Abstract
The neurotransmitters/neuromodulators galanin (GAL) and galanin-like peptide (GALP) are known to operate through three G protein-coupled receptors, GALR1, GALR2 and GALR3. The aim of this study was to investigate changes in expression of mRNA for galanin, GALP and GALR1–3 in the hypothalamus and pituitary gland, of male and female sheep, to determine how expression changed in association with growth and the attainment of reproductive competence. Tissue samples from the hypothalami and pituitary glands were analysed from late foetal and pre-pubertal lambs and adult sheep. Although mRNA for galanin and GALR1-3 was present in both tissues, at all ages and in both genders, quantification of GALP mRNA was not possible due to its low levels of expression. mRNA expression for both galanin and its receptors was seen to change significantly in both tissues as a function of age. Specifically, hypothalamic galanin mRNA expression increased with age in the male, but decreased with age in the female pituitary gland. mRNA expression for all receptors increased between foetal and pre-pubertal age groups and decreased significantly between pre-pubertal and adult animals. The results indicate that the expression of mRNA for galanin and its receptors changes dynamically with age and those significant differences exist with regard to tissue type and gender. These changes suggest that galaninergic neuroendocrine systems could be involved in the regulation of ovine growth and or the development of reproductive competence. The roles played by these systems in the sheep, however, may differ from other species, in particular the neuroendocrine link between nutrition and reproduction and GALR1's role in pituitary signalling.
Introduction
Galanin was initially isolated from porcine gut, and the expression of this 29 amino acid peptide has since been reported in a variety of species and tissues, including the central and peripheral nervous systems (Melander et al. 1986, Skofitsch & Jacobowitz 1986, Chaillou et al. 1999). Due to its widespread distribution within the central nervous system (CNS), it has been proposed that galanin is involved in the regulation of a number of physiological processes, including nutrition, growth and reproduction (Crawley et al. 1993, Cheung et al. 1996, Baratta et al. 1997, Shen et al. 1998, Chaillou & Tillet 2005). Reported changes in galanin expression during foetal and post-natal development in the rat, human and opossum also suggest that galanin may be involved in the developmental differentiation of the foetal brain (Sizer et al. 1990, Elmquist et al. 1992, Bhide & Puranik 2005).
Galanin is highly conserved across species (Smith et al. 1998) and is able to bind to and activate at least three G protein-coupled receptors; galanin receptor 1 (GALR1), galanin receptor 2 (GALR2) and galanin receptor 3 (GALR3). mRNA for each receptor isoform has been reported in numerous peripheral tissues, in a variety of species (Urbanski & Ojeda 1990, Bartfai et al. 1993, Lorimer & Benya 1996, Wang et al. 1997). In addition, the receptor mRNA has been reported to be expressed within developing neural tissue (Tarasov et al. 2002) and within the adult CNS (Waters & Krause 2000).
In addition to galanin, the endogenous peptide galanin-like peptide (GALP) is also able to activate galanin receptors. GALP is a 60 amino acid peptide, originally isolated from porcine hypothalamic extracts (Ohtaki et al. 1999). GALP has a high structural homology with galanin (Ohtaki et al. 1999, Cunningham et al. 2002) and is able to bind to all three receptor isoforms. The greatest binding affinity being with GALR3 and the lowest with GALR1 (Lang et al. 2005). GALP-immunopositive cells exhibit a more limited distribution within the hypothalamo-pituitary axis (Ohtaki et al. 1999) and species-specific differences have been reported (Larm & Gundlach 2000, Jureus et al. 2001, Takatsu et al. 2001, Cunningham et al. 2002, Iqbal et al. 2005).
A number of experimental observations have suggested that galanin is particularly important in the regulation of growth and reproduction with studies showing galanin and its receptors co-expressed within both hypothalamic GnRH1 and GHRH neurones (Murakami et al. 1989, Hohmann et al. 1998). In addition, hypothalamic galanin mRNA expression has been reported to increase with age through the juvenile period, reaching a peak during puberty, in GnRH1 and other hypothalamic neurones of both male and female rats (Planas et al. 1994, 1995, Rossmanith et al. 1994). The number of galaninergic synapses onto GnRH1 neuronal cell bodies has also been reported to be increased in adult, compared with juvenile female mice (Rajendren & Li 2001).
Support for a specific role for galaninergic systems in the steroidogenic control of the reproductive and growth axes comes from the observation that many of the reported developmental changes in galanin expression are gonad dependent (Rossmanith et al. 1994). In addition, it has been reported that galanin expression in both the hypothalamus and pituitary gland is sexually dimorphic, with higher levels of expression within the hypothalamus and specifically within hypothalamic GnRH1 neurones of female compared with male rats (Mitchell et al. 1999, Todman et al. 2005). The reported sexually dimorphic patterns of hypothalamic galanin expression are of particular interest with regard to the steroidogenic regulation of GnRH1 secretion, as they mean that galanin could be one of the neurotransmitter systems involved in mediating the indirect, positive feedback effects of high concentrations of oestradiol on GnRH1 neurones (Herbison et al. 1993, Lehman & Karsch 1993, Evans et al. 1997, Skinner et al. 2001).
There is also evidence to suggest that GALP could be involved with the regulation of reproduction, as GALP-positive fibres have been shown to make contact with GnRH1 cell bodies (Takatsu et al. 2001). In addition, intracerebroventricular injections of GALP have been shown to stimulate fos activity in GnRH1 neurones, and subsequent LH secretion, via an action within the hypothalamus that can be blocked with the competitive GnRH1 antagonist, cetrorelix (Matsumoto et al. 2001).
Previous studies in sheep have reported that galanin is expressed within the hypothalamus and pituitary gland (Tempel et al. 1988, Leibowitz et al. 1998, Chaillou et al. 1999), and GALR1 is expressed within the hypothalamus (Tempel et al. 1988, Leibowitz et al. 1998, Chaillou et al. 1999), is co-expressed within GnRH1 neurones and is dynamically regulated by oestradiol (Merchenthaler et al. 1993, Rossmanith et al. 1994, Mitchell et al. 1999, Dufourny & Skinner 2005, Tourlet et al. 2005). GALR2-immunopositive neurones have also been reported to be present within the ovine hypothalamus and a proportion of the GALR2-immunopositive cells have been reported to be oestradiol sensitive (Chambers et al. 2007). However, to date, no studies have characterised age-related changes in mRNA expression for galanin and its three receptor isoforms in the hypothalamo-pituitary axis of both male and female animals.
Given the differences in the steroidogenic regulation of GnRH1 secretion in mammals such as sheep and humans, compared with rodents (Freeman 1993), and the detailed knowledge of the effects of steroids on the patterns of ovine hypothalamic GnRH1 secretion (Clarke et al. 1987, Moenter et al. 1992, 1993, Evans et al. 1995a, 1995b, Skinner et al. 1998), this study aimed to increase our understanding of age-related changes in hypothalamo-pituitary galanin, GALP and GALR mRNA expression, in male and female sheep.
The approach used in this study was to quantify the expression of mRNA for GALP, galanin and its three receptor subtypes in the hypothalamus and pituitary gland of late foetal, pre-pubertal and adult male and female sheep. To facilitate this work, partial mRNA sequences for ovine galanin, GALP, GALR1, GALR2 and GALR3 were identified using RTPCR with primers designed against the conserved regions of published rat, mouse and human sequences.
Results
mRNA sequences for ovine galanin, GALP, GALR1, GALR2 and GALR3
Partial mRNA sequences were obtained and published on the GenBank database, for ovine galanin (
GALP mRNA expression
Despite identification of an mRNA sequence for ovine GALP and the design and use of ovine primers and probes for use in qPCR and extensive optimisation studies, the levels of GALP mRNA expression were consistently too low to allow quantification. Analysis of samples by repeated RT-PCR (2× 35 cycles) resulted in a faint band of an appropriate size, indicating very low expression (data not shown). As such, GALP mRNA expression was not quantified in the samples in this experiment.
Hypothalamic galanin mRNA expression
Mean galanin mRNA expression in the hypothalamus of the foetal, pre-pubertal and adult animals of both genders is shown in Fig. 1A. Residual maximal likelihood (REML) indicated no significant effect of gender on hypothalamic galanin mRNA expression. A trend (P=0.077) was noted for galanin mRNA to change with age, wherein levels of expression observed in the foetal animals were lower than in the pre-pubertal or adult animals, these effects being most pronounced in the males.
Pituitary galanin mRNA expression
Mean galanin mRNA expression in the pituitary glands of the foetal, pre-pubertal and adult animals of both genders is shown in Fig. 1B. In the pituitary gland galanin mRNA expression differed significantly as a function of both age (P<0.05) and gender (P<0.001). Expression of galanin mRNA being significantly (P<0.05) lower in the adult animals compared with the pre-pubertal animals and the expression in females being significantly (P<0.001) lower than that in males. The effects of age were most pronounced in the females and the effects of gender were most pronounced in the foetal and adult animals.
Hypothalamic GALR1 mRNA expression
Mean GALR1 mRNA expression in the hypothalamus of the foetal, pre-pubertal and adult animals of both genders is shown in Fig. 2A. There was a significant (P<0.005) effect of age on GALR1 mRNA expression within the hypothalamus, specifically expression being the lowest in the adults. The difference was the greatest in the males where expression in the adults was significantly lower (P<0.005) than the two other ages tested whereas in the females significance (P<0.005) was only seen between the adult and the pre-pubertal animals. The effect of gender was not statistically significant (P=0.087).
Pituitary GALR1 mRNA expression
Mean GALR1 mRNA expression in the pituitary glands of the foetal, pre-pubertal and adult animals of both genders is shown in Fig. 2B. The pattern of change in the pituitary gland matched that of the hypothalamus, with a significant (P<0.05) effect of age, levels of expression being the lowest in the adult and the highest in the pre-pubertal animals of both genders.
Hypothalamic GALR2 mRNA expression
Mean GALR2 mRNA expression in the hypothalamus of the foetal, pre-pubertal and adult animals of both genders is shown in Fig. 3A. Age significantly (P<0.05) affected GALR2 mRNA expression. Expression in the pre-pubertal females was significantly (P<0.05) greater than that in both foetal and adult animals but in the males there was only a significant difference (P<0.05) between the pre-pubertal and adult animals.
Pituitary GALR2 mRNA expression
Mean GALR2 mRNA expression in the pituitary glands of the foetal, pre-pubertal and adult animals of both genders is shown in Fig. 3B. In the males, pre-pubertal and foetal expression was significantly (P<0.05) greater than adult expression. However, in the females, significantly (P<0.05) greater expression was seen in the pre-pubertal animals relative to the two other ages tested.
Hypothalamic GALR3 mRNA expression
Mean GALR3 mRNA expression in the hypothalamus of the foetal, pre-pubertal and adult animals of both genders is shown in Fig. 4A. There was a significant (P<0.05) effect of age on hypothalamic GALR3 expression. Expression was the lowest in the adults, levels of expression being significantly (P<0.05) higher in both foetal and pre-pubertal males and in pre-pubertal females compared with their respective adults.
Pituitary GALR3 mRNA expression
Mean GALR3 mRNA expression in the pituitary glands of the foetal, pre-pubertal and adult animals of both genders is shown in Fig. 4B. The patterns of expression and the observed significant differences were similar to those observed in the hypothalamus; namely, an effect of age (P<0.005), whereby expression in the adult was again low relative to the other two ages tested.
Discussion
Using quantitative RT-PCR, this study characterises the patterns of expression of genes involved in galaninergic neurotransmission within the ovine hypothalamus and pituitary gland. The results are consistent with a role for galaninergic systems in the regulation of growth and reproduction at the levels of the hypothalamo-pituitary gland complex. The expression patterns suggest that the role played by these systems, in sheep, may differ in several ways from those reported in other species (Chaillou et al. 1999) and reflect underlying differences in the physiology of the sheep compared with the other studied species.
Initial characterisation of mRNA sequences for ovine galanin, GALP and the three galanin receptor subtypes GALR1, GALR2 and GALR3 indicated that they were highly conserved, relative to published sequences for other species. Galanin and galanin receptor mRNAs (Melander et al. 1985, Skofitsch & Jacobowitz 1986, Morris et al. 1989) were found to be present in both the hypothalamus and pituitary gland, in both sexes, at all of the ages tested. The absence of significant amounts of GALP mRNA in the ovine hypothalamus and pituitary gland was surprising given its reported expression in the other species (pigs (Ohtaki et al. 1999), rats (Takatsu et al. 2001), mice (Jureus et al. 2001) and macaques (Scarlett et al. 2001)) where it has been proposed to act as a link between metabolic/nutritionally sensitive and reproductive neuroendocrine systems (Cunningham et al. 2002, Castellano et al. 2006) but agrees with the results of ovine studies using in situ hybridisation (Adam Personal communication). It should be noted, that one previous study has reported GALP immunoreactivity within the ovine hypothalamus, although again expression appeared to be at a very low level (Iqbal et al. 2005). Greater expression of GALP and GALP mRNA in the other studied species may reflect distinct physiological differences between these species with regard to nutrient utilisation and signalling. Species previously reported to express high levels of GALP within the hypothalamus and pituitary gland are monogastric and, thus, may depend upon GALP to detect acute variations in circulating metabolic mediators such as glucose and insulin, whereas acute changes in such metabolic markers are less variable in ruminants. Further studies, however, would be required to confirm this proposal.
The presence of galanin mRNA expression within the ovine hypothalamus, supports previous data in which galanin immunopositive cells have been reported in the ovine hypothalamus (Chaillou et al. 1999, Dufourny et al. 2003). Hypothalamic galanin mRNA expression was seen to change as a function of age, as has been reported in other species (Mitchell et al. 1999, Hull & Harvey 2002) and thus is consistent with a role for ovine galanin as a potential hypothalamic neurotransmitter. This study provides the first report of galanin mRNA expression within the ovine pituitary gland, and supports reports of both galanin protein and mRNA expression within the pituitary gland in other species (Vrontakis et al. 1989, Selvais et al. 1995, Hyde et al. 1998) and the reported role that galanin may play as a neuromodulator (Baratta et al. 1997, Cai et al. 1998). The age-related increase in galanin mRNA expression seen in the hypothalamus in this study, corroborates changes in protein (Gabriel et al. 1989) and mRNA reported within GHRH (Delemarre-van de Waal et al. 1994) and GnRH1 neurones (Rossmanith et al. 1994) in other species. These earlier reports suggested that the observed age-related increase in mRNA expression was dependent upon the presence of the gonad and reflected attainment of sexual maturity (Rossmanith et al. 1994) which would be accompanied by an increase in the circulating gonadal steroid concentrations. This possibility is supported by the results of this study, as in both genders the age at which galanin mRNA expression was the lowest, was in the foetal animals, when steroid secretion would also be expected to be low. The larger change in mRNA expression in the male, as opposed to the female animals in this study, between foetal life and 8 weeks of age, also supports a link between galanin mRNA expression and maturational changes within the hypothalamo-pituitary gonadal axis that are required to achieve reproductive competence, as this time period precedes the pre-pubertal increase in LH secretion, normally seen in male sheep (Evans et al. 1991), that is thought to be a key factor in the initiation of sperm production and thus puberty. The comparable activation of the hypothalamic–pituitary–gonadal axis is delayed in female sheep (Wood et al. 1991). It is worth noting, however, that galanin has also been reported to be co-expressed in GHRH neurones in both the rat and monkey (Niimi et al. 1990, Hohmann et al. 1998) and, therefore, the increase in hypothalamic galanin mRNA expression seen in the current study could be due to activity within the GHRH neurosecretory axis. The possible actions of galanin on the hypothalamic regulation of both the reproductive and growth axes are supported by data which have shown that infusion of galanin into the brain results in a dose-dependent increase in growth hormone, prolactin and LH secretion (Baranowska-Bik et al. 2005).
Changes in pituitary expression of galanin have been reported in other species in response to steroids (Vrontakis et al. 1989, Wynick et al. 1993) and it is interesting to note that in this study, as reported in the rat (Vrontakis et al. 1989, Wynick et al. 1993), the levels of pituitary galanin mRNA expression did not change as a function of testosterone exposure, levels remaining constant with age, prior to and after puberty in the males. However, in the females, pituitary galanin mRNA expression was affected by puberty but in the opposite direction to that which would be predicted based upon studies in rats where oestradiol has been shown to stimulate galanin expression (Wynick et al. 1993, Hammond et al. 1997). However, it is important to note that the current study did not look at the cellular localisation of pituitary galanin expression that has been reported to differ between species (Hsu et al. 1991).
In this regard, it is interesting to note that the age-related changes in galanin mRNA expression observed in the present study were tissue and gender specific, a significant increase being seen in the male hypothalamus, and a significant decrease in the female pituitary. This difference would suggest that the regulation of galanin gene expression might be different in the two tissues and or genders.
Our finding of mRNA expression for all three galanin receptor subtypes in the hypothalamus and pituitary gland, at all of the ages tested, again supports a role for galanin as a neurohormone or a neuromodulator. Expression of GALR1 mRNA within the ovine pituitary gland was of particular interest, as previous work in the rat (Hohmann et al. 1998, Hull & Harvey 2002) failed to demonstrate its presence in this tissue. While previous ovine studies have characterised GALR1 expression within the hypothalamus (Dufourny & Skinner 2005), no previously published work has documented the presence of GALR1 mRNA within the ovine pituitary gland.
It is apparent from the results of this study that, as in other species (Smith et al. 1998, Waters & Krause 2000), GALR1 and GALR2 are expressed at higher levels than GALR3, which is the least abundant, receptor isoform present in both tissues in either gender. Less information is available with regard to GALR2 and GALR3 but the lack of an effect of gender on the expression of mRNA for any of the receptor isoforms in this study, contrasts with reported gender-specific differences in GALR1 expression in the rat (Faure-Virelizier et al. 1998). This species difference, however, may be the result of technical differences between these two studies as, while the current study used an overall measure of hypothalamic mRNA expression, the rat study used in situ hybridisation to look at dynamic changes in GALR1 mRNA expression within specific hypothalamic nuclei. Alternatively, however, it could reflect differences in the role played by galaninergic systems in the control of species-specific regulatory processes. This proposal is supported by other species differences, such as the presence of GALR1 mRNA in the ovine pituitary and the lack of significant GALP mRNA expression in either the ovine hypothalamus or pituitary gland found in this study.
Receptor mRNA expression was seen to change in both the ovine hypothalamus and pituitary gland as a function of age. The observed age-related changes in receptor mRNA expression were similar across the three receptor isoforms, expression being the highest in the pre-pubertal animals and the lowest in the adults, with the exception of GALR3 in the male pituitary gland, where foetal mRNA expression was the highest. As discussed above with regard to galanin, this pattern of receptor mRNA expression could support a role for galanin sensitive systems in the regulation of growth and/or in the initiation of reproductive activity, as these both occur during the pre-pubertal stage of development. This possibility is supported by a variety of evidence relating to galanin receptor expression. For example, galanin receptor knockout studies, conducted in mice (Hohmann et al. 2003, Krasnow et al. 2004) demonstrated that GALR1 and GALR2 are required for normal LH, FSH and testosterone secretion. Similarly, GALR1 has been shown to be the predominant receptor subtype involved with the effects of galanin on feeding behaviour (Bartfai et al. 1993) and GALR2 has been implicated in the regulation of processes that induce growth and cell proliferation (Wang et al. 1998). Both of these receptors have also been implicated in reproduction via GnRH1 neurones in the preoptic area of the hypothalamus in rats (Faure-Virelizier et al. 1998, Bouret et al. 2000) and sheep (Dufourny et al. 2003). Very little has been reported with regard to GALR3. While the pattern of expression of GALR3 mRNA expression overlaps that of both GALR1 and GALR2, in the rat (Mennicken et al. 2002), the relatively low levels of expression compared with GALR1 and GALR2 and its lower affinity for galanin (Smith et al. 1998) may indicate a lesser physiological role. Changes in GALR3 mRNA expression were seen in this study and, as they changed in a similar manner to those of GALR1 and GALR2, it is possible that its expression is regulated such as to supplement or complement the actions of the other galanin receptor isoforms. In this respect, if we look at the actions of the three receptor subtypes we see that they are functionally different as they link to different G protein complements within the cells. GALR1 and GALR3 are both linked to Gi and Gi/o proteins, which decrease intracellular adenylate cyclase activity and hyperpolarise cells respectively, (Berridge & Irvine 1989, Wang et al. 1998) thus GALR1 and GALR3 may have complementary actions to decrease cell activity. By contrast, GALR2 has both inhibitory actions, through Gi and Go proteins and stimulatory actions through a Gq protein, via increased MAP kinase activity within the hypothalamus and intracellular calcium release within the pituitary gland (Depczynski et al. 1998, Tsaneva-Atanasova et al. 2007). Thus, GALR2 may mediate both inhibitory and stimulatory effects on cell function (Wang et al. 1998).
Finally, comparison of expression levels of mRNA for galanin and its receptors, in the foetal and pre-pubertal animals, indicated that in both genders and both tissues, expression of the mRNAs for the three galanin receptor subtypes substantially exceeded that of galanin. In the adults, regardless of tissue type expression of mRNA for all the receptor isoforms decreased dramatically. In the females, however, receptor isoform mRNA expression continued to exceed that of the ligand whereas in the males this relationship was reversed. This result would suggest that the regulation of mRNA expression for the components of galaninergic systems changes dramatically in association with the change from reproductive immaturity to reproductive maturity. It also provides evidence that galaninergic regulatory systems in the sheep may, as in other species, be sexually dimorphic (Hull & Harvey 2002).
While an argument could be made that the age-related changes observed in both galanin and galanin receptor mRNA expression, within the ovine hypothalamus and pituitary gland are related to seasonal changes in gene expression, the design of this experiment was not sufficient to allow investigation of specific seasonal effects on galaninergic systems, that would be independent of both age and reproductive status. However, as no specific effects of season have been reported on galanin mRNA expression in other species, and age-related changes have been reported in both galanin and galanin receptor expression, a more parsimonious explanation of the results obtained in this study is with a role for ovine galanin as a potential hypothalamic neurotransmitter that is modulated as a function/consequence of the age and reproductive status of the animal.
While we had hypothesised that there might be differences in gene expression between the two hypothalamic slices analysed, one of which encompassed the preoptic area, and the other the mediobasal hypothalamus, statistical analysis of mRNA expression did not indicate significant differences between the two slices. The final results, however, exhibited significant variation in gene expression with age and gender, between each group/tissue. However, it is possible that the high variability in gene expression between samples within each group may have obscured observation of some biologically significant age, gender and or tissue specific differences in target gene expression.
In conclusion, the results of this study demonstrate that the mRNAs for galanin and its three identified receptor isoforms are expressed within the ovine hypothalamus and pituitary gland in foetal, pre-pubertal and adult animals and support a role for galanin as both a neurotransmitter and a neuromodulator. The lack of significant amounts of GALP mRNA expression within the ovine hypothalamo-pituitary gland complex, contrasts with other species and would suggest that GALP does not fulfil the same proposed role as part of a hypothalamic nutritional signalling system in the sheep compared with other species. The expression of the mRNA for galanin and GALR1–3 all showed changes with age, which are consistent with a role(s) in the regulation of growth and/or the initiation of reproduction but suggest that their role in the regulation of neuroendocrine process, in the sheep, may differ from those seen in rats, mice and monkeys.
Materials and Methods
Tissue
Samples of the hypothalamus and pituitary gland were collected, following a lethal dose of barbiturates (20 mg/kg BW, i.v.; Lethobarb, Duphar Vet, UK), from sheep of mixed breed (predominantly Scotch mules), at a series of defined ages. Immediately following death, the brains were removed and a tissue block containing the preoptic area/hypothalamus dissected out. Pituitary glands were also collected at this time and all tissues were frozen at −70 °C until processed. Tissues were collected from foetal lambs, ∼110 days of gestation (males n=5, females n=4); pre-pubertal lambs, 8 weeks of age (females and males n=6); reproductively active adult, more than 2 and less than 5 years of age, males (n=4) and cycling females (as the aim of this study was to examine the effects of age, the adult females were of mixed oestrous cycle stage n=16). Tissue from the adult sheep and foetal lambs were collected in the autumn and early spring respectively, whereas tissue from the pre-pubertal lambs was collected in the early summer. All procedures were approved by the Faculty's Ethics and Welfare committee and were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986.
Whilst still frozen, each hypothalamic block was cut into coronal slices using external landmarks, with the most rostral cut ∼1 mm in front of the optic chiasma, such that the first slice encompassed the preoptic area and the second the mediobasal hypothalamus/stalk median eminence (∼4 mm in adult, 3 mm in pre-pubertal and 2 mm in foetal lambs). For RNA extraction, 100–200 mg of tissue were then harvested from an area close to the ventricle and near the base of the hypothalamus from each tissue block. Pituitary glands were cut along the mid-sagittal plane (both posterior and anterior glands) and ∼100–200 mg tissue harvested from the mid-sagittal face for RNA extraction.
RNA extraction
Total RNA was extracted from the tissue using Trizol (Invitrogen) according to the manufacturer's instructions. cDNA was obtained by reverse transcription of the resultant mRNA using random hexamers (Promega), M-MLV reverse transcriptase (Invitrogen) and RNasin (Promega), as described previously (O'Shaughnessy & Murphy 1993). mRNA and cDNA purity and quantity were assessed by spectroscopy (yield averaged 1000 ng/ul and 260/280 ratio 2.0±0.2).
mRNA sequencing and quantification
Partial mRNA sequences for ovine galanin, GALP, GALR1, GALR2 and GALR3 were obtained as follows; primer sequences for the genes of interest were designed based upon regions of high interspecies (rat, mouse, cow, human) homology using available (GenBank) sequences. Synthesised primers were used with samples of ovine hypothalamic and pituitary gland cDNA in standard RTPCR protocols and the RTPCR products separated on a 1% agarose gel. Where bands of an appropriate size were visualised, the associated cDNA was isolated and purified using Biorad Micro-Bio-spin-chromatography columns according to the manufacturers protocol and sequenced using BigDye Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems 3100 Genetic Analyser). Primer sequences (MWG-BIOTECH AG, Ebersberg, Germany) used for RTPCR and cDNA sequencing are listed in Table 1.
Primer and probe sequences used to isolate and quantify ovine galanin, GALP and galanin receptor mRNAs.
Forward 5′–3′ | Probe | Reverse 3′–5′ | |
---|---|---|---|
RT-PCR | |||
β-actin | TCCTTCCTGGGCATGGAATC | GGGCGCGATGATCTTGATCT | |
Galanin | TACCTTCTCGGACCACATGC | TGCAGGAAAGTGAGAAACTC | |
GALR1 | CCTTGGCATAGCAGAAGCAG | ATGTCGGTGGACGCGTACGT | |
GALR2 | CGCTCATCTTCCTCGTGGG | AGCCGTCCAGGGTGTAGATG | |
GALR3 | TAGACAGCCCAGGGAGTATG | GTCTTTGCCCTCATCTTCCT | |
GALP | CGAGGAGGCTGGACCCTCAA | CAGGTCTAGGATCTCAAG | |
qPCR | |||
β-actin | CCCTGAGGCTCTCTTCCA | CTTCCTTCCTGGGCATGGAATCC | GGAATTGAAGGTAGTTTCGTGAAT |
Galanin | GAGAGGCTGGACCCTGAACA | TGCCGGCTACCTTCTCGGACCA | CGTGAAATGACCTGTGGTTGTC |
GALR1 | CACACCACGTAGGCCTTCTTG | CGCTGGTTGGGCCACTGCTCC | GACGTCAGCAACCAGACCTTCT |
GALR2 | AGCCGTCCAGGGTGTAGATG | CTTGGAAGGGCACGCAG | CGACCTGTGTTTCATCGTGTG |
GALR3 | ACGACGGATCTATTCATACCTCAAC | CTGGCGGCAGCTGACCTCTGCT | GATGGCGGCCTGGAAAG |
Semi-quantitative real-time taqman PCR (qPCR) was performed on duplicate samples of cDNA using Amplitaq Gold kits (Applied Biosciences, Beaconsfield, Bucks, UK) using ovine β-actin as the housekeeping gene, in a Stratagene MX 3000P thermal cycler. Due to the high GC content and low levels of mRNA expression of the expected products, qPCR conditions were as follows; 97 °C for 10 min – 1 cycle, 95 °C for 45 s, 60 °C for 1 min – 45 cycles.
Primer-Probe sets (Eurogentecs S.A, Liege, Belgium) for qPCR were designed using Primer Express (Applied Biosystems) software and used FAM as the 5′-reporter and DDQ1 as the 3′-quencher.
Statistical analysis
For relative quantification of mRNA concentrations the comparative CT method was used, wherein the expression of each gene of interest was quantified relative to the expression of the housekeeping gene (β-actin; User Bulletin no. 2, PE Biosystems, UK). Validation experiments (data not shown) confirmed that the amplification efficiencies of the galaninergic genes and β-actin were comparable, the slope of the difference between CT values for the standard curves for each gene were <0.1 and within 5% of the β-actin slope. All results are expressed as the mean±s.e.m.
Relative expression levels of galanin and the three receptor isoforms were compared between areas of the hypothalamus by ANOVA (Genstat, release 10, VSN International, Hemel Hempstead, UK). As the expression of galanin and the three receptor isoforms was not found to differ significantly between the two hypothalamic slices, hypothalamic expression was averaged for each animal. Values were then multiplied by 1000 (for ease of data handling) prior to further statistical analysis. To equalise variance between groups the data were log transformed and, due to the non-orthogonal nature of the data (largely due to the disparity in numbers between groups), differences in expression between sexes and ages studied within each tissue type by REML (Genstat, release 10, VSN International), using a linear mixed model analysis where the fixed model was sex x age and individual animals were included as the random model term. Differences between treatment groups were calculated using least significant differences analysis, calculated from the standard error of the difference (s.e.d.) between means obtained from the REML analysis.
Declaration of interest
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported in this manuscript.
Funding
This work was supported by the Biotechnology and Biological Sciences Research Council (Grant number S18947).
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