PCR. The other 6 were used to confirm the expression pattern in DDRT-PCR. Rumen and abomasum tissues from 12 Holstein calves were also used; these are the same samples as described in a previous report (Yonekura et al., 2002). Briefly, 12 male Holstein calves were fed for 1 wk after birth to ensure the consumption of colostrum before beginning the experimental feeding. Six were fed a commercial milk replacer and calf starter and were slaughtered at 3 wk of age. The other 6 Holstein calves were fed on calf starter and hay until they were 13 wk old and slaughtered. They were allowed free access to timothy hay from 5 wk of age. All calves were fed diets at 1000 and 1600 (twice daily) and were slaughtered 18 h after the last meal.
Tissue Collection
Cattle entering commercial abattoirs were stunned with a captive bolt gun. Tissues were available within 15 min after decapitation, and each tissue sample was quickly removed and washed with PBS at 37 ° C to remove microbial and fungal contaminants. Tissue was sampled from several places in each organ, and rumen tissue was obtained from the ventral sac. The epithelial mucosa in rumen, reticulum, and omasum was stripped from the muscle layers using arterial forceps, scissors, and scalpel. The abomasal mucosa was scraped from the underlying musculature. All tissue samples (1 to 2 g) were frozen immediately in liquid nitrogen and stored at –80 ° C until RNA extraction.
Total RNA Extraction
Total RNA was prepared according to the manufacturer’s instructions. Tissue in TRIzol (Gibco/Invitrogen, Carlsbad, CA) was homogenized using an Ultra Turrax T25 Homogenizer (Ika Labortechnik, Janke and Kunkel, Staufen, Germany) starting at 5,000 rpm and gradually increasing to approximately 16,000 rpm over a period of 30 to 60 s at room temperature. The TRIzol/tissue homogenate was centrifuged at 12,000 x g for 15 min at 4 ° C in the presence of chloroform. The upper aqueous phase was collected, and total RNA was precipitated by the addition of isopropanol and centrifugation at 7,500 x g for 5 min at 4 ° C. Pellets of RNA were washed with 75% ethanol, dried, and reconstituted with sterile water. To remove contaminating DNA from the RNA preparations, the samples were incubated with ribonuclease-free deoxyribonuclease I (DNase-I; Roche, Indianapolis, IN) for 30 min at room temperature and then extracted with phenol/chloroform. The final concentration of DNase-I-treated RNA was quantified spectrophotometrically. Samples were stored at – 80 ° C.
DDRT-PCR
Total RNA from 6 Japanese Black cattle used in Figure 1 was pooled and mixed for DDRT-PCR. Differential display was performed using the differential display kit (Takara Co. Ltd., Tokyo, Japan) as described previously (Hishikawa et al., 2005). A total of 216 combinations of primers were assayed for each tissue sample, using 24 upstream primers and 9 downstream primers. Total RNA (250 ng) was subjected to reverse transcription (RT) in a 10-µL reaction mixture containing 1X, first-strand synthesis buffer, 1 mM each of dNTP, 0.1 µM anchored oligo (dT) primer, and 20 U of Avian Myeloblastosis Virus-reverse transcription (AMV-RT). The reaction mixture was incubated for 3 min at 70 ° C followed by 1 h at 42 ° C after the addition of AMV-RT, and the reaction was terminated by incubation for 10 min at 75 ° C.
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Figure 1. Representative differential display, reverse transcription PCR band pattern of genes highly expressed in the rumen, reticulum, and omasum compared with the abomasum. Total RNA pooled from 4 tissue samples of 6 Japanese Black cattle (18 to 24 mo of age) were reverse transcribed to cDNA, amplified, and run side-by-side on a 6% denaturing polyacrylamide gel. (a) RNA of lanes 1, 2, 3, and 4 were reverse transcribed with downstream primer No. 3 and amplified with upstream primer No. 11 and downstream primer No. 3. Arrow indicates the band identified as NADH dehydrogenase flavoprotein 2 (NDUFV2) after reamplification and analysis of the band. (b) Reverse transcription-PCR was performed with downstream primer No. 9 and amplified with upstream primer No. 7 and downstream primer No. 9. Arrow indicates the band identified as ribosomal protein 23 (RPS23) after reamplification and analysis of the band. Lanes: 1 = rumen; 2 = reticulum; 3 = omasum; and 4 = abomasum.
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The PCR amplification was performed in a 20-µL reaction mixture composed of 1 x PCR reaction buffer, 15 mM MgCl2, 20 µM each of dNTP, 1 U of Taq DNA polymerase, 1 µM of 1 of 24 anchor primers, and 1 µM of 1 of 9 arbitrary primers. The PCR protocol consisted of an initial denaturation at 94 ° C for 3 min; 35 cycles of denaturation at 94 ° C for 2 min, annealing at 40 ° C for 5 min, and extension at 72 ° C for 5 min; and a final extension at 68 ° C for 5 min. All reactions were performed in duplicate. Each sample was loaded onto a 6% nondenaturing polyacrylamide gel and separated by electrophoresis. The gel was stained with ethidium bromide and exposed to UV light.
The PCR bands not expressed in the abomasum were selected using the image capture system and analysis by NIH image software. Differentially displayed PCR bands were excised from the gel, washed twice with 100 µL of RNase-free water, and boiled for 5 min in a water bath. The DNA fragments were then immediately subjected to reamplification or frozen at – 20 ° C. A gene expression difference was confirmed from 2 replicates from each group. Selected bands showing the same intensity in replicate DDRT-PCR were excised from the gel, washed twice with 100 µL of RNase-free water, and boiled for 5 min in a water bath.
Re-amplification and Analysis of the Band of Interest
Ten microliters of eluent was taken from each band and reamplified with 10 µL of PCR reaction mixture [2 µ L 10 x PCR buffer (MBI Fermentas, Hanover, MD), 2 µL of 100 µM dNTP, 1 µL of each primer (upstream and downstream primer; 20 µM), 1 µL of Taq polymerase mixture (1 unit/µL; MBI Fermentas), and 4 µL of H2O]. The primer set used was the same as in the corresponding complementary DNA (cDNA) display. Ten microliters of the reamplified PCR sample was analyzed on a 1.5% agarose gel in 1 x TBE [44.5 mM Tris, 44.5 mM boric acid, and 1 mM Na2EDTA (pH 8)].
The PCR band of interest was cut from the gel under UV illumination. The DNA was purified using a QIA-quick gel extraction kit (Qiagen KK, Tokyo, Japan). The purified cDNA was eluted in 10 µM Tris-HCl (pH 8.5) and cloned into pGEM-T Easy vector (Promega, Madison, WI). After extraction and cloning of 5 single bands, 5 independent clones for each candidate were selected. An automated sequencing reaction was performed using an ABI PRISM Big Dye Terminator Cycle sequencing Ready Reaction Kits version 2.0 (PE Biosynthesis, CA) with M13 forward sequencing primer or reverse primer. Sequencing products are analyzed on the ABI 3100 Genetic Analyzer (Applied Biosystems, Tokyo, Japan).
The nucleotide sequences obtained were compared with known sequences by searching the National Center for Biotechnology Information databases with the basic local alignment search tool (BLAST) program. Comparison of the sequences of 5 independent colonies for each amplified differential band illustrated one of the pitfalls of DDRT-PCR. In most cases, 5 different sequences were obtained, making it impossible to predict which sequence represented the true differential band. Only 12 cloned differential cDNA sequences revealed a consensus sequence of the putative differentially expressed band.
Semiquantitative RT-PCR with Specific Primers
Semiquantitative RT-PCR was performed on samples of 4 tissues from 3- and 13-wk-old and adult Holstein cattle. Tissue samples of Japanese Black cattle used in Figure 2 were different from those used from DDRT-PCR. One microgram of total RNA was reverse transcribed to cDNA in a 20-µL RT reaction system containing oligo dT primers and AMV-RT. The RT reaction was carried out at 42 ° C. One microliter of the RT products was used for subsequent PCR amplification. Primers used for semiquantitative RT-PCR were targeted to the identified clones and were designed to contain 19 to 21 bases and have a melting temperature of 56 to 60 ° C. Preliminary experiments showed that for all of the primers used for PCR, the linear amplification range occurred at 27 to 33 cycles; all subsequent amplifications were therefore carried out using this cycle range.
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Figure 2. Expression pattern of ribosomal protein 19 (RPS19; 387 bp), basic helix-loop-helix domain containing class B2 (BHLHB2; 454 bp), NADH dehydrogenase flavoprotein 2 (NDUFV2; 415 bp), exosome component 9 (EXOSC9; 447 bp), and ribosomal protein 23 (RPS23; 354 bp) in the rumen, reticulum, omasum, and abomasum of (a) adult Japanese Black cattle and (b) adult Holstein cattle. Total RNA was pooled from the same concentration of RNA samples from 5 Holstein and 6 Japanese Black cattle; these were different animals from those used for the DDRT-PCR. The RT-PCR results shown are representative of 3 to 4 independent experiments with the same protocol. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 424 bp) was used as an internal control. Lanes: M = 100 bp DNA ladder; 1 = rumen; 2 = reticulum; 3 = omasum; 4 = abomasum; and no cDNA = no cDNA added to the lane.
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Products from PCR were resolved on a 1.2% agarose gel; the DNA was visualized by ethidium bromide staining and analyzed using NIH image software, in which band intensity is expressed in pixels. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal expression control. Our preliminary data showed that GAPDH did not change according to the age of the animal or across tissues. Relative gene expression was calculated as the ratio of the band intensity of the cloned gene to that of GAPDH. Amplified cDNA were subcloned into pGEM-T Easy vector (Promega), and the nucleotide sequences were confirmed using an ABI 3100 Genetic Analyzer (Applied Biosystems).
Statistical Analysis
Data in Figures 3 to 7 are presented as means ± SEM from 5 to 6 animals in each experimental group. Statistical analyses of the differences between growth stages were performed by ANOVA followed by the Duncan multiple range test when the F-test was significant using StatView (SAS Inst. Inc., Cary, NC).
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Figure 3. The levels of ribosomal protein 19 (RPS19; 387 bp) mRNA in the rumen and abomasum of 3- and 13-wk-old and adult (18- to 20-mo-old) Holstein cattle. The reverse transcription-PCR results shown for RPS19 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 424 bp) are representative of 5 to 6 independent experiments with the same protocol. The levels of RPS19 mRNA were corrected against those of the GAPDH mRNA. Data were expressed as percentages of the values obtained from the rumen of 3-wk-old calves. The column represents the mean ± SEM of 5 to 6 animals. a,bIndicates a significant difference between each tissue of 3- and 13-wk-old and adult Holstein cattle (P < 0.05). Lanes: M = 100 bp DNA ladder; 1 = rumen; 4 = abomasum; and no cDNA = no cDNA added to the lane.
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Figure 4. The levels of basic helix-loop-helix domain containing class B2 (BHLHB2; 454 bp) mRNA in the rumen and abomasum of 3- and 13-wk-old and adult (18-to 20-mo-old) Holstein cattle. The reverse transcription-PCR results shown for BHLHB2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 424 bp) are representative of 5 to 6 independent experiments with the same protocol. The levels of BHLHB2 mRNA were corrected against those of the GAPDH mRNA. Data were expressed as percentages of the values obtained from the rumen of 3-wk-old calves. The column represents the mean ± SEM of 5 to 6 animals. a,bIndicates a significant difference between each tissue of 3- and 13-wk-old and adult Holstein cattle (P < 0.05). Lanes: M = 100 bp DNA ladder; 1 = rumen; 4 = abomasum; and no cDNA = no cDNA added to the lane. ND = not detected.
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Figure 5. The levels of NADH dehydrogenase flavo-protein 2 (NDUFV2; 415 bp) mRNA in the rumen and abomasum of 3- and 13-wk-old and adult (18- to 20-mo-old) Holstein cattle. The reverse-transcription-PCR results shown for NDUFV2 and glyceraldehyde-3-phosphate de-hydrogenase (GAPDH; 424 bp) are representative of 5 to 6 independent experiments with the same protocol. The levels of NDUFV2 mRNA were corrected against those of the GAPDH mRNA. Data were expressed as percentages of the values obtained from rumen of 3-wk-old calves. The column represents the mean ± SEM of 5 to 6 animals. a–cIndicates a significant difference between each tissue of 3- and 13-wk-old and adult Holstein cattle (P < 0.05). Lanes: M = 100 bp DNA ladder; 1 = rumen; 4 = abomasum; and no cDNA = no cDNA added to the lane.
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Figure 6. The levels of exosome component 9 (EXOSC9; 447 bp) mRNA in the rumen and abomasum 3- and 13-wk-old and adult (18- to 20-mo-old) Holstein cattle. The reverse-transcription-PCR results shown for EXOSC9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 424 bp) are representative of 5 to 6 independent experiments with the same protocol. The levels of EXOSC9 mRNA were corrected against those of the GAPDH mRNA. Data were expressed as percentages of the values obtained from rumen of 3-wk-old calves. The column represents the mean ± SEM of 5 to 6 animals. a,bIndicates a significant difference between each tissue of 3- and 13-wk old and adult Holstein cattle (P < 0.05). Lanes: M = 100 bp DNA ladder; 1 = rumen; 4 = abomasum; and no cDNA = no cDNA added to the lane.
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Figure 7. The levels of ribosomal protein 23 (RPS23; 354 bp) mRNA in the rumen and abomasum of 3- and 13-wk-old and adult (18- to 20-mo-old) Holstein cattle. The reverse transcription-PCR results shown for RPS23 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 424 bp) are representative of 5 to 6 independent experiments with the same protocol. The levels of RPS23 mRNA were corrected against those of the GAPDH mRNA. Data were expressed as percentages of the values obtained from rumen of 3-wk-old calves. The column represents the mean ± SEM of 5 to 6 animals. a–dIndicates a significant difference between each tissue of 3- and 13-wk-old and adult Holstein cattle (P < 0.05). Lanes: M = 100 bp DNA ladder; 1 = rumen; 4 = abomasum; and no cDNA = no cDNA added to the lane. M: 100 bp DNA ladder; 1: rumen; 4: abomasum.
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RESULTS
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Identification of Differentially Expressed Genes
After image analysis, 32 bands that appeared to be differentially expressed were excised from 40 DDRT-PCR gels. Figure 1
shows the band pattern typical of a DDRT-PCR gel. Of the 32 products excised, 18 that were judged to show similar expression in the rumen, reticulum, and omasum, but a considerably different level of expression in the abomasum, were selected for reamplification and sequencing. The 18 sequenced clones were subjected to a BLAST search of the National Center for Biotechnology Information databases. This search identified 12 genes; 8 were present only in a single clone, and the remaining 4 were present in 2 or more clones (Table 1).
Confirmation of Differential Gene Expression Using RT-PCR
We used RT-PCR to confirm differential gene expression of the 12 identified genes in pooled RNA samples from Japanese Black and Holstein cattle. Table 2 shows the primer sequences and PCR conditions used in RT-PCR. Only 5 of the 12 genes showed differential expression among the 4 tissues: ribosomal protein 19 (RPS19), basic helix-loop-helix domain containing class B2 (BHLHB2), NADH dehydrogenase flavoprotein 2 (NDUFV2), exosome component 9 (EXOSC9), and ribosomal protein 23 (RPS23; Figure 2). In Japanese Black cattle, gene expression levels were less in the abomasum than in the other 3 fore-stomach tissues (Figure 2a). Holstein cattle additionally had lower levels of expression in the reticulum and omasum compared with the rumen (Figure 2b). These analyses were carried out in different animals to those used in the differential display experiment to ensure that the observed differential expression was not due to a particular genetic background or response of 1 animal. We confirmed the sequences of the PCR products to monitor whether our RNA samples were contaminated with rumen microbes. The sequences of bovine PCR products of RPS19, BHLHB2, NDUFV2, EXOSC9, and RPS23 had at least 99% homology with those of the bovine gene, indicating that these sequences were not of rumen bacterial origin (Table 2). The 5 genes were selected for further analysis because of their potential for involvement in the control of rumen development.
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Table 2. Genes subjected to semiquantitative reverse transcription-PCR analysis of differential expression in bovine rumen, reticulum, omasum, and abomasum
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Developmental Changes in Gene Expression in the Rumen and Abomasum of Holstein Cattle
Because the age of cattle is an important factor in the development of rumen and abomasum, the patterns of expression of the 5 genes described above were analyzed in 2 tissues of 3- and 13-wk-old and adult Holstein cattle; the sampling intervals represent preweaning, postweaning, and adult stages, respectively. There was no difference in the levels of RPS19 mRNA expression in the rumen or abomasum of 3-wk-old calves compared with 13-wk-old Holsteins (Figure 3). Expression of mRNA for RPS19 was high (P < 0.05) in the rumen compared with the abomasums, and the expression pattern did not change at weaning or in adulthood (Figure 3). The expression of BHLHB2 was consistently greater (P < 0.05) in the rumen than abomasum at all developmental stages studied (Figure 4). There was no age-related change in the levels of BHLHB2 mRNA in the rumen of Holstein cattle; however, there was a considerable fall (P < 0.05) in expression level of the gene in the abomasum from 3-wk-old to 13-wk-old and adult cattle (Figure 4). There was no indication of any change at weaning in the expression pattern of NDUFV2 in the rumen; however, in the abomasum, the level of expression decreased (P < 0.05) from the age of 3 to 13 wk and fell (P < 0.05) further in adulthood (Figure 5). Figure 6 shows the gene expression level of EXOSC9 in 3- and 13-wk-old and adult Holstein cattle. Expression of EXOSC9 mRNA did not vary significantly in the rumen between the 3 developmental stages tested. Interestingly, although the level of expression was similar at 3 and 13 wk in the abomasum, it fell (P < 0.05) sharply in adulthood (Figure 6). The transcripts of RPS23 showed considerable variation in expression levels in each age group; moreover, the changes in patterns of expression differed (P < 0.05) between the rumen and abomasum. In the rumen, the expression level of RPS23 mRNA was relatively low at 3 wk and increased (P < 0.05) with age (Figure 7). In contrast, in the abomasum, RPS23 mRNA levels at 3 wk were higher (P < 0.05) than in the rumen; however, in older cattle, expression levels in the abomasum fell sharply (P < 0.05; Figure 7).
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DISCUSSION
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Gene expression profiles in the rumen, reticulum, omasum, and abomasum of cattle were investigated in this study, and 5 genes showing differential expression in these tissues were identified. Four of these genes, BHLHB2, NDUFV2, EXSOC9, and RPS23, had expression patterns that were influenced by weaning and the age of the animal. The fifth gene, RPS19, was apparently unaffected by these factors. Only with RPS23 was the expression level in the rumen increased after weaning. The level of expression of 4 genes, BHLHB2, NDUFV2, EXOSC9, and RPS23, declined in the abomasum after weaning. Weaning, accompanied by the start of rumination, is important in cattle because the absorption of VFA from the rumen provides essential energy for growth and development. In addition to the developmental changes we describe here, it is known that the physiology of the rumen and abomasum can be changed by the foodstuff given to cattle. Our data support the hypothesis that this alteration must involve several genes.
The protein of RPS19 is part of the 40S ribosomal unit and shows a high degree of sequence conservation across mammalian species at the protein level (Draptchinskaia et al., 1999). The RPS19–/– genotype in C57BL/6J mice is lethal before implantation, whereas mice with disruption of 1 RPS19 allele have a normal phenotype, including the hematopoietic system (Matsson et al., 2004). Expression of RPS19 mRNA decreases during terminal erythroid differentiation (Da Costa et al., 2003). In addition to its involvement in erythropoiesis, there are indications that RPS19 has extraribosomal functions. Free RPS19 protein interacts with fibroblast growth factor (FGF)-2, which may indicate that the interaction between RPS19 and FGF-2 plays a key role in the control of signal transduction by FGF-2 and in the decision between differentiation and proliferation pathways (Soulet et al., 2001). Although it is not known which proteins bind RPS19 on the external surface of the 40S ribosomal subunit, RPS19 appears to be a normal part of the maintenance of rumen function from birth because its expression does not change with age. However, unlike RPS19, expression of RPS23 mRNA is upregulated in the rumen with age. The level of expression of RPS23 increased in the rumen after weaning; conversely, the level declined in the abomasum. In adult cows compared with 13-wk-old animals, expression of RPS23 increased in the rumen but declined further in the abomasum. Gene expression of RPS23 is greater in the abomasum than in the rumen in 3- and 13-wk-old calves. In other words, developmental age is an influential factor in the control of expression of the RPS23 gene in the rumen and abomasum. The pattern of expression of RPS23 mRNA is consistent with the possibility that the gene may be involved with rumen development. These observations indicate that, despite the presence of other ribosomal proteins, RPS23 and RPS19 each have an important, albeit functionally different, role in rumen development.
The expression level of BHLHB2 mRNA decreased remarkably in the abomasum after weaning. This decrease can be thought to be a cause or result of weaning because the difference in expression between the rumen and the abomasum is not changed between 13-wk-old and adult cattle. The basic helix-loop-helix transcriptional regulators are critical for controlling the activity of gene expression networks in many fundamental biological processes, such as early cell determination and differentiation, cell cycle maintenance, and homeostasis or stress response pathways (Massari and Murre, 2000). The consistent level of gene expression of BHLHB2 in the rumen at all developmental stages examined indicates that this gene is not important to the change in rumen physiology associated with weaning. However, because expression declined in the abomasum after weaning, we suggest that the requirement for the function of the BHLHB2 gene disappears in the abomasum as part of cell determination and differentiation associated with the onset of rumination.
The gene expression of NDUFV2 declined in the abomasum after weaning. Although the levels of expression in the rumen and abomasum were the same at 3 wk, its expression level in the abomasum was less than that in the rumen at 13 wk. In adult cattle, the expression of the NDUFV2 gene decreased further in the abomasum, whereas that in the rumen gene did not change. Our results indicate that the NDUFV2 gene is not part of the regulatory pathway involved in the development of the rumen after weaning because there is no age-related change in expression levels. However, in the abomasum, it is possible that energy production from complex I decreased after weaning as a result of the reduction in NDUFV2 expression.
The level of gene expression of EXOSC9 in the abomasum decreased after weaning. In the rumen, there is no evidence of an age-related change in the expression of EXOSC9 mRNA, which indicates that its decrease in the abomasum is a factor in the physiological changes associated with weaning. The protein of EXOSC9 is known to be a subunit of the exosome that is involved in the processing and degradation of RNA (Gelpi et al., 1990; Alderuccio et al., 1991; Butler, 2002). We suggest on the basis of consistent expression patterns in the 3 development stages sampled that EXOSC9 is not concerned with the development of the rumen. However, because expression declined in the abomasum after weaning, we suggest that the requirement for the function of the EXOSC9 gene disappears in the abomasum.
As the calf grows and begins to consume a variety of foods, its stomach compartments also grow or shrink, changing the way it digests its food. Within the first 2 mo, rumen development depends primarily on how soon dry feed is given and the amount of milk and dry feed the calf consumes. The abomasum increases in absolute mass, but a large increase in rumen mass occurs. The goal of raising a calf effectively is to ensure that the rumen develops correctly by feeding it the proper mixtures of feeds in the first few months of life. The purpose of this investigation was to determine whether there was evidence of differences in gene expression profiles between the rumen and abomasum of adult cattle raised under a normal feeding program. Our data indicate that such differences are present between rumen and abomasum examined in fattened adult cattle. The primary findings of our study are, first, the differences in expression pattern in the 2 tissues in cattle and, second, the influence of developmental states on differentially expressed genes. Although the functional unit of these tissues is the epithelium coordinated with connective tissue, muscle, and extracellular matrix, the specific signals for different expression of 5 genes was mainly derived from epithelium. However, laser capture microscopy or dissections may clarify the specific tissues differentially expressed by these genes.
Analysis of RPS19, BHLHB2, NDUFV2, EXOSC9, and RPS23 showed that expression was generally higher in the rumen. Moreover, we found an age-related change in expression in the rumen and the abomasum. We suggest that these 5 genes play an important role in the function of the rumen of adult cattle. It was also observed that, after weaning, the levels of expression of RPS19, BHLHB2, NDUFV2, and EXOSC9 were reduced in the abomasum although not in the rumen. The expression patterns of these genes may indicate that they are involved in functions that are no longer required in the abomasum after the start of rumination. Interestingly, we found that the expression of RPS23 changed with age and at weaning. Therefore, we believe that our data on those 5 genes will contribute to an understanding of the different characteristics of rumen and abomasum in adult animals. The function of this gene is not clear, but its increase in expression with age suggests the possibility that it may be involved in the metabolic changes associated with rumen development.
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Footnotes
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1 This work was partly supported by a grant-in-aid (No. 17380168 to Sang-Gun Roh) for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan. 
2 Corresponding author: sangroh{at}shinshu-u.ac.jp
Received for publication April 12, 2006.
Accepted for publication September 12, 2006.
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LITERATURE CITED
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Alderuccio, F., E. K. Chan, and E. M. Tan. 1991. Molecular characterization of an autoantigen of PM-scl in the polymyositis/scleroderma overlap syndrome: A unique and complete human cdna encoding an apparent 75-kD acidic protein of the nucleolar complex. J. Exp. Med. 173:941–952.[Abstract/Free Full Text]
Butler, J. S. 2002. The yin and yang of the exosome. Trends Cell Biol. 12:90–96.[CrossRef][Medline]
Da Costa, L., G. Narla, T. N. Willig, L. L. Peters, M. Parra, J. Fixler, G. Tchernia, and N. Mohandas. 2003. Ribosomal protein S19 expression during erythroid differentiation. Blood 101:318–324.[Abstract/Free Full Text]
Draptchinskaia, N., P. Gustavsson, B. Andersson, M. Pettersson, T. N. Willig, I. Dianzani, S. Ball, G. Tchernia, J. Klar, H. Matsson, D. Tentler, N. Mohandas, B. Carlsson, and N. Dahl. 1999. The gene encoding ribosomal protein S19 is mutated in diamond-blackfan anaemia. Nat. Genet. 21:169–175.[CrossRef][Medline]
Gelpi, C., A. Alguero, M. Angeles Martinez, S. Vidal, C. Juarez, and J. L. Rodriguez-Sanchez. 1990. Identification of protein components reactive with anti-PM/SCL autoantibodies. Clin. Exp. Immunol. 81:59–64.[Medline]
Hishikawa, D., Y. H. Hong, S. G. Roh, H. Miyahara, Y. Nishimura, A. Tomimatsu, H. Tsuzuki, C. Gotoh, M. Kuno, K. C. Choi, H. G. Lee, K. K. Cho, H. Hidari, and S. Sasaki. 2005. Identification of genes expressed differentially in subcutaneous and visceral fat of cattle, pig, and mouse. Physiol. Genomics 21:343–350.[Abstract/Free Full Text]
Massari, M. E., and C. Murre. 2000. Helix-loop-helix proteins: Regulators of transcription in eucaryotic organisms. Mol. Cell. Biol. 20:429–440.[Free Full Text]
Matsson, H., E. J. Davey, N. Draptchinskaia, I. Hamaguchi, A. Ooka, P. Leveen, E. Forsberg, S. Karlsson, and N. Dahl. 2004. Targeted disruption of the ribosomal protein S19 gene is lethal prior to implantation. Mol. Cell. Biol. 24:4032–4037.[Abstract/Free Full Text]
Soulet, F., T. Al Saati, S. Roga, F. Amalric, and G. Bouche. 2001. Fibroblast growth factor-2 interacts with free ribosomal protein S19. Biochem. Biophys. Res. Commun. 289:591–596.[CrossRef][Medline]
Wang, L., R. L. t. Baldwin, and B. W. Jesse. 1996. Identification of two cDNA clones encoding small proline-rich proteins expressed in sheep ruminal epithelium. Biochem. J. 317:225–233.
Yonekura, S., K. Kitade, G. Furukawa, K. Takahashi, N. Katsumata, K. Katoh, and Y. Obara. 2002. Effects of aging and weaning on mRNA expression of leptin and CCK receptors in the calf rumen and abomasum. Domest. Anim. Endocrinol. 22:25–35.[CrossRef][Medline]
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Abstract
INTRODUCTION
