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RESEARCH |
Reproduction, Genes and Development Group, Department of Veterinary Basic Sciences, Royal Veterinary College, London, AL9 7TA, UK1 Animal Production Research Centre, Mellows Campus, Athenry, Co. Galway, Ireland2 School of Agriculture, Food Science and Veterinary Medicine, University College, Dublin, Ireland and3 Teagasc Moorepark, Dairy Production Research Centre, Fermoy, Co. Cork, Ireland
Correspondence should be addressed to D C Wathes; Email: dcwathes{at}rvc.ac.uk
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Abstract |
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/β. Expression of IGFBP-2 and IGFBP-6 (both of which have a high affinity for IGF-II) was decreased in SNEB relative to MNEB (P<0.05). No other gene was altered by NEB, but IGF-II, IGFBP-3, IGFBP-5 and IGFBP-6 all showed differential expression in different regions of the oviduct. These results indicate that, in addition to low circulating IGF-I after calving, NEB may also influence IGF availability in the oviduct indirectly through changes in specific IGFBP expression. It is possible that the predicted increased signalling by IGF-II may perturb embryo development, contributing to the high rates of embryonic mortality in dairy cows. |
Introduction |
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Previous work has shown that amongst the metabolites and metabolic hormones that change in relation to NEB, insulin-like growth factor-I (IGF-I) is strongly associated with the calving to conception interval and pregnancy outcome (Wathes et al. 2003, Taylor et al. 2004a). The nutritional sensitivity of IGF-I is widely reported (Thissen et al. 1994) and we and others have shown that severe NEB (SNEB) during early lactation attenuates gene transcription of IGF-I and also a number of IGF-related members in the liver, producing an endocrine environment of reduced IGF-I stability and bioavailability (Lucy et al. 2001, Fenwick et al. 2006). IGFs have a broad range of functions to promote differentiation, stimulate mitogenesis and inhibit apoptosis (Jones & Clemmons 1995). The IGF system comprises a second ligand (IGF-II), two receptors (IGF-1R and IGF-2R) and at least six binding proteins (IGFBP). This complexity serves to enable differential regulation of the IGF system within different tissues (Clemmons 1997).
Transcripts for IGF-I and IGF-II have been detected in the oviduct of ruminants (Schmidt et al. 1994, Stevenson & Wathes 1996), primates (Gabler et al. 1998) and rodents (Carlsson et al. 1993). Specific localisation studies have further shown that IGF-I expression mainly predominates in the mucosa and IGF-II in the muscularis (Pfeifer & Chegini 1994, Pushpakumara et al. 2002). Purified isolates of oviductal epithelial cells produce and secrete both IGF-I and IGF-II (Wiseman et al. 1992) as well as the regulatory binding proteins IGFBP-2 and IGFBP-3 (Giudice et al. 1992). IGF-I and IGF-II in a variety of species are generally regulated in a positive manner with ovarian oestradiol production (Wiseman et al. 1992, Carlsson et al. 1993, Stevenson & Wathes 1996, Gabler et al. 1998). The presence of an IGF system in the oviduct suggests that the IGFs may act as important modulators of oviductal function.
For many tissue types, support and maintenance rely on the presence of an adequate IGF system which may be derived from both an endocrine source like the liver or produced locally in a paracrine/autocrine manner. Circulating IGF-I concentrations fall in the immediate postpartum period in dairy cows and may not return to pre-partum levels for more than 12 weeks in some cows (Taylor et al. 2004a). Restoration of oviductal activity therefore occurs in an endocrine environment of low IGF-I. Waves of follicular activity resume after about 10–14 days (Roche & Diskin 2001) but progesterone concentrations remain low until after the first ovulation. In dairy cows from the same herd as that used for the present study, the interval to commencement of luteal activity was 31±15 days (mean±S.D.) and was shorter in cows having higher IGF-I concentrations in the first 2 weeks after calving (Patton et al. 2007). The uterus is also undergoing involution during this period, a process which is normally complete in 30–50 days (Gier & Marion 1968). Microbial contamination of the bovine uterus is almost universal during the first week postpartum, and the rate of bacterial clearance also affects the return to normal cyclicity (Sheldon et al. 2006).
At present, virtually nothing is known about the potential regulatory effects of NEB on oviduct function and we postulated that the global effects of NEB might influence local gene expression during the recovery period. The specific aims of this study were thus to determine the effects of NEB on oviductal IGF transcript expression during the early postpartum interval.
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Effect of NEB on the expression of IGF family members and related hormone receptors in the oviduct
The real-time RT-PCR data, based on whole oviducts, revealed that expression of IGFBP-2 and IGFBP-6 was significantly lower in the SNEB cows (Table 2). There were no other differences according to EB status for other members of the IGF family or in the other receptors examined (insulin receptor type A/B, IR-A/B, oestrogen receptor /β, ER/β, glucocorticoid receptor , GR and growth hormone receptor, GHR). The concentration of 18SrRNA measured was similar between groups. IGF-2R was only detected in 3 out of 12 oviducts, and IGFBP-1 was undetectable in 10 out of 12 samples (data not shown). These findings were closely confirmed by analysis of the in situ hybridisation data from the opposite oviduct (Table 3). Using this technique, there was a highly significant (P=0.009) reduction in IGFBP-6 expression in the SNEB cows, and a strong tendency towards reduced IGFBP-2 expression in the same cows (P=0.068). Overall, levels of the IGFBP-6 transcript were reduced in SNEB cows by more than twofold relative to MNEB cows as detected by both real-time PCR and in situ hybridisation. No other members of the IGF family examined showed significant differences according to EB status.
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Gene expression relationships in the postpartum oviduct
When values obtained from quantitative RT-PCR were considered regardless of treatment, a number of traits were identified that exhibited positive relationships in the postpartum oviduct (Fig. 3; all P=0.01). In particular, expression levels of IGF-I and GHR were strongly associated with each other (r=0.921) and levels of both of these gene transcripts were individually associated with IGF-1R and ERβ (see Fig. 3). Expression of the IGF-I transcript was also associated with IGF-II and GR transcript levels and GHR was associated with IGFBP-6 transcript levels. ERβ levels were positively associated with GR (r=0.912) and IGF-1R (r=0.761), while IR-B levels were positively associated with IR-A (r=0.835) and IGFBP-5 (r=0.772) (all P=0.01, not shown). No relationships were detected between gene expression and circulating oestradiol concentrations in individual cows.
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Discussion |
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The liver is the major source of IGF-I and IGF-II in the circulation (Zapf & Froesch 1986). In our model, the production of hepatic IGF-I was severely restricted in SNEB cows in association with a reduction in GHR but hepatic IGF-II mRNA was not significantly changed, so the ratio of IGF-II:IGF-I mRNA within the liver was increased. These data are consistent with those of others which showed a decline in GHR and IGF-I in the periparturient period (Radcliff et al. 2003). Circulating IGF-II was not measured in the present experiment, but concentrations of IGF-II were about fourfold higher than those of IGF-I during pregnancy in heifers, and showed a lesser response to alterations in dietary protein (Perry et al. 2002). In our animals, these changes occurred alongside elevated hepatic concentrations of IGFBP-2 mRNA and a reduction in both IGFBP-3 and the acid-labile subunit (Fenwick et al. 2006).
One assumption is that, under these circumstances, IGFBP-2 forms a binary complex with IGF-II in order to facilitate circulatory transport to peripheral tissues and allow translocation of IGF-II through the capillary endothelium (Bar et al. 1990). Within the oviduct, there was local expression of both IGF-I and IGF-II mRNAs, although in this tissue neither differed in relation to EB status. Oestradiol appears to be the major regulator of IGF-I within the reproductive tract (Murphy & Friesen 1988) and in oviduct IGF-I mRNA increases at oestrus (Stevenson & Wathes 1996). In rats, GH can also increase uterine IGF-I expression (Murphy & Friesen 1988) although Pershing et al. (2002) could detect no effect of bovine somatotrophin (bST) treatment on bovine endometrial IGF-I expression. However, in this latter experiment, bST did increase IGF-II mRNA in the oviduct on day 3 of the cycle. Our results across EB groups showed highly significant positive correlations between RT-PCR values for IGF-I, ERβ and GHR mRNAs, suggesting that both oestradiol and GH may be involved in the control of oviductal IGF-I production. The IGF-I mRNA concentration was also highly correlated with that of the glucocorticoid receptor. Follicular fluid contains high concentrations of cortisol, and it has been suggested that when this is released at ovulation it may influence the activity of the oviduct (Yding Anderson 2002). In late pregnant ewes, the surge release of cortisol induces a switch from IGF-II to IGF-I expression in foetal liver (Fowden 1995), but this effect has not, to our knowledge, been investigated in the reproductive tract.
Previous evidence suggests that expression of IGF-I in the bovine oviduct is comparatively low, with greater production of IGF-II (Watson et al. 1999, Pershing et al. 2002). Isolated oviduct epithelial cells are capable of producing large amounts of IGF-II in culture (Wiseman et al. 1992, Xia et al. 1996, Winger et al. 1997), and in the pig IGF-II levels in the oviductal fluid fluctuate in a cyclical manner and reach a peak just prior to oestrus (Wiseman et al. 1992). Given the intrinsic ability of IGF-II to stimulate mitogenesis and prevent apoptosis, locally produced IGF-II may promote proliferation and metabolic activity of the oviduct itself (Tieman & Hansen 1995) and is also likely to be secreted into the lumen to influence development of the early embryo. The IGF-1R is detectable within bovine embryos from the two-cell stage onwards (Lonergan et al. 2000).
The local expression of specific IGFBPs within individual tissues can provide information on IGF availability due to their intrinsic binding preferences. IGFBP-2 exhibits a relatively stronger affinity for IGF-II over IGF-I and likewise IGFBP-6 has an extreme (20- to 100-fold) preference for IGF-II (Bach 1999). Although the functional consequences of these relationships may vary, degradation of bound IGFBPs by proteolytic cleavage allows ‘free IGF’ to gain access to their receptors. IGFBP-6 is more resistant to proteolysis and studies have shown that the effects of IGF-II are generally inhibited by the presence of IGFBP-6 (Grellier et al. 2002, Bach 2005). A relative reduction of oviductal IGFBP-6 and IGFBP-2 expression during SNEB may thus increase IGF-II signalling in the oviduct to counteract the low levels of circulating IGF-I during this time. This could act as a safety mechanism by allowing IGF-II to counteract the effects of NEB. Alternatively, as discussed below, this switch might have adverse consequences for fertility.
The concurrent reduction in IGFBP-2 and IGFBP-6 mRNA expression from opposing oviducts provides support for a systemic effect of NEB on the reproductive tract. Both IGFBP-2 and IGFBP-6 are sensitive to changes in reproductive hormones as shown by concurrent up-regulation during induced luteolysis in the bovine ovary (Neuvians et al. 2003). A temporal effect on uterine IGFBP-6 expression levels during the oestrous cycle has also been demonstrated in the sheep, with maximal concentrations around ovulation and the early luteal phase (Gadd et al. 2002). In the rat, a putative oestrogen receptor-binding site has been characterised in the promoter region of the IGFBP-6 gene (Zhu et al. 1993). Furthermore, ovariectomy can attenuate IGFBP-6 expression in bone and uterus (Higashi et al. 1996, Gadd et al. 2002), whereas administration of oestrogen increased IGF-II in embryonic cells (Kuhl et al. 2003). In the study presented here, however, there were no differences in either plasma oestradiol or oviductal ER or ERβ expression according to EB status. Across treatments, there was a significant positive relationship between IGFBP-6 and expression of GHR, however, neither GH concentrations nor GHR differed between the groups. Another potential regulatory agent is retinoic acid, which increases IGFBP-6 mRNA synthesis in a number of cell lines (Bach 1999). Major changes in retinol metabolism occur in the immediate postpartum period in dairy cows (Horst et al. 1995, Van Merris et al. 2004), but were not assessed in the present study. In relation to IGFBP-2, there is considerable evidence that it is nutritionally responsive in certain tissues (Vicini et al. 1991, Clemmons 1997). In support of this, we recently showed that IGFBP-2 mRNA was down-regulated in granulosa cells of SNEB cows, further implicating the regulation of this binding protein by energy balance in reproductive tissues (Llewellyn et al. 2007).
In general, there was excellent agreement between the PCR and in situ hybridisation data, even though the results for each technique were obtained from the opposing oviducts in each cow. For the PCR analysis, oviducts had been frozen whole, whereas the latter technique offered the additional benefit that it was possible to distinguish the different regions. When data from both treatments were pooled, overall analysis indicated that IGF-II expression was stronger in the isthmus when compared with the remainder of the oviduct, while IGFBP-6 showed the opposite with the weakest expression in the isthmus. Furthermore, the relative reduction of IGFBP-6 in SNEB oviducts was specifically attributable to a change in expression in the ampullary region. Based on the in situ hybridisation data, IGFBP-3 expression levels were significantly higher in the isthmus relative to the infundibulum. In the liver, IGFBP-3 expression is regulated concurrently with IGF-I (Woelfle & Rotwein 2004) and therefore elevated levels of IGFBP-3 suggest a relationship with relatively high levels of uterine IGF-I as previously reported (Pershing et al. 2002). These differences in expression patterns along the oviduct are likely to reflect the different structure and functions of these two regions. The ampulla has more secretory tissue and is functionally important for sperm capacitation and fertilisation, whereas the isthmus contains more smooth muscle and is important for sperm transport (Ellington 1991, Hunter 2003).
Several studies have detected IGFBP-1 expression in human oviduct samples (Pfeifer & Chegini 1994, Qiu et al. 2003), which was reduced in response to progesterone in culture (Davies et al. 2004). The undetectable levels of IGFBP-1 transcript in the bovine in this study and others (Winger et al. 1997, Pushpakumara et al. 2002) therefore appear to reflect species variation. The type-2 IGF receptor (IGF-2R) has not been studied in the oviduct previously and was only detected in 3 out of the 12 oviducts by quantitative RT-PCR. Transcript expression for other genes that can affect or mediate IGF transcription and signalling such as the receptors for insulin, IGF-I, GH, cortisol and oestrogen all remained unaltered by NEB.
In conclusion, the IGF system is relevant to many aspects of reproductive physiology and the involvement of these growth factors in the oviduct provides a potential link between nutritional status and fertility. We have shown that SNEB reduces both circulating IGF-I and the local expression of IGFBP-2 and IGFBP-6 (but not IGF-I or IGF-II) within the oviduct. This would predict enhanced IGF-II signalling, although conception rates are reduced in cows in NEB (Wathes et al. 2007). Many studies have investigated the effects of different culture systems for in vitro produced embryos. These have highlighted the fact that rates of embryo development and relative transcript abundance are both strongly influenced by the culture conditions (e.g. Thompson 2000). Furthermore, a suboptimal environment for as little as 1 day can perturb subsequent long-term development potential (Lonergan et al. 2003). IGF-II is an imprinted gene that mediates growth of early embryos and is known to have many profound effects on the formation of the foetus and placenta (Fernandez-Gonzalez et al. 2007). Foetal calf serum has been a common constituent of embryo culture medium. It contains active IGF-II (Motoike & Unsicker 1999), causes faster development to the blastocyst stage (Gutierrez-Adan et al. 2001), but ultimately results in birth of fewer normal calves (Farin et al. 2001). The fine control of energy metabolism by the embryo at this stage is also of crucial importance (Thompson 2000). In the light of our results, we speculate that embryos from cows in SNEB experience altered IGF signalling within the oviduct, which likewise has adverse effects on their development, contributing to the high rates of embryo mortality seen in high-yielding dairy cows.
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Materials and Methods |
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Animals and tissue collection
All procedures were carried out under license in accordance with the European Community Directive, 86-609-EC. From an initial pool of 24 Holstein–Friesian cows with an average previous lactation yield of 6477±354 kg, 12 multiparous cows were randomly allocated 2 weeks prior to expected calving to either a MNEB or SNEB treatment, taking into account their parity, body condition score and previous lactation yield in a randomised block design. The mean parity numbers were 4.3 and 5.2 for the MNEB and SNEB groups respectively and the mean body condition scores (5-point scale with 0.25 increments) at calving were 3.5 and 4.0, with an overall ranges of 3.0–4.5. The treatments began on the morning after the second or third milking following parturition. MNEB cows were fed grass silage ad libitum with 8 kg/day of a 21% crude protein dairy concentrate and milked once daily; SNEB cows were fed 25 kg/day silage with 4 kg/day concentrate and milked thrice daily. Similar treatment groupings were shown in a previous study to produce differences in EB in early lactation (Patton et al. 2006).
Energy balance was estimated as the difference between energy intake and the sum of energy for maintenance and milk production, based on measurements of milk yield, milk composition, liveweight and feed intake. The French NE system was used, where 1 unité fourragère lait (UFL) is the net energy for lactation equivalent of 1 kg standard air-dry barley (Jarrige 1989). The diameter of the first dominant follicle was determined for each cow from day 7 postpartum via daily transrectal ovarian ultrasonography (Aloka SSD-500 7.5 MHz transducer; Aloka Ltd, Tokyo, Japan). Cows were slaughtered on days 6–7 of the first follicular wave after calving. This design was chosen to collect tissue at a fixed time in relation to ovarian function to allow direct comparisons between tissues at the same stage of follicle development. Oviducts were collected at slaughter by manual separation from the ovary and cutting through the utero-tubal junction to separate from the uterus. Connective tissue was removed by dissection. The oviduct ipsilateral to the dominant follicle was frozen whole in liquid nitrogen for subsequent RNA extraction. For in situ hybridisation studies, the contralateral oviduct was coiled into a cardboard ring containing Cryo-M-Bed (Bright Instruments Co. Ltd, Huntingdon, UK) and placed on a sheet of metal cooled on dry ice. Once this had solidified, the rings were put into isopentane cooled in liquid N2 to freeze. Both sets of samples were then stored at –80 °C. The diameter of both uterine horns was measured 
5 cm anterior to the bifurcation of the uterus, as an indicator of the process of uterine involution.
Blood samples were collected just prior to slaughter into lithium–heparin-primed vials and immediately placed on ice before centrifugation at 2000 g for 10 min. Plasma was decanted and stored at –20 °C for subsequent analysis. Samples of plasma were analysed for glucose, NEFA, BHB and urea using appropriate kits and an ABX Mira autoanalyser (ABX Mira, Montpellier, France). Concentrations of plasma IGF-I, insulin, GH and oestradiol were assayed as described previously (Taylor et al. 2004b, Fenwick et al. 2006, Llewellyn et al. 2007). Briefly, IGF-I concentrations were determined using the human OCTEIA IGF-I kit (IDS, Tyne and Wear, UK), the inter-assay coefficient of variation (CV) was 8.7% and the intra-assay CV values were 8.7 and 16.8% for samples with mean values of 81.5 and 4.7 ng/ml respectively. The sensitivity of the assay was 1.9 ng/ml. Insulin concentration in plasma was assayed using a solid-phase RIA (Coat-a-count; Diagnostics Products Corp., Los Angeles, CA, USA). The inter- and intra-assay CV values were 14.2 and 9.8% respectively. The minimum detectable limit of the assay was 1.6±0.03 µIU/ml. A double antibody RIA was used for GH determination where bovine GH (a gift from Dr J Gong, Roslin Institute, Edinburgh) was used for iodination and preparation of the standard curve (Taylor et al. 2004b). The antiserum was raised in a guinea pig against bovine GH and was used at 1:10 000 dilution. The sensitivity of the assay was 1.6 ng/ml. The intra-assay coefficient of variation was 12.9%. Oestradiol concentrations were assayed using the Oestradiol MAIA assay kit (BioStat Diagnostic Systems, Stockport, UK). The intra-assay CV for samples with a mean of 1.1 and 4.6 oestradiol pg/ml of serum were 5.8 and 7.3% respectively and the sensitivity was 0.2 pg/ml. The intra-assay CV values for the same samples were 0.6 and 5.6% respectively. Cortisol concentrations were measured by RIA. Aliquots of 100 µl plasma were extracted with 3 ml diethyl ether in glass tubes, the organic phase was dried down and the sample was reconstituted in 300 µl assay buffer. Next, 100 µl aliquots of standard or extracted plasma were incubated with first antibody (100 µl sheep anti-bovine cortisol; Diagnostics Scotland; diluted 1:1000 in assay buffer) and [1, 2, 6, 7-3H]-cortisol (Amersham Biosciences UK Ltd; 100 µl, 10 000 c.p.m.) overnight at 4 °C. Separation was by dextran–charcoal. The intra-assay CV values for samples with means of 3.6 and 51.4 cortisol nmol/l of plasma were 5.6 and 6.1% respectively and the sensitivity of the assay was 0.7 nmol/l.
Real-time RT-PCR
Optimised PCR assays were used to detect transcripts for members of the IGF family including IGF-I/II, IGF receptors (IGF-1R/2R) and IGF-binding proteins (IGFBP-1/2/3/4/5/6), and also related factors including the receptors for IR-A/B, GHR, GR and ER/β. Primer sequence information is given in Table 4. A detailed description and applied conditions and protocols for these assays have been described and validated previously in bovine liver (Fenwick et al. 2006). Briefly, total RNA was extracted from frozen whole oviduct tissue ipsilateral to the dominant ovarian follicle and homogenised in TRI reagent (Molecular Research Centre Inc., Cincinnati, OH, USA). Sample RNA concentration and purity was determined by spectrophotometry (NanoDrop Technologies Inc., Wilmington, DE, USA) where all samples had a 260/280 ratio of absorbance between 1.8 and 2.1. Any potential genomic DNA contamination was treated by incubating samples in the presence of DNase I in accordance with the manufacturer's instructions (Promega Corporation). Precisely, 1 µg DNase-treated RNA was converted to cDNA in a randomly primed RT reaction using reagents and instructions supplied by Promega. Standards for quantitative PCR were prepared from purified PCR products that were quantified by spectroscopy (NanoDrop) and diluted over at least eight orders of magnitude. Standards and samples were quantified by PCR in 20 µl reactions with SYBR green (Absolute QPCR SYBR green mix, ABgene, Surrey, UK) using the DNA Engine Opticon 2 (MJ Research Inc., Waltham, MA, USA). For all unknown oviduct samples, 50 ng reverse-transcribed RNA was added to each reaction in duplicate. To minimise variation, all samples included in each analysis were derived from the same RT batch, prepared under the same conditions throughout and were analysed on a single plate. A melting curve analysis was performed for each reaction to confirm specificity with previously sequenced amplification products. Data were recorded with the Opticon Monitor Analysis Software (V2.02; MJ Research, Inc.) and transcript expression levels were derived from standard curves of amplicons as described previously in bovine oviduct tissues (Bauersachs et al. 2004). Transcript levels for the ribosomal gene 18S were also analysed to account for sample variation through processing. Negative control samples were prepared as above minus the reverse transcriptase enzyme or cDNA to confirm the absence of genomic DNA and extraneous contamination respectively.
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To aid cellular localisation of hybridised probes, selected slides were subsequently coated with photographic emulsion LM1 (Amersham Biosciences UK Ltd) according to the manufacturer's instructions and stored for 28, 30 or 42 days at 4 °C in darkness (Table 5). The slides were developed in 20% phenisol (ILFORD Imaging UK Ltd, Cheshire, England), fixed in 1.9 M sodium thiosulphate and counterstained with haematoxylin and eosin. All other slides were also stained with haematoxylin and eosin to aid identification of tissue region. The oviducts were characterised by tubal region (isthmus, ampulla and infundibulum) according to Hunter (2003).
OD measurements
The relative expression of mRNA for components of the oviduct IGF system was quantified from the autoradiographs using the public domain NIH ImageJ program (available through the NIH website – http://www.nih.gov), which calculated the average OD over the selected area of film based on a linear grey scale of 0.01–2.71. For each oviduct, the isthmus, ampulla and infundibulum regions were measured separately. Autoradiographs were scanned into a computer using a Hewlett–Packard Scanjet 5400c (Sheffield, UK) and OD measurements were recorded from digital images. Readings were obtained from at least two sections per tissue for each of the antisense (AS) and sense (S) probes. The background OD, from a blank area of film, was also measured and subtracted from both AS and S OD measurements. Finally, the S values were subtracted from AS values to give an average OD value for specific hybridisation (Perks et al. 1995). The detection limit was taken as an OD value of 0.01.
Statistical analysis
Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS for Windows, V13.0; SPSS Inc., Chicago, IL, USA). Data obtained by real-time PCR were expressed as fg/µg RT RNA and differences at the time of tissue collection between MNEB and SNEB groups were analysed using an independent samples t-test. Levene's test was used to account for variance and if homogeneity was not achieved an unequal variance t-test was used.
For OD measurements from the in situ hybridisation studies, the fixed effects of treatment and oviduct region on the level of mRNA expression for each probe were analysed by Linear Mixed Model analysis. Cow was entered as a random effect. For this purpose, data from oviducts in which a particular probe showed no detectable specific hybridisation (OD of <0.01) were given an OD of 0.01, which equated to the lower limit of detection.
Relationships between absolute levels of expression for all genes measured by real-time PCR were determined from values pooled across treatment groups. The spread of data was tested for normality using the Shapiro–Wilk test and square root transformations were made (if appropriate) before deriving Pearson correlations. For the correlation data, only those relationships with P<0.01 were considered. Results from all other analyses were considered statistically significant when P<0.05.
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Footnotes |
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Received 25 May 2007
Revision received 27 July 2007
3 October 2007.
Accepted 17 October 2007
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