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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Division of Nutrition and Physiology, Institute of Animal Genetics, Nutrition, and Housing, Vetsuisse Faculty, University of Berne, CH-3012 Berne, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Key Words: Calf • Messenger RNA • Small Intestine • Somatotropic Axis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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; Georgieva et al., 2003; Ontsouka et al., 2004a,c). Expression of these genes in the GIT of neonatal calves varies according to intestinal site and ontogenesis, is modified by nutrition and other endocrine systems (Georgiev et al., 2003; Georgieva et al., 2003; Ontsouka et al., 2004a,c), and is thought to exert important physiological functions in the GIT (Blum and Baumrucker, 2002; Howarth, 2003).
To our knowledge, there have been no studies on the abundance of mRNA coding for the various members of the somatotropic axis and for the InsR in mucosal layers of the GIT in calves. Binding studies in rats indicated different numbers of IGF receptors in villi and crypts (Laburthe et al., 1988; Heinz-Erian et al., 1991; MacDonald, 1999). In laboratory animals, cryostat sectioning has been used for gene expression studies in different intestinal layers (Goda et al., 1983). This method promised to be advantageous compared with more traditional methods in which fractionation of intestinal layers is achieved by incubation in buffer solutions (Traber et al., 1991; Fan et al., 1999), but our experience is that mRNA is highly susceptible to degradation when using this procedure.
Based on these premises, we have measured mRNA levels of IGF-I, IGF-2, IGF-1R, IGF-2R, GHR, InsR, IGFBP-2, and IGFBP-3 in jejunal fractions, mainly composed of villus tips or crypts, from 5-d-old calves. Additionally, ileal fractions consisting mainly of villus tips, crypts, and lamina propria (LP) with Peyer’s patches (PP) were also examined. In this context, suitability of the sectioning technique of Goda et al. (1983) for jejunal and ileal tissues was tested.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Nine calves (Holstein Friesian and Red Holstein x Simmental), born after a normal gestation length (290 ± 2 d) at the Experimental Station in Posieux, were used for the study. Calves were held on straw in individual boxes for 4 d. Calves received the first meal within 2 h after birth and the next meals at 8, 24, and 32 h after the first meal. From d 3 on, calves were fed at 0800 and at 1600. On d 4, all calves received a milk replacer. Amounts (as-fed basis) of colostrum fed were 6% of BW on d 1, 8% of BW on d 2, and 10% of BW from d 3 on. On d 4, milk replacer was fed at 10% of BW.
Colostrum fed to calves was derived from approximately 70 dairy cows and consisted of the first, third, and fifth milkings obtained on d 1, 2, and 3 of life, respectively. The colostrum fed to calves on d 1 contained 251 g of DM/kg, and contained (per kilogram of DM) 24.7 MJ of GE, 634 g of CP, 211 g of crude fat, 108 g of N-free extract (mainly lactose), and 47 g of crude ash. The colostrum fed to calves on d 2 and 3 contained 164 and 144 g of DM and contained (per kilogram of DM) 24 and 23.8 MJ of GE, 470 and 403 g of CP, 238 and 250 g of crude fat, 232 and 285 g of N-free extract, and 61 and 63 g of crude ash, respectively. The milk replacer fed to calves on d 4 contained 127 g of DM/kg and contained (per kilogram of DM) 23.6 MJ of GE, 378 g of CP, 236 g of crude fat, 354 g of N-free extract, and 32 g of crude ash. Before feeding, colostrum was warmed to 40°C and then fed immediately. The milk replacer (UFA natura, without antibiotics; UFA AG, Sursee, Switzerland) was prepared as a 100-g/L solution and contained 979 g of DM/kg and contained (per kilogram of DM) 18.3 MJ of GE, 220 g of CP, 210 g of crude fat, 311 g of N-free extract, and 263 g of crude ash.
Calves were protected against infections by a s.c. injection of 2 g of a bovine colostral immunoglobulin preparation (Gammaserin; Gräub AG, Berne, Switzerland) before the first meal.
Tissue Preparation
Calves were killed on d 5 of life by i.v. injection of 80 mg of pentobarbital/kg BW (Eutha 77; Serum- and Impfinstitut, Berne, Switzerland). Immediately after slaughter, the GIT was removed and flushed with chilled PBS (consisting of 8 g/L of NaCl, 200 mg/L of KCl, 2.6 g/L of Na2HPO4 •7 H2O, and 240 mg/L of KH2PO4, pH = 7.4) and then placed into ice-cold diethyl-pyrocarbonate-treated NaCl (154 mmol/L). Segments of approximately 20 cm of the middle parts of jejunum or ileum, respectively, were opened longitudinally and flattened on a glass slide, serosa side down, and were then frozen immediately in liquid nitrogen. Frozen tissue pieces (5 mm x 5 mm or 25 mm2) were covered with a supporting medium (i.e., O.C.T. compound; Tissue-Tek, Zoeterwoude, Netherlands) and were again placed in liquid N and then transferred onto preflattened supporting surface of 1% agar within the cryostat at a temperature of –20°C. The mucosa was cut horizontally into 10-µm slices for later determination of mRNA levels of lactase and of components of the somatotropic axis in the different intestinal layers (Goda et al., 1983). Fifteen consecutive and morphologically similar slices (10 µm thick) were collected as fractions for the villus, crypt, and LP layers, respectively. The first and last slices were stained with hematoxylin and eosin and evaluated microscopically to check for homology of the fraction. The slice before the last cut was used to assay the incorporation of 5'-bromo-2-deoxyuridine (BrdU; Roche Diagnostics, Rotkreuz, Switzerland) into dividing cells, whereas the remaining 12 slices were combined and were stored at –80°C for extraction of total RNA.
Evaluation of Cell Proliferation and Characterization of Gut Fractions
As described by Blättler et al. (2001), calves were injected i.v. with 500 mg of BrdU dissolved in 20 mL of saline 60 min before slaughter. Slides were stained using a mouse monoclonal antiBrdU antibody (catalog No. 1 170 376, Roche Applied Science) for the detection of BrdU incorporation into DNA. The BrdU incorporation was visualized using biotinylated goat antimouse immunoglobulin (Dako, Glostrup, Denmark), Strept-ABComplex/AP (Dako), and Fast Red TR/Naphtol AS-Mix (Sigma, St. Louis, MO).
The evidence that fractions consisted of mainly villus tips, crypts, or lymph follicles of PP in the LP fraction was based on the presence of typical morphological features. Furthermore, analysis of BrdU incorporation showed that epithelial cells of the villus tip fraction were not labeled, in contrast to epithelial cells of crypt fractions and lymphocytes of PP in LP fractions. Villus tip and LP fractions were very pure. Crypt layers contained at least 60% crypts to be acceptable for further evaluation.
To evaluate cell proliferation, photographs of slices before the last cut of villus, crypt, and LP layers, assayed for BrdU incorporation, were made randomly through a microscope with a digital camera (Axio Cam HR with the software Axio Vision v3.1; Carl Zeiss Vision GmbH, Munich-Hallbergmoos, Germany). The number of typical morphological structures for villus tips, crypts, and LP were evaluated by the grid method using standard graphics software (Coral Draw 9, Version 9.337; Corel Corp., Ottawa, Ontario, Canada). The numbers of typical morphological structures were counted in tissue sections of the villus, crypt, and LP fractions. In each fraction, morphological structures were evaluated at three locations, and at each of these three locations, eight 4 cm x 4 cm squares at the same magnification were evaluated. These eight squares were distributed along a line drawn from the left upper to the right lower corner of the computer screen. Structures that crossed the upper and right line of the square were counted, but not those that crossed the left and lower line of the square. One centimeter on the computer screen corresponded to 40 µm of the original tissue. For each fraction, the mean value of three photographs was used to quantify the number of target structures. In addition, the abundance of lactase mRNA served mainly to evaluate the purity of villus and crypt fractions relative to the LP fraction.
Extraction of RNA and cDNA Production
Total RNA was extracted from the combined 12 sections of villus tips, crypts, and LP fractions (12 µm x 10 µm), respectively, as recently described in detail (Pfaffl et al., 2002). Because a pool of 12 slices was used to extract total RNA, there was no effect of a single slice on amounts of total RNA that could be extracted. To decrease variation in the extraction procedure from one fraction to the other and from one intestinal site (jejunum, ileum) to the other, we have always extracted RNA from one calf from all fractions within a single run. Thus, within-calf variations in the extraction of total RNA could be kept to a minimum, thereby allowing comparisons between fractions within one intestinal site (i.e., within jejunum or within ileum).
Fractions were homogenized for 30 s with an Ultra-Turrax T 8 homogenizer (IKA Werke GmbH & Co. KG, Staufen, Germany) in Eppendorf tubes using 500 µL of Trizol (Gibco BRL, Basel, Switzerland) and incubated for 10 min at room temperature. After the addition of 100 µL of chloroform and vortexing for 15 s, the homogenate was incubated for 10 min at room temperature for phase separation, and was centrifuged at 12,000 x g for 15 min at 4°C. Then, 300 µL of the upper (aqueous) phase, containing exclusively RNA, was transferred into a new microcentrifuge tube. After addition of one volume of isopropanol and incubation for 30 min at room temperature, RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4°C. The recovered total RNA was washed twice with 75% ethanol, followed each time by centrifugation at 9,200 x g for 8 min at 4°C. The pellet containing the RNA was resuspended in diethylpyrocarbonate-treated RNase-free water. The concentration of RNA was measured photometrically (Biophotometer, Eppendorf, Netheler-Hinz, Hamburg, Germany) at an optical density (OD) of 260 nm. The integrity and purity of the RNA were verified by gel electrophoresis with ethidium bromide staining and by photometrical absorption (ratio of OD at 260 nm to that at 280 nm being > 1.9 and, ideally, 1.9 to 2.0). No extraction was accepted that differed from this range. The RNA solution was diluted to the working concentration of 100 ng/µL and was measured in triplicate. Then, 1 µg of RNA from the working solution was reverse transcribed into cDNA with 100 U of MMLV Plus RNase H–Reverse Transcriptase (Promega Corp., Madison, WI) using 100 pmol of random hexamer primers (Pharmacia Biotech; Buckinghamshire, U.K.).
To avoid differences in the reverse transcription PCR between different fractions within an intestinal site (jejunum or ileum) and between intestinal sites (jejunum or ileum) from one calf, we always performed the reverse-transcription PCR (RT-PCR) on all samples from a given calf within the same run. There was no genomic DNA present.
Real-Time Reverse-Transcription PCR of the Somatotropic Axis and of InsR
The mRNA abundance of IGF-I, IGF-2, IGFBP-2, IGFBP-3, IGF-1R, IGF-2R, GHR, and InsR was related to four housekeeping (control) genes, as described below. Procedures followed for the design of primers used for the amplification of listed traits were described in detail by Pfaffl et al. (2002). Primers were located in high homologous regions of multiple species (bovine, mouse, ovine, and rat) by clustal alignment and spanned at least two exons. Primers were optimized with regard to primer dimer formation, self priming formation, and primer melting temperature.
The PCR were performed with a LightCycler (Roche Diagnostics) with 25 ng of reverse-transcribed total RNA. Reaction components for the LightCycler reactions were 1.0 µL of LightCycler FastStart Mastermix (Roche Diagnostics) containing Taq DNA polymerase reaction buffer, 10 mM MgCl2, deoxynucleotide triphosphate mix, and SYBR Green I dye, 4 mM MgCl2, 4 pmol forward primer, 4 pmol reverse primer, and sterile water up to a final volume of 10 µL. Conditions for the amplification and specificity of PCR products of the somatotropic axis were as reported (Pfaffl et al., 2002).
Real-Time Reverse-Transcription PCR of Lactase
For the amplification of bovine lactase mRNA, primers were designed in 100% homology regions of multiple species (human, rabbit, and rat) clustal alignment and spanned exons 10 and 11 (Ontsouka et al., 2004b). The sequences of primers used and length of PCR products were as follows: forward = 5'-tggagagcagatggcaaagg-3', reverse = 5'-cagcctctggaagagcacatc-3'; 397bp. The specificity of lactase primers used in the current study has been recently reported (Ontsouka et al., 2004b).
Real-Time Reverse-Transcription PCR of Housekeeping Genes
Glyceraldehyde phosphate dehydrogenase (GAPDH), ubiquitin, ribosomal RNA (18S), and ß-actin, which are involved in different cell functions and frequently used as housekeeping genes, were selected and used as internal control genes for the normalization of gene expression for members of the somatotropic axis, InsR, and lactase. The sequences of primers used for amplification of housekeeping genes were identical to those of previous studies (Inderwies et al., 2003; Reist et al., 2003).
Mathematical and Statistical Analyses
The mRNA abundance of members of the somatotropic axis, of InsR, of lactase, and of housekeeping genes was evaluated by amplification curve analysis of the LightCycler real-time RT-PCR. The SYBR Green I (DNA binding dye) incorporated into double-stranded DNA during PCR amplification emits fluorescence of increasing intensity with each cycle number. The exponential phase of the PCR begins when the fluorescence signal from the accumulated PCR product is greater than the background fluorescence. To exclude noninformative fluorescence background points, a fixed fluorescence threshold line is set to the exponential portion of the amplification curve as low as possible without including any background points. The intersection of the threshold line and the amplification curve represents the crossing point value (Rasmussen, 2001).
The mRNA levels of members of the somatotropic axis, InsR, and lactase were related to those of housekeeping genes. By definition, housekeeping gene expression should be constant in a given tissue. This may not strictly be the case in rapidly growing animals and especially in transition periods, such as during early postnatal periods. If housekeeping genes are stable, however, levels of different housekeeping genes should be closely correlated. Because this was the case in our study, the mean values for the crossing points of GAPDH, UBC, ß-actin, and 18S were calculated for jejunum and ileum to get the most constant level. The 
1 crossing point value of housekeeping genes represents the difference between the mean crossing point value of the tissue and the individual sample crossing point value. The 1 crossing point value corresponds to the number of cycles to be added or subtracted from the individual crossing point value to get the virtually constant housekeeping gene expression in tissues. The lactase crossing point value was accordingly normalized in individual samples as 2 crossing point value. The lactase expression level was calculated as (2[–2 crossing point]) x 100, where 2 is the efficiency of PCR leading to amplicon duplication at every cycle (Medhurst et al., 2001). Data are presented as means ±SEM.
Because there was a nonnormal distribution of data in some intestinal layers, differences among small intestinal fractions in jejunum and ileum were tested for significance with the nonparametric Kruskall-Wallis test. Effects were considered to be significant at P < 0.05, and data are presented as means ±SE.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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; Fan et al., 1999). In addition, the method used in the current study allows histological and histochemical analyses of the same tissues to be performed.
The mean crossing point values of ß-actin, ubiquitin, GAPDH, and 18S and their mutual relationships (coefficients of correlation) are presented in Table 1. Abundance of mRNA for these four housekeeping genes was highly correlated. Therefore, it can be assumed that housekeeping genes were stable and not regulated in this situation. If they had been regulated, they would not have been correlated so closely because they represent such different functions that it would be very unlikely that their expression would occur in a uniform manner.
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; Sauter et al., 2004). In the ileum, there was also marked BrdU incorporation into lymphocytes of PP in LP fractions, which agrees with previous studies (David et al., 2003; Norrman et al., 2003). One additional criterion was the level of lactase mRNA, which was greater on tips of villi than in crypt fractions in jejunum (P < 0.05) and ileum (P < 0.07). Importantly, lactase mRNA was absent in the LP fraction. These findings agreed with results of studies in milk-fed calves (Ontsouka et al., 2004b) and in other species (Duluc et al., 1993; Hansen et al., 1994; Goda et al., 1999; Fan et al., 2001). Because lactase mRNA could not be detected in the LP fraction, it allowed for the differentiation of this fraction from the other two mucosal fractions. Based on these methods (histomorphology, histomorphometry, presence or absence of BrdU incorporation, presence or absence of lactase mRNA), the jejunal villus and crypt fractions and the ileal villus, crypt, and LP fractions clearly contained different cell types (i.e., exclusively villi in the villus fraction, mostly crypt cells in the crypt fraction, and exclusively LP fraction that consisted mainly of lymphocytes of PP; Table 2). The villus fractions (tips of villi) in both jejunum and ileum were extremely pure (100%; i.e., they did not contain crypt cells and lymphocytes of PP or of other parts of the gut-associated lymphoid tissues). The crypt fractions were relatively pure because they consisted mainly, but not exclusively, of crypt cells. Contamination of the crypt fraction by villus epithelial cells and by cells of the LP fraction was obviously unavoidable and not surprising, given the morphological situation. The LP fractions were also very pure (100%; i.e., they did not contain cells of crypt or villus fractions). To conduct further analyses (determination of mRNA levels of members of the somatotropic axis and InsR), a particular fraction was required to contain at least 60% of typical histological structures. As shown in Table 2, this condition was met.
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. In the jejunum and ileum, there were differences (P < 0.05) among fractions of total RNA (in jejunum, crypts > villi; in ileum, LP and crypts > villi; Tables 3 and 4). Differences in total RNA levels among fractions likely indicated differences in cellular turnover rates and likely also of protein synthesis. If so, it was not surprising to find greater total RNA concentrations in the jejunal and ileal crypt than in villus fractions (P < 0.05) or to find the greatest RNA concentrations in LP fractions (containing rapidly dividing lymphocytes and other immune cells of the gut-associated lymphoid tissues; David et al., 2003; Norrman et al., 2003).
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). There were differences (P < 0.05) among fractions of total RNA levels (crypts > villi) and of mRNA levels of InsR (crypts > villi), IGFBP-2 (crypts > villi), and IGFBP-3 (crypts > villi). In the ileum there were also marked differences in mRNA levels of members of the somatotropic axis expressed relative to housekeeping genes (Table 4). There were differences (P < 0.05) among fractions of total RNA concentrations (LP, crypts > villi) and of mRNA levels of IGF-I (LP > villi, crypts), IGF-2 and IGFBP-3 (villi > crypts, LP), GHR and InsR (crypts > LP), and IGFBP-2 (crypts > villi, LP).
Differences in the abundance of mRNA of members of the somatotropic axis and of InsR in different small intestinal layers can result from differences in production and degradation rates (i.e., turnover rates). Although, there may be positive associations, the extent to which mRNA levels of members of the somatotropic axis and InsR are related to respective protein levels was not the subject of this study. However, there may also be negative relationships, as we have recently shown for IGF1-R, IGF-2R, and InsR (Georgiev et al., 2003). Thus, the current study does not allow final conclusions about the biological importance of our findings. Nevertheless, the relatively great abundance of IGF-I mRNA in the LP fraction of the ileum is of interest. If mirrored by correspondingly high local IGF-I production, this may be evidence that IGF-I is involved in the stimulation of proliferation and differentiation of lymphocytes in PP. The greater levels of IGF-2 mRNA in jejunual villus fractions compared with crypt fractions suggest that this growth factor may exert its effects mainly on villus cells. The relatively high mRNA levels of the GHR in crypts of the ileum suggest that GH may exert its effects through stimulation of epithelial growth, consistent with data in neonatal calves showing deeper crypts after GH treatment (Bühler et al., 1998). Whether the greater mRNA levels of InsR in crypts indicate that insulin exerts effects mainly on crypt cells is an open question because numbers of insulin receptors were found to be negatively correlated with mRNA levels in neonatal calves (Georgiev et al., 2003). The relatively great abundance of IGFBP-2 in the crypt fraction also is of interest, but speculation as to its possible biological importance is not immediately evident. The same holds for mRNA levels of IGFBP-3, which were greatest in the villus fraction of the jejunum and in the crypt and LP fraction of the ileum. High levels of IGFBP-3 in the villus fraction could be expected as the mRNA of IGFBP-3 was shown by in situ hybridization to be localized within the LP of villi of the small intestine (Winesett et al., 1995; Shoubridge et al., 2001).
This study shows that there are differences between the jejunum and ileum with respect to abundance of mRNA of members of the somatotropic axis in villus and crypt fractions. Thus, mRNA levels of IGF-I, IGF-2, and GHR differed between villus and crypt fractions in the ileum, but not in the jejunum. This confirms previous data showing that there are site-specific differences in abundance of mRNA for members of the somatotropic axis in the GIT.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Footnotes |
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2 This study was in part supported by Swiss National Science Foundation (Grants 32-56823.99 and 32-67205.01).
3 Correspondence: Bremgartenstrasse 109a (phone: +41-31-6312324; fax: +41-31-6312640; e-mail: juerg.blum{at}itz.unibe.ch).
Received for publication April 5, 2004. Accepted for publication July 30, 2004.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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