Neuromuscular stimulation causes muscle phenotype-dependent changes in the expression of the IGFs and their binding proteins in developing slow and fast muscle of chick embryos

Authors


Abstract

Insulin-like growth factor (IGF) -1 and -2 and binding protein (IGFBP-2, -4, and -5) expression can be affected by several environmental factors, but the impact of movement on the IGF axis during late embryogenesis has yet to be fully characterized. Movement was promoted in chick embryos during mid-embryogenesis using 4-aminopyridine (4-AP). The results indicate an increase in IGF-1 (P < 0.01) and a decrease in IGFBP-2 (P < 0.05) mRNA expression in slow muscle of the stimulated group compared with the control group. In fast muscle, there was a decrease in IGF-2 (P < 0.01) on embryonic day (ED) 16 and an increase in IGFBP-2 (P < 0.01) and IGFBP-4 (P < 0.05) and in IGFBP-5 (P < 0.05) expression on ED18 in the stimulated group compared with the control group. These results indicate that neuromuscular stimulation with 4-AP influences IGF axis gene expression in a muscle fiber type-dependent manner. Consequences of the changes in the IGF system for each muscle are discussed. Developmental Dynamics 235:1777–1784, 2006. © 2006 Wiley-Liss, Inc.

INTRODUCTION

Our previous experiments have indicated that the developmental process of proliferation and differentiation during secondary myogenesis can be manipulated by means of the stimulation of movement in ovo during mid-embryogenesis and that excess movement in ovo increased the oxidative capacity as well as altered the fiber number immediately pre-hatch (Heywood et al., 2005). Studies in mammals have also shown that various factors, such as movement (Willis et al., 1998) and overloading of mature skeletal muscle (Awede et al., 1999), affect the regulation of the insulin-like growth factor (IGF) system. To date, there is relatively little research exploring the effects of stimulation-induced movement during mid-to-late embryonic development in vivo on the expression of genes associated with the IGF system.

Numerous studies have demonstrated that the IGF system is developmentally regulated and plays a key role in the regulation of myoblast proliferation and differentiation (for a review, see Florini et al., 1996). In chicken, IGF-1 mRNA has been detected as early as E14 in the pectoralis major muscle and its expression has been shown to decrease from embryonic day (ED) 14 until hatching and then increase from hatching to post-hatch day 28 (Guernec et al., 2004). IGF-1 treatment on myoblasts has been shown to enhance proliferation and repress the expression of myogenin, which delays differentiation (Engert et al., 1996). Overexpression experiments in chick embryos have shown that IGF-1 expands the undifferentiated myoblast population, resulting in an increase in muscle fiber number through the process of proliferation (Mitchell et al., 2002). Additionally, in ovo administration of IGF-1 has been shown to promote postnatal muscle growth (Kocamis et al., 1998).

The developmental regulation of IGF-2 in chicken muscle is less well-documented than that of IGF-1. In rodents, IGF-2 is highly expressed in fetal muscle during polyneural innervation but is markedly down-regulated postnatally from day 5 when superfluous synapses are eliminated (Ishii, 1989). An in vitro study, using avian (turkey) satellite cells, has shown that IGF-2 mRNA was expressed at higher levels during the proliferative phase, followed by a marginal reduction during differentiation (Ernst et al., 1996). Like IGF-1, IGF-2 is also a potent regulator of chicken muscle cell proliferation (Duclos et al., 1991) and differentiation (Schmid et al., 1983). However, unlike IGF-1, IGF-2 administration does not promote lean growth in young chicken (Tomas et al., 1998). It is also interesting to note that, in chicken, there is only one type of IGF receptor; therefore, both IGF-1 and IGF-2 exert their actions through the same receptor (Duclos et al., 1991).

The insulin-like growth factor binding proteins (IGFBP) are members of the IGF system. Their exact functions are still largely unclear, but they are thought to play an important role in modulating the regulatory actions of the IGFs on fetal muscle growth and development at the protein level (Delhanty et al., 1993; Jones and Clemmons, 1995; Ernst et al., 1996). There are very few studies on the IGFBPs in chicken muscle. In turkey, IGFBP-2 is expressed at higher levels during myoblast proliferation and its expression is reduced during cell differentiation in vitro (Ernst et al., 1996); similar observations have been made in a mouse cell line (Bayol et al., 2000) and in pig (Gerrard et al., 1999). IGFBP-2 has been shown predominantly to reduce both IGF-1 and IGF-2 bioactivity (Jones and Clemmons, 1995; Fligger et al., 1998).

In vitro mouse studies have shown that IGFBP-4 is expressed at higher levels during myoblast proliferation and is down-regulated during differentiation, whereas IGFBP-5 is not detectable in myoblasts but is markedly up-regulated during differentiation, suggesting that these two IGFBPs may play specialized functions in promoting either muscle proliferation or differentiation (Ewton and Florini, 1995). IGFBP-4 is believed to inhibit most IGF actions, whereas IGFBP-5 can both enhance and reduce the IGF actions (Jones and Clemmons, 1995). IGFBP-2 and IGFBP-5 have a greater affinity for IGF-2 over IGF-1, whereas IGFBP-4 binds IGF-1 and IGF-2 with the same affinity (Jones and Clemmons, 1995).

Neuromuscular stimulation in vivo has been achieved by means of the application of 4-aminopyridine (4-AP), a potassium channel blocker, that increases the release of the neurotransmitter acetylcholine (ACh) and enhances its availability at the synaptic cleft (Burley and Jacobs, 1981). Studies using this drug in mammals and birds have revealed potent effects in very small doses. Dekkers et al. (2001) demonstrated that treatment with 4-AP improves reinnervation and force production in rat muscle, whereas in avian embryonic chicks, stimulation in ovo alters the metabolism of developing muscles (Keresztes et al., 1985). At low doses, 4-AP also has been shown to have a marked effect on neuromuscular stimulation and to induce synaptic transmission in rats (Smith et al., 2000), thus increasing skeletal muscle twitch tension.

The aim of this study is to explore the effect of 4-AP–induced neuromuscular stimulation on the IGF axis in skeletal muscle of different phenotypes during a crucial phase of embryonic development in the chick, just before the onset of secondary fiber formation. In addition, we examine the role of the IGF axis as a potential mediator for movement-induced hyperplasia and determine whether the response of IGF axis gene expression is muscle type-specific.

RESULTS

mRNA Expression of the IGFs and Their Binding Proteins in Slow Muscle

There was significantly more IGF-1 mRNA expressed in the stimulated group compared with the control group in the anterior latissimus dorsi (ALD) muscle (P < 0.01) in the pooled analysis. IGF-1 mRNA expression was significantly higher 5 days post-hatch compared with ED15, 16, 19, and 20 in the control group, but only significantly higher on ED15 compared with ED16 in the stimulated group (P < 0.05; Fig. 1a). There was significantly more IGF-1 mRNA expressed in the ALD muscle of the stimulated group compared with the control group on ED15 (P < 0.01) (Fig. 1a).

Figure 1.

ae: Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses on 2μg of total RNA extracted from the anterior latissimus dorsi (ALD) muscle separated to show expression levels across time from embryonic day (ED) 15–ED20 and 5 days post-hatch (PH), illustrating insulin-like growth factor (IGF) -1 (a), IGF-2 (b), insulin-like growth factor binding proteins (IGFBP) -2 (c), and IGFBP-4 (d). Low relative expression of IGFBP-5 (e) means data from ED17 to ED20 only are presented. The control group is the solid line; the stimulated group is the broken line. Y axis is log copy number; X axis is embryonic day (ED). Results are presented as log-transformed means ± SEM (n = 4–6 per treatment group). Asterisks represent *P < 0.05, **P < 0.01 within sample days and between treatment groups. Different letters indicate statistical differences (P < 0.05) between development stages within the same treatment group: a and b represent the control group, and c and d represent the stimulated group.

There was no significant difference in total IGF-2 mRNA expression of the control compared with the stimulated group in the ALD muscle on any of the developmental stages examined (Fig. 1b). In the control group, there was no significant change in IGF-2 mRNA expression during late embryonic development. Expression was significantly higher on ED20 compared with the previous EDs in the stimulated group (P < 0.05; Fig. 1b).

There was significantly more IGFBP-2 mRNA expression observed in the control compared with the stimulated group of the ALD muscle in the pooled analysis (P < 0.01). Across sample days in the control group, there was significantly more IGFBP-2 mRNA expressed in the embryonic muscle on ED16, 17, and 18 compared with the post-hatch muscle, and this difference was only significant on ED16 compared with 5 days post-hatch of the stimulated group (P < 0.05). Within-sample day–between-treatment comparisons indicated significantly higher IGFBP-2 expression in the control compared with the stimulated group on ED18 (P < 0.01) and ED19 (P < 0.05; Fig. 1c).

There was significantly more IGFBP-4 expressed in the ALD muscle of the stimulated group compared with the control group in the pooled analysis (P < 0.01; Fig. 1d). IGFBP-4 was more highly expressed on ED18 and 5 days post-hatch compared with ED17 in the control group (P < 0.05), whereas in the stimulated group, there was significantly more IGFBP-4 mRNA expression on ED15 and 5 days post-hatch compared with ED18 and ED19. Within-sample–between-treatment comparisons indicated that there was more IGFBP-4 expressed in the stimulated group compared with the control group on ED15 and ED17 (P < 0.05), whereas the opposite occurred on ED18 (P < 0.01; Fig. 1d).

Due to an unquantifiable amount of IGFBP-5 expressed on ED15 and 16, and post-hatch day (PH) 5, these sample days are not included in the analyses. Total mRNA expression of IGFBP-5 was relatively low compared with expression levels of the other IGFBPs in the ALD muscle, and there was no significant treatment effect on the stimulated group compared with the control group of ALD muscle (Fig. 1e).

mRNA Expression of the IGFs and Their Binding Proteins in Fast Muscle

In the posterior latissimus dorsi (PLD) muscle, there was a significant down-regulation of IGF-1 expression on ED20 compared with the other pre-hatch sampling points (ED15–ED19), after which at 5 days post-hatch, IGF-1 mRNA expression recovered to a similar expression level observed pre-ED19 in both the control and stimulated groups (P < 0.05; Fig. 2a). However, between-treatments–within-sample day comparisons revealed no significant difference due to 4-AP treatment. There was more IGF-2 mRNA present in the PLD muscle of the control group compared with the stimulated group (P < 0.01) on ED16 (Fig. 2b). Within the control group, there was no significant difference across sample days in IGF-2 mRNA expression. There was a significant down-regulation of expression from ED15 to ED16 in the stimulated group (P < 0.01), which is recovered and maintained from ED17 through to 5 days post-hatch (Fig. 2b).

Figure 2.

ae: Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses on 2 μg of total RNA extracted from the posterior latissimus dorsi (PLD) muscle separated to show expression levels across time from embryonic day (ED) 15–ED 20 and 5 days post-hatch (PH), illustrating insulin-like growth factor (IGF) -1 (a), IGF-2 (b), insulin-like growth factor binding proteins (IGFBP) -2 (c), and IGFBP-4 (d). Low relative expression of IGFBP-5 (e) means data from ED15 to ED18 only are presented. The control group is the solid line; the stimulated group is the broken line. The Y axis is the log copy number; the X axis is the embryonic day. Results are presented as log-transformed means ± SEM (n = 4–6 per treatment group). Asterisks represent *P < 0.05, **P < 0.01 within sample days and between treatment groups. Different letters indicate statistical differences (P < 0.05) between development stages within the same treatment group: a and b represent the control group, and c and d represent the stimulated group, except for Figure 2a, where different letters indicate statistical differences between developmental stages for both control and stimulated groups as expression profiles were similar in control and stimulated groups.

Total IGFBP-2 mRNA expression was up-regulated in the PLD muscle of the stimulated group compared with the control group in the pooled analysis. Expression was maintained at a relatively constant level in the stimulated group throughout developmental stages, but through normal development, expression fluctuated and a sharp increase was observed on ED17 compared with ED16, 18, and 19 (P < 0.05). IGFBP-2 mRNA expression was significantly higher in the stimulated group on ED16 (P < 0.01) and ED18 (P < 0.01) compared with the control group (Fig. 2c). There was significantly more IGFBP-4 mRNA expressed in the PLD of the stimulated group compared with the control group in the pooled analysis. IGFBP-4 expression was significantly up-regulated in the control group at ED20 compared with ED19, and this level was maintained through to PH5, whereas its expression was down-regulated from ED16 to ED20 in the stimulated group (Fig. 2d). Within-sample day–between-treatment comparisons, indicated more IGFBP-4 expression in the stimulated group compared with the control group on ED17 and ED19 (P < 0.05; Fig. 2d).

IGFBP-5 mRNA expression was significantly elevated in the stimulated fast muscle compared with the control group on ED18 (P < 0.05) and followed a similar pattern of expression in both the control and stimulated groups (Fig. 2e). However, post-ED18, IGFBP-5 expression was too low to be quantified using the analytical procedures used in the current study (Fig. 2e).

DISCUSSION

Given that increased movement in ovo affects skeletal muscle development and growth (Heywood et al., 2005) and that the IGF system is largely involved in the regulation of myogenesis (Florini et al., 1996), we examined whether the effects of movement on muscle development could be partially mediated through the IGF system. We also took into account that the IGF system may respond differently to movement, depending on the phenotype of the muscle. In effect, the experiment has demonstrated that (1) neuromuscular stimulation during a key phase of myogenesis can alter the level and timing of the expression of genes associated with the IGF axis and, (2) the expression of genes associated with the IGF axis undergoing neuromuscular stimulation is muscle phenotype-specific.

IGF System Is Differently Regulated During the Development of Slow and Fast Muscles

To date, very little information is available regarding the developmental regulation of the IGF system in chicken. In this study, results revealed that IGF-1 mRNA expression was not significantly altered between ED15 and ED20 in the slow muscle, whereas it was down-regulated from ED19 until ED20 and up-regulated again at PH5 in the fast muscle. The IGF-1 expression profile in the fast muscle followed a pattern previously described by Guernec et al. (2004), who also reported a down-regulation of IGF-1 expression around the time of hatching in the fast breast muscle. Given the role of IGF-1 in regulating myoblast proliferation and differentiation (Florini et al., 1996) as well as promoting neurite growth (Caroni et al., 1994), different expression profiles in the slow and fast chicken muscles may reflect the differences associated with the development of distinct slow and fast phenotypes.

The levels of IGF-2 mRNA remained constant between ED15 and PH5 in both the slow and fast muscles, indicating that the developmental regulation of IGF-2 may not be linked to the development of slow and fast phenotypes. The pattern of expression of IGF-2 in developing chicken muscles differed from observations made in the developing rodent muscles, whereby IGF-2 expression correlated with patterns of muscle innervation (Ishii, 1989), suggesting that IGF-2 might play different functions in rodents and chicken. This explanation could be attributed to the fact that, in rodents, the IGF-2 actions are mediated through two different receptors, namely, the IGF-1 receptor and the IGF-2 receptor, for which IGF-2 has a greater affinity (Florini et al., 1996), whereas chicken only possesses one type of IGF receptor, which is similar to the IGF-1 receptor (Duclos et al., 1991).

In the slow muscle, IGFBP-2 mRNA was expressed at higher levels during the early stages of development (E16–E18) compared with the PH5 stage, which is consistent with previous reports showing a down-regulation of IGFBP-2 during muscle differentiation in mammals (Ernst et al., 1996; Gerrard et al., 1999; Bayol et al., 2000). However, the pattern of IGFBP-2 mRNA expression differed somewhat in the fast muscle, highlighting differences in the developmental regulation of IGFBP-2 in slow and fast muscles.

There was some fluctuation in IGFBP-4 mRNA expression throughout the developmental stages examined in both muscles; however, the pattern of expression was different between the slow and fast muscles, suggesting that again the developmental regulation of IGFBP-4 differed between these two muscles. IGFBP-4 has been reported to be expressed at higher levels in proliferating myoblasts than in differentiating myotubes in vitro (Ewton and Florini, 1995), and the increased expression from ED17 to ED18 may reflect a wave of proliferation in the slow muscle at this particular stage of development.

IGFBP-5 was not detected during the early stages of development (E15–E16) in the slow muscle, which is consistent with previous in vitro reports of very low IGFBP-5 expression in myoblasts followed by a marked up-regulation during myotube differentiation (Ewton and Florini, 1995; Bayol et al., 2000). In both slow and fast chick muscles, IGFBP-5 was no longer detectable at PH5, which again is consistent with the marked down-regulation observed around the time of birth in the pig (Gerrard et al., 1999). In the fast muscle, IGFBP-5 became detectable earlier during development, i.e., from ED15 instead of ED17, but was also down-regulated earlier than in the slow muscle, i.e., at ED18 instead of ED20. This finding suggests that IGFBP-5 expression was delayed in the slow muscle compared with the fast muscle. The significance of this result is unclear at this stage, but because IGFBP-5 is associated with myotube differentiation, this finding may indicate delayed muscle differentiation in the slow muscle compared with fast muscle. This study therefore confirms that the genes that constitute the IGF system are developmentally regulated in chicken and shows that their pattern of expression differs between slow and fast muscles.

Increased Movement Differently Alters the IGF System in Slow and Fast Muscles

In the slow muscle, increased movement was accompanied by an overall increase in IGF-1 mRNA with a specific up-regulation at ED15, i.e., shortly after 4-AP treatment. As IGF-1 is known to promote cell proliferation and differentiation, it thus appears that an up-regulation of this gene upon treatment may constitute a mechanism for the increased nuclear and fiber number previously reported (Heywood et al., 2005). The increase in IGF-1, however, was not accompanied by an increase in IGF-2 in the slow muscle, suggesting that the effect of movement on muscle development are unlikely to be directly mediated through IGF-2 expression levels.

The marked up-regulation of IGF-1 at ED15 upon treatment in the slow muscle was followed by a progressive down-regulation of IGFBP-2 mRNA compared with the control group, which reached statistical significance at ED18 and ED19 before returning to control levels from ED20. Because IGFBP-2 is believed to predominantly inhibit the IGF actions, a down-regulation of this inhibitor would contribute to increased IGF bioavailability in the treated muscle. In contrast, IGFBP-4, another inhibitor of IGFs, was up-regulated in the treated group. Of interest, a similar up-regulation of IGFBP-4 has been observed in electrically stimulated rodent myotubes (Bayol et al., 2005) and stretched muscles (Awede et al., 1999) as well as during denervation in vivo (Bayol et al., 2000), indicating that IGFBP-4 expression is increased in response to both increased and decreased muscle activity. It has been proposed that IGFBP-4 may protect cells from IGF overstimulation or allow the activation of alternate signaling pathways, which are inhibited by the IGFs (Jones and Clemmons, 1995). Our results indicate that this finding may also be the case in developing chicken muscles in which increased movement is induced. Unlike IGFBP-2 and IGFBP-4, IGFBP-5 expression was not affected by 4-AP treatment in the slow muscle, suggesting that this binding protein may have little function in the slow muscle adaptations to increased movement. Overall, the data suggest that increased movement may trigger an IGF-1 response locally regulated by IGFBP-2 and IGFBP-4, which may contribute to the physiological adaptation of the slow muscle in response to 4-AP treatment.

In the fast muscle, the IGF system was differently affected upon 4-AP treatment compared with the slow muscle. IGF-1 was not affected by the treatment, and IGF-2 was down-regulated after 4-AP exposure, particularly at ED16. This finding was accompanied by the up-regulation of IGFBP-2 and IGFBP-4, both predominant inhibitors of IGFs, in the stimulated group. Based on the current knowledge of the IGF system, unchanged IGF-1 and decreased IGF-2 combined with increased IGFBP-2 and IGFBP-4 would indicate a reduction in IGF bioavailability in the stimulated group, which surprisingly may indicate decreased cell proliferation and differentiation in the fast muscle after treatment. Nevertheless, IGFBP-5, a regulator of muscle differentiation (Ewton and Florini, 1995), was markedly up-regulated at ED18 in the stimulated group compared with the control group, suggesting possible enhanced muscle differentiation upon treatment from ED17 and ED18. Taken together, these results suggest a down-regulation of the IGF actions in the fast muscle upon treatment, which is almost the opposite situation of that observed in the slow muscle. Based on the current knowledge of the IGF functions, a diminution of IGF bioavailability in the fast muscle would lead to impaired muscle development and growth, which is contradictory to the myotrophic effects of the drug as previously observed in the mixed semitendinosus muscle (Heywood et al., 2005). There is no clear explanation for this finding, but the data suggest that the effects of increased movement on muscle development and growth may be differently mediated in fast and slow muscles.

In conclusion, neuromuscular-stimulated movement during embryonic development, therefore, may have a significant influence on the post-hatch muscle phenotype and potential post-hatch growth. Neuromuscular stimulation leads to an altered timing (through either up- or down-regulation) of the expression of genes associated with the IGF axis that is muscle phenotype dependent.

EXPERIMENTAL PROCEDURES

Animals

A total of 148 Fertile White Leghorn (Gallus domesticus) eggs (Joice & Hill Poultry Ltd., Norfolk, UK) were incubated in a static air incubator at 37.5°C ± 0.5 and relative humidity of approximately 65–70% throughout embryonic growth. Eggs were turned four times each day (every 3–4 hr) to promote normal growth and development. On ED9, the eggs were windowed above the air sac (5 mm diameter) and sealed with adhesive tape to limit possible infection of the embryo from the outer environment. After the windowing procedure, the eggs were divided into a control (C) group, which received 100 μl of phosphate buffered saline (PBS) on ED10, 11, 12, and 13, and a stimulated (S) group, which received 0.1 mg of 4-AP in 100 μl of sterile PBS on ED10 and 11, and 0.2 mg of 4-AP in 100μl of sterile PBS on ED12 and 13. The window was sealed with fresh adhesive tape after each treatment, and the eggs were returned to the incubator for the remainder of their embryonic development. The concentration of 4-AP used was based on the drug's half-life and on work done by Osborne (2000) in which a stimulatory effect on movement was observed. In the present study, the concentration of the drug was modified and reduced by 50% for the first 2 days of treatment (days 10 and 11). This concentration proved to be adequate to promote excess muscular twitching, which was observed shortly after treatment by candling the eggs, while significantly reducing the percentage of treatment-related mortality noted previously (Heywood et al., 2005). Table 1 summarizes the number of embryos used and the mortality rates over the course of the experiment.

Table 1. Total Sample Number and Mortality Rates of the Control and 4-Aminopyridine Stimulated Embryos
GroupNumber of eggsInfertile/did not develop (both groups)Pre-day 10 mortalityPost-day 10 mortality% mortality in control and treated embryos post-day 10
Control65120816%
Stimulated83 016 

Pre-hatch Sampling

Embryos from E15 to E20 were killed daily by decapitation, and the body mass of embryos (including the yolk sac) at all ages was recorded (n = 4–5 per day; per group). On each day of sampling, the right and left (ALD) muscle and the right and left (PLD) muscle were dissected from each embryo, representing the slow and fast muscle types, respectively (Gardahaut et al., 1988). The muscles from the right side were wrapped in ethanol-sprayed foil and snap frozen immediately in liquid nitrogen for mRNA expression analysis.

Post-hatch Material

The remaining 20 eggs were transferred to the Biological Science Unit, Royal Veterinary College, for hatching and rearing. Hatched chicks were sexed at 1-day-old and given feed and water ad libitum. Chicks were tagged for individual identification and weighed daily to assess rate of daily body mass gain. On day 5 post-hatch, three males and three females from each group were killed by cervical dislocation and the anterior and PLD muscles were dissected from each chick and treated in the same way as the embryonic muscles.

RNA Extraction

Total RNA was extracted from the muscle tissue samples using tri-reagent as recommended by the supplier (Sigma-Aldrich, UK). The RNA was dissolved in RNAse-free water and a subsample of the RNA extract was run on an ethidium bromide gel to ensure that there was no RNA degradation. The RNA samples were purified using the RNeasy mini purification kit with DNAse treatment to remove genomic DNA contamination following the manufacturer's recommended protocol (Qiagen, UK). The purified RNA was quantified at 260 nm using gene spectrophotometry (GeneSpec I, Naka Instruments, Japan) to measure the concentration of total RNA in each sample.

cDNA Synthesis

Using the Omniscript reverse transcription kit (Qiagen, Crawley, UK), reverse transcription was performed on all the samples to produce cDNA. All samples for each muscle group across all sample days were processed on the same day using the same master mix. A total of 2 μg of total RNA were used in each reverse transcription reaction in RNAse-free water in a total volume of 10 μl and denatured by heating to 65°C for 5 min before transfer to ice. The samples were then mixed with 2 μl of first-strand buffer (×10), 2 μl of deoxynucleotide triphosphates (dNTPs; 5 mM each), 50 pMol of random primers, 1 μl of RNase inhibitor (10 units μl−1), and 1 μl of Omniscript reverse transcriptase enzyme (4 units μl−1). The reaction volume was made up to 20 μl using RNAse-free water. The samples were then incubated at 37°C for 1 hr followed by heating for 5 min at 95°C to inactivate the reverse transcriptase (RT). The synthesized cDNA was stored at −20°C until subsequent analysis by real-time polymerase chain reaction (PCR).

Real-Time Quantitative PCR

RT-PCR was performed using LightCycler technology as described by Hameed et al. (2003) and further developed according to Bayol et al. (2004). A 20-μl reaction mix contained 10 μl of a SYBR green mix (QuantiTect, Qiagen, Crawley, UK), 10 pMol of each forward and reverse primer, 2 μl of cDNA (made from 1 μg of RNA), and nuclease-free water to make up the reaction volume. The primers used for real-time PCR were designed from the mRNA sequences available on the NCBI nucleotide database, using the Primer3 Web interface (Rozen and Skaletsky, 2000) and primers were synthesized by MWG Biotech (Ebersberg, Germany). Where possible, the sequences amplified were checked for the spanning of two exons to prevent amplification of introns. The sequences of the primers used are given in Table 2. The Quantitect SYBR Green PCR kit (Qiagen, Crawley, UK) was used, and the reactions amplified using the DNA Opticon Engine (GRI, Braintree, UK) as recommended by the manufacturers. SYBR Green I dye was used to detect double-stranded DNA (dsDNA). PCR data were calculated as copy number in 2μg of total RNA relative to the standard curve of known concentrations. Target specificity was further confirmed by running samples on an agarose gel. PCR runs that yielded a standard curve with an r value greater than 0.95 were used for analysis, as a good r value was deemed to reflect only marginal pipetting error.

Table 2. Forward and Reverse Primer Sequences Used in the Real-Time RT-PCR Analyses for IGF-1 and IGF-2 and IGFBP-2, IGFBP-4, IGFBP-5a
Primer nameSequence (5′ to 3′)Product size (base pair number)Accession no.
  • a

    RT-PCR, reverse transcriptase-polymerase chain reaction; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; For, forward; Rev, reverse.

IGF-1_ForCCC AGA AAC ACT GTG TGG TG126XM_416323
IGF-1_RevATT CCC TTG TGG TGT AAG CG  
IGF-2_ForCAA CGT GTG GCA GAA GAA GA240AF218827
IGF-2_RevTCC CCA GGA GAT CAC AAA TC  
IGFBP-2_ForGAG CAT CCT TGC TGA GAA CC177NM_205359
IGFBP-2_RevTTT GAG TCC TCG TGG TTG TG  
IGFBP-4_ForGAG AAG GAG GAG ATC GAG CA253AB192413
IGFBP-4_RevGGA TGG GGA TGA CGT AGA GA  
IGFBP-5_ForCAA CCT GGG CCT TGT ACT GT172L27560
IGFBP-5_RevAGG AGG CCA GAG TCT GTG AA  

Statistical Analysis

All data sets were checked using residuals to ensure a normal distribution of the data before statistical analysis. PCR data that deviated from a normal distribution were log-transformed before analysis of variance (ANOVA) and are presented as log copy number in the tables below. Data were analyzed for treatment, day, and interaction effects using the general linear model (ANOVA) in Minitab Release Software 12.21 (1998). The software identified data outliers that were more than two standard deviations from the mean and these data were removed from further analysis. Data analysis for treatment × day interaction allowed us to generate means and standard errors for each treatment group on each embryonic day for the PCR data. Where an interaction effect was found, the control and the stimulated groups were further analyzed using Tukey's pairwise comparisons to pinpoint true statistical differences between experimental groups within days. These data, indicating the change in gene expression across time, are presented in the figures. The level of statistical significance was taken as P < 0.05. In all cases, data are presented as means ± SEM.

Acknowledgements

The input of Dr. Esther Walters and Jane Heywood in the early stages of this work is gratefully acknowledged. The BBSRC is acknowledged for its funding of this research.

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