REPRODUCTIONRESEARCH

Expression of mRNA for , galanin-like and galanin receptors 1–3 in the ovine and : effects of age and gender

Christine Margaret Whitelaw, Jane Elizabeth Robinson, George Ballantine Chambers, Peter Hastie, Vasantha Padmanabhan1, Robert Charles Thompson1 and Neil Price Evans Division of Cell Sciences, Faculty of Veterinary Medicine, Institute of Comparative Medicine, University of Glasgow, Glasgow G61 1QH, UK and 1Department of Pediatrics and Psychiatry, University of Michigan, Ann Arbor, Michigan 48103, USA Correspondence should be addressed to N P Evans; Email: [email protected]

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. Reproduction (2009) 137 141–150

Introduction three G protein-coupled receptors; galanin 1 (GALR1), (GALR2) and galanin Galanin was initially isolated from porcine gut, and the receptor 3 (GALR3). mRNA for each receptor isoform expression of this 29 amino acid peptide has since been has been reported in numerous peripheral tissues, in a reported in a variety of species and tissues, including the variety of species (Urbanski & Ojeda 1990, Bartfai et al. central and peripheral nervous systems (Melander et al. 1993, Lorimer & Benya 1996, Wang et al. 1997). In 1986, Skofitsch & Jacobowitz 1986, Chaillou et al. addition, the receptor mRNA has been reported to be 1999). Due to its widespread distribution within the expressed within developing neural tissue (Tarasov et al. central nervous system (CNS), it has been proposed that 2002) and within the adult CNS (Waters & Krause 2000). galanin is involved in the regulation of a number of In addition to galanin, the endogenous peptide physiological processes, including nutrition, growth and galanin-like peptide (GALP) is also able to activate reproduction (Crawley et al. 1993, Cheung et al. 1996, galanin receptors. GALP is a 60 amino acid peptide, Baratta et al. 1997, Shen et al. 1998, Chaillou & Tillet originally isolated from porcine hypothalamic extracts 2005). Reported changes in galanin expression during (Ohtaki et al. 1999). GALP has a high structural foetal and post-natal development in the rat, human and homology with galanin (Ohtaki et al. 1999, Cunningham opossum also suggest that galanin may be involved in the et al. 2002) and is able to bind to all three receptor developmental differentiation of the foetal brain (Sizer isoforms. The greatest binding affinity being with GALR3 et al. 1990, Elmquist et al. 1992, Bhide & Puranik 2005). and the lowest with GALR1 (Lang et al. 2005). Galanin is highly conserved across species (Smith GALP-immunopositive cells exhibit a more limited et al. 1998) and is able to bind to and activate at least distribution within the hypothalamo-pituitary axis

q 2009 Society for Reproduction and Fertility DOI: 10.1530/REP-08-0266 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access 142 C M Whitelaw and others

(Ohtaki et al. 1999) and species-specific differences be present within the ovine hypothalamus and a have been reported (Larm & Gundlach 2000, Jureus et al. proportion of the GALR2-immunopositive cells have 2001, Takatsu et al. 2001, Cunningham et al. 2002, been reported to be oestradiol sensitive (Chambers et al. Iqbal et al. 2005). 2007). However, to date, no studies have characterised A number of experimental observations have age-related changes in mRNA expression for galanin and suggested that galanin is particularly important in the its three receptor isoforms in the hypothalamo-pituitary regulation of growth and reproduction with studies axis of both male and female animals. showing galanin and its receptors co-expressed within Given the differences in the steroidogenic regulation both hypothalamic GnRH1 and GHRH neurones of GnRH1 secretion in mammals such as sheep and (Murakami et al. 1989, Hohmann et al. 1998). In humans, compared with rodents (Freeman 1993), and addition, hypothalamic galanin mRNA expression has the detailed knowledge of the effects of steroids on the been reported to increase with age through the juvenile patterns of ovine hypothalamic GnRH1 secretion (Clarke period, reaching a peak during puberty, in GnRH1 and et al. 1987, Moenter et al. 1992, 1993, Evans et al. other hypothalamic neurones of both male and female 1995a, 1995b, Skinner et al. 1998), this study aimed to rats (Planas et al. 1994, 1995, Rossmanith et al. 1994). increase our understanding of age-related changes in The number of galaninergic synapses onto GnRH1 hypothalamo-pituitary galanin, GALP and GALR mRNA neuronal cell bodies has also been reported to be expression, in male and female sheep. increased in adult, compared with juvenile female mice The approach used in this study was to quantify the (Rajendren & Li 2001). expression of mRNA for GALP, galanin and its three Support for a specific role for galaninergic systems in receptor subtypes in the hypothalamus and pituitary the steroidogenic control of the reproductive and growth gland of late foetal, pre-pubertal and adult male and axes comes from the observation that many of the female sheep. To facilitate this work, partial mRNA reported developmental changes in galanin expression sequences for ovine galanin, GALP, GALR1, GALR2 and are gonad dependent (Rossmanith et al. 1994). In GALR3 were identified using RTPCR with primers addition, it has been reported that galanin expression designed against the conserved regions of published in both the hypothalamus and pituitary gland is sexually rat, mouse and human sequences. dimorphic, with higher levels of expression within the hypothalamus and specifically within hypothalamic GnRH1 neurones of female compared with male rats Results (Mitchell et al. 1999, Todman et al. 2005). The reported mRNA sequences for ovine galanin, GALP, GALR1, sexually dimorphic patterns of hypothalamic galanin GALR2 and GALR3 expression are of particular interest with regard to the steroidogenic regulation of GnRH1 secretion, as they Partial mRNA sequences were obtained and published mean that galanin could be one of the neurotransmitter on the GenBank database, for ovine galanin (EF192581, systems involved in mediating the indirect, positive 290 bp), GALP (EF192585, 114 bp), GALR1 (EF192582, feedback effects of high concentrations of oestradiol on 208 bp), GALR2 (EF192583, 100 bp) and GALR3 GnRH1 neurones (Herbison et al. 1993, Lehman & (EF192584, 196 bp). The mRNA sequence for ovine Karsch 1993, Evans et al. 1997, Skinner et al. 2001). galanin encompasses that which encodes the secreted There is also evidence to suggest that GALP could be peptide. Those obtained for the three receptors only involved with the regulation of reproduction, as GALP- encoded that part of the predicted amino acid sequence positive fibres have been shown to make contact with that could be identified specifically for each receptor, to GnRH1 cell bodies (Takatsu et al. 2001). In addition, enable design of specific ovine qPCR primers and probes intracerebroventricular injections of GALP have been for the three galanin receptor isoforms. The mRNA shown to stimulate fos activity in GnRH1 neurones, and sequences identified, all showed high homology with subsequent LH secretion, via an action within the those reported in other species: galanin, bovine 98%, hypothalamus that can be blocked with the competitive porcine 90% and murine 89%; GALR1, equine 94%, GnRH1 antagonist, cetrorelix (Matsumoto et al. 2001). murine 93% and human 90%; GALR2, rat 89%, murine Previous studies in sheep have reported that galanin is 89% and human 89%; GALR3, bovine 97%, human expressed within the hypothalamus and pituitary gland 90% and murine 85%; and GALP, murine 96%, human (Tempel et al. 1988, Leibowitz et al. 1998, Chaillou et al. 82% and rat 100%. 1999), and GALR1 is expressed within the hypothalamus (Tempel et al. 1988, Leibowitz et al. 1998, Chaillou et al. GALP mRNA expression 1999), is co-expressed within GnRH1 neurones and is dynamically regulated by oestradiol (Merchenthaler et al. Despite identification of an mRNA sequence for ovine 1993, Rossmanith et al. 1994, Mitchell et al. 1999, GALP and the design and use of ovine primers and Dufourny & Skinner 2005, Tourlet et al. 2005). GALR2- probes for use in qPCR and extensive optimisation immunopositive neurones have also been reported to studies, the levels of GALP mRNA expression were

Reproduction (2009) 137 141–150 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access Galaninergic mRNA in the ovine hypothalamus/pituitary 143

Figure 1 Effects of age and gender on predicted mean levels of galanin Figure 2 Effects of age and gender on predicted mean levels of GALR1 mRNA expression, (relative to b-actin, derived from the log- mRNA expression, (relative to b-actin, derived from the log- transformed data following REML analysis) in the (A) hypothalamus and transformed data following REML analysis) in the (A) hypothalamus and (B) pituitary gland. Solid bars depict data from male animals and open (B) pituitary gland. Solid bars depict data from male animals and open bars female animals. S.E.D. for effects of age, gender and age–gender bars female animals. S.E.D. for effects of age, gender and age–gender interaction are provided at the top of each panel. Significance levels are interaction are provided at the top of each panel. Significance levels are indicated for each of the three effects, NS, not significant. indicated for each of the three effects, NS, not significant. consistently too low to allow quantification. Analysis of within the hypothalamus, specifically expression being samples by repeated RT-PCR (2! 35 cycles) resulted in a the lowest in the adults. The difference was the greatest faint band of an appropriate size, indicating very low in the males where expression in the adults was expression (data not shown). As such, GALP mRNA significantly lower (P!0.005) than the two other ages expression was not quantified in the samples in this tested whereas in the females significance (P!0.005) experiment. was only seen between the adult and the pre-pubertal animals. The effect of gender was not statistically significant (PZ0.087). Hypothalamic galanin mRNA expression Mean galanin mRNA expression in the hypothalamus of Pituitary GALR1 mRNA expression the foetal, pre-pubertal and adult animals of both genders is shown in Fig. 1A. Residual maximal Mean GALR1 mRNA expression in the pituitary glands of likelihood (REML) indicated no significant effect of the foetal, pre-pubertal and adult animals of both gender on hypothalamic galanin mRNA expression. A genders is shown in Fig. 2B. The pattern of change in trend (PZ0.077) was noted for galanin mRNA to change the pituitary gland matched that of the hypothalamus, ! with age, wherein levels of expression observed in the with a significant (P 0.05) effect of age, levels of foetal animals were lower than in the pre-pubertal or expression being the lowest in the adult and the highest adult animals, these effects being most pronounced in in the pre-pubertal animals of both genders. the males. Hypothalamic GALR2 mRNA expression Pituitary galanin mRNA expression Mean GALR2 mRNA expression in the hypothalamus of Mean galanin mRNA expression in the pituitary glands the foetal, pre-pubertal and adult animals of both of the foetal, pre-pubertal and adult animals of both gendersisshowninFig. 3A. Age significantly (P!0.05) affected GALR2 mRNA expression. Expression genders is shown in Fig. 1B. In the pituitary gland galanin ! mRNA expression differed significantly as a function of in the pre-pubertal females was significantly (P 0.05) greater than that in both foetal and adult animals but in 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.

Figure 3 Effects of age and gender on predicted mean levels of GALR2 Hypothalamic GALR1 mRNA expression mRNA expression, (relative to b-actin, derived from the log transformed Mean GALR1 mRNA expression in the hypothalamus of data following REML analysis) in the (A) hypothalamus and (B) pituitary gland. Solid bars depict data from male animals and open bars female the foetal, pre-pubertal and adult animals of both animals. S.E.D. for effects of age, gender and age–gender interaction are genders is shown in Fig. 2A. There was a significant provided at the top of each panel. Significance levels are indicated for (P!0.005) effect of age on GALR1 mRNA expression each of the three effects, NS, not significant. www.reproduction-online.org Reproduction (2009) 137 141–150

Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access 144 C M Whitelaw and others

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 Figure 4 Effects of age and gender on predicted mean levels of GALR3 GALR1, GALR2 and GALR3 indicated that they were mRNA expression, (relative to b-actin, derived from the log- highly conserved, relative to published sequences for other transformed data following REML analysis) in the (A) hypothalamus and species. Galanin and galanin receptor mRNAs (Melander (B) pituitary gland. Solid bars depict data from male animals and open bars female animals. S.E.D. for effects of age, gender and age–gender et al. 1985, Skofitsch & Jacobowitz 1986, Morris et al. interaction are provided at the top of each panel. Significance levels are 1989) were found to be present in both the hypothalamus indicated for each of the three effects, NS, not significant. and pituitary gland, in both sexes, at all of the ages tested. The absence of significant amounts of GALP mRNA in the the males there was only a significant difference ovine hypothalamus and pituitary gland was surprising (P!0.05) between the pre-pubertal and adult animals. 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 Pituitary GALR2 mRNA expression has been proposed to act as a link between metabolic/ Mean GALR2 mRNA expression in the pituitary glands of nutritionally sensitive and reproductive neuroendocrine the foetal, pre-pubertal and adult animals of both genders systems (Cunningham et al.2002, Castellano et al. 2006) is shown in Fig. 3B. In the males, pre-pubertal and foetal but agrees with the results of ovine studies using in situ expression was significantly (P!0.05) greater than adult hybridisation (Adam Personal communication). It should expression. However, in the females, significantly be noted, that one previous study has reported GALP (P!0.05) greater expression was seen in the pre-pubertal immunoreactivity within the ovine hypothalamus, animals relative to the two other ages tested. 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 Hypothalamic GALR3 mRNA expression distinct physiological differences between these species Mean GALR3 mRNA expression in the hypothalamus of with regard to nutrient utilisation and signalling. Species the foetal, pre-pubertal and adult animals of both previously reported to express high levels of GALP within genders is shown in Fig. 4A. There was a significant the hypothalamus and pituitary gland are monogastric and, (P!0.05) effect of age on hypothalamic GALR3 thus, may depend upon GALP to detect acute variations in expression. Expression was the lowest in the adults, circulating metabolic mediators such as glucose and levels of expression being significantly (P!0.05) higher , whereas acute changes in such metabolic markers in both foetal and pre-pubertal males and in pre-pubertal are less variable in ruminants. Further studies, however, females compared with their respective adults. would be required to confirm this proposal. The presence of galanin mRNA expression within the ovine hypothalamus, supports previous data in which Pituitary GALR3 mRNA expression galanin immunopositive cells have been reported in the Mean GALR3 mRNA expression in the pituitary glands of ovine hypothalamus (Chaillou et al. 1999, Dufourny the foetal, pre-pubertal and adult animals of both et al. 2003). Hypothalamic galanin mRNA expression genders is shown in Fig. 4B. The patterns of expression was seen to change as a function of age, as has been and the observed significant differences were similar to reported in other species (Mitchell et al. 1999, Hull & those observed in the hypothalamus; namely, an effect of Harvey 2002) and thus is consistent with a role for ovine age (P!0.005), whereby expression in the adult was galanin as a potential hypothalamic neurotransmitter. again low relative to the other two ages tested. 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 Discussion et al. 1989, Selvais et al. 1995, Hyde et al. 1998) and the Using quantitative RT-PCR, this study characterises the reported role that galanin may play as a neuromodulator patterns of expression of genes involved in galaninergic (Baratta et al. 1997, Cai et al. 1998). The age-related neurotransmission within the ovine hypothalamus and increase in galanin mRNA expression seen in the pituitary gland. The results are consistent with a role for hypothalamus in this study, corroborates changes in galaninergic systems in the regulation of growth and protein (Gabriel et al. 1989) and mRNA reported within

Reproduction (2009) 137 141–150 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access Galaninergic mRNA in the ovine hypothalamus/pituitary 145

GHRH (Delemarre-van de Waal et al. 1994) and GnRH1 hypothalamus, and a significant decrease in the female neurones (Rossmanith et al. 1994) in other species. pituitary. This difference would suggest that the These earlier reports suggested that the observed age- regulation of galanin gene expression might be different related increase in mRNA expression was dependent in the two tissues and or genders. upon the presence of the gonad and reflected attainment Our finding of mRNA expression for all three galanin of sexual maturity (Rossmanith et al. 1994) which would receptor subtypes in the hypothalamus and pituitary gland, be accompanied by an increase in the circulating at all of the ages tested, again supports a role for galanin as a gonadal steroid concentrations. This possibility is neurohormone or a neuromodulator. Expression of GALR1 supported by the results of this study, as in both genders mRNA within the ovine pituitary gland was of particular the age at which galanin mRNA expression was the interest,aspreviousworkintherat(Hohmann et al. 1998, lowest, was in the foetal animals, when steroid secretion Hull & Harvey 2002) failed to demonstrate its presence in would also be expected to be low. The larger change in this tissue. While previous ovine studies have charac- mRNA expression in the male, as opposed to the female terised GALR1 expression within the hypothalamus animals in this study, between foetal life and 8 weeks of (Dufourny & Skinner 2005), no previously published age, also supports a link between galanin mRNA work has documented the presence of GALR1 mRNA expression and maturational changes within the within the ovine pituitary gland. hypothalamo-pituitary gonadal axis that are required to It is apparent from the results of this study that, as in achieve reproductive competence, as this time period other species (Smith et al. 1998, Waters & Krause 2000), precedes the pre-pubertal increase in LH secretion, GALR1 and GALR2 are expressed at higher levels than normally seen in male sheep (Evans et al. 1991), that is GALR3, which is the least abundant, receptor isoform thought to be a key factor in the initiation of sperm present in both tissues in either gender. Less information is production and thus puberty. The comparable activation available with regard to GALR2 and GALR3 but the lack of the hypothalamic–pituitary–gonadal axis is delayed in of an effect of gender on the expression of mRNA for any female sheep (Wood et al. 1991). It is worth noting, of the receptor isoforms in this study, contrasts with however, that galanin has also been reported to be reported gender-specific differences in GALR1 expression co-expressed in GHRH neurones in both the rat and in the rat (Faure-Virelizier et al. 1998). This species monkey (Niimi et al. 1990, Hohmann et al. 1998) and, difference, however, may be the result of technical therefore, the increase in hypothalamic galanin mRNA differences between these two studies as, while the expression seen in the current study could be due to current study used an overall measure of hypothalamic activity within the GHRH neurosecretory axis. The mRNA expression, the rat study used in situ hybridisation possible actions of galanin on the hypothalamic to look at dynamic changes in GALR1 mRNA expression regulation of both the reproductive and growth axes within specific hypothalamic nuclei. Alternatively, are supported by data which have shown that infusion of however, it could reflect differences in the role played galanin into the brain results in a dose-dependent by galaninergic systems in the control of species-specific increase in , and LH secretion regulatory processes. This proposal is supported by other (Baranowska-Bik et al. 2005). species differences, such as the presence of GALR1 Changes in pituitary expression of galanin have been mRNA in the ovine pituitary and the lack of significant reported in other species in response to steroids GALP mRNA expression in either the ovine hypo- (Vrontakis et al. 1989, Wynick et al. 1993) and it is thalamus or pituitary gland found in this study. interesting to note that in this study, as reported in the rat Receptor mRNA expression was seen to change in both (Vrontakis et al. 1989, Wynick et al. 1993), the levels of the ovine hypothalamus and pituitary gland as a function pituitary galanin mRNA expression did not change as a of age. The observed age-related changes in receptor function of testosterone exposure, levels remaining mRNA expression were similar across the three receptor constant with age, prior to and after puberty in the isoforms, expression being the highest in the pre-pubertal males. However, in the females, pituitary galanin mRNA animals and the lowest in the adults, with the exception of expression was affected by puberty but in the opposite GALR3 in the male pituitary gland, where foetal mRNA direction to that which would be predicted based upon expression was the highest. As discussed above with studies in rats where oestradiol has been shown to regard to galanin, this pattern of receptor mRNA stimulate galanin expression (Wynick et al.1993, expression could support a role for galanin sensitive Hammond et al. 1997). However, it is important to systems in the regulation of growth and/or in the initiation note that the current study did not look at the cellular of reproductive activity, as these both occur during the localisation of pituitary galanin expression that has been pre-pubertal stage of development. This possibility is reported to differ between species (Hsu et al. 1991). supported by a variety of evidence relating to galanin In this regard, it is interesting to note that the age- receptor expression. For example, galanin receptor related changes in galanin mRNA expression observed in knockout studies, conducted in mice (Hohmann et al. the present study were tissue and gender specific, a 2003, Krasnow et al. 2004) demonstrated that GALR1 and significant increase being seen in the male GALR2 are required for normal LH, FSH and testosterone www.reproduction-online.org Reproduction (2009) 137 141–150

Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access 146 C M Whitelaw and others secretion. Similarly, GALR1 has been shown to be the sufficient to allow investigation of specific seasonal predominant receptor subtype involved with the effects of effects on galaninergic systems, that would be indepen- galanin on feeding behaviour (Bartfai et al. 1993) and dent of both age and reproductive status. However, as no GALR2 has been implicated in the regulation of processes specific effects of season have been reported on galanin that induce growth and cell proliferation (Wang et al. mRNA expression in other species, and age-related 1998). Both of these receptors have also been implicated changes have been reported in both galanin and galanin in reproduction via GnRH1 neurones in the preoptic area receptor expression, a more parsimonious explanation of of the hypothalamus in rats (Faure-Virelizier et al. 1998, the results obtained in this study is with a role for ovine Bouret et al. 2000) and sheep (Dufourny et al. 2003). Very galanin as a potential hypothalamic neurotransmitter little has been reported with regard to GALR3. While the that is modulated as a function/consequence of the age pattern of expression of GALR3 mRNA expression and reproductive status of the animal. overlaps that of both GALR1 and GALR2, in the rat While we had hypothesised that there might be (Mennicken et al. 2002), the relatively low levels of differences in gene expression between the two hypo- expression compared with GALR1 and GALR2 and its thalamic slices analysed, one of which encompassed the lower affinity for galanin (Smith et al. 1998) may indicate preoptic area, and the other the mediobasal hypo- a lesser physiological role. Changes in GALR3 mRNA thalamus, statistical analysis of mRNA expression did not expression were seen in this study and, as they changed in indicate significant differences between the two slices. a similar manner to those of GALR1 and GALR2,itis The final results, however, exhibited significant variation possible that its expression is regulated such as to in gene expression with age and gender, between each supplement or complement the actions of the other group/tissue. However, it is possible that the high galanin receptor isoforms. In this respect, if we look at the variability in gene expression between samples within actions of the three receptor subtypes we see that they are each group may have obscured observation of some functionally different as they link to different G protein biologically significant age, gender and or tissue specific complements within the cells. GALR1 and GALR3 are differences in target gene expression. both linked to Gi and Gi/o proteins, which decrease In conclusion, the results of this study demonstrate that intracellular adenylate cyclase activity and hyperpolarise the mRNAs for galanin and its three identified receptor cells respectively, (Berridge & Irvine 1989, Wang et al. isoforms are expressed within the ovine hypothalamus and 1998) thus GALR1 and GALR3 may have complementary pituitary gland in foetal, pre-pubertal and adult animals actions to decrease cell activity. By contrast, GALR2 has and support a role for galanin as both a neurotransmitter both inhibitory actions, through Gi and Go proteins and and a neuromodulator. The lack of significant amounts of stimulatory actions through a Gq protein, via increased GALP mRNA expression within the ovine hypothalamo- MAP kinase activity within the hypothalamus and pituitary gland complex, contrasts with other species and intracellular calcium release within the pituitary gland would suggest that GALP does not fulfil the same proposed (Depczynski et al. 1998, Tsaneva-Atanasova et al. 2007). role as part of a hypothalamic nutritional signalling system Thus, GALR2 may mediate both inhibitory and stimu- in the sheep compared with other species. The expression latory effects on cell function (Wang et al. 1998). of the mRNA for galanin and GALR1–3 all showed changes Finally, comparison of expression levels of mRNA for with age, which are consistent with a role(s) in the galanin and its receptors, in the foetal and pre-pubertal regulation of growth and/or the initiation of reproduction animals, indicated that in both genders and both tissues, but suggest that their role in the regulation of neuroendo- expression of the mRNAs for the three galanin receptor crine process, in the sheep, may differ from those seen in subtypes substantially exceeded that of galanin. In the rats, mice and monkeys. adults, regardless of tissue type expression of mRNA for all the receptor isoforms decreased dramatically. In the females, however, receptor isoform mRNA expression Materials and Methods continued to exceed that of the ligand whereas in the males Tissue this relationship was reversed. This result would suggest Samples of the hypothalamus and pituitary gland were that the regulation of mRNA expression for the components collected, following a lethal dose of barbiturates (20 mg/kg of galaninergic systems changes dramatically in associ- BW, i.v.; Lethobarb, Duphar Vet, UK), from sheep of mixed ation with the change from reproductive immaturity to breed (predominantly Scotch mules), at a series of defined ages. reproductive maturity. It also provides evidence that Immediately following death, the brains were removed and a galaninergic regulatory systems in the sheep may, as in tissue block containing the preoptic area/hypothalamus otherspecies,besexuallydimorphic(Hull & Harvey 2002). dissected out. Pituitary glands were also collected at this While an argument could be made that the age-related time and all tissues were frozen at K70 8C until processed. changes observed in both galanin and galanin receptor Tissues were collected from foetal lambs, w110 days of mRNA expression, within the ovine hypothalamus and gestation (males nZ5, females nZ4); pre-pubertal lambs, 8 pituitary gland are related to seasonal changes in gene weeks of age (females and males nZ6); reproductively active expression, the design of this experiment was not adult, more than 2 and less than 5 years of age, males (nZ4)

Reproduction (2009) 137 141–150 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access Galaninergic mRNA in the ovine hypothalamus/pituitary 147 and cycling females (as the aim of this study was to examine was isolated and purified using Biorad Micro-Bio-spin- the effects of age, the adult females were of mixed oestrous chromatography columns according to the manufacturers cycle stage nZ16). Tissue from the adult sheep and foetal protocol and sequenced using BigDye Terminator v1.1 Cycle lambs were collected in the autumn and early spring Sequencing Kits (Applied Biosystems 3100 Genetic Analyser). respectively, whereas tissue from the pre-pubertal lambs was Primer sequences (MWG-BIOTECH AG, Ebersberg, Germany) collected in the early summer. All procedures were approved used for RTPCR and cDNA sequencing are listed in Table 1. by the Faculty’s Ethics and Welfare committee and were Semi-quantitative real-time taqman PCR (qPCR) was per- carried out in accordance with the UK Animals (Scientific formed on duplicate samples of cDNA using Amplitaq Gold Procedures) Act 1986. kits (Applied Biosciences, Beaconsfield, Bucks, UK) using Whilst still frozen, each hypothalamic block was cut into ovine b-actin as the housekeeping gene, in a Stratagene MX coronal slices using external landmarks, with the most rostral 3000P thermal cycler. Due to the high GC content and low cut w1 mm in front of the optic chiasma, such that the first slice levels of mRNA expression of the expected products, qPCR encompassed the preoptic area and the second the mediobasal conditions were as follows; 97 8C for 10 min – 1 cycle, 95 8C hypothalamus/stalk median eminence (w4 mm in adult, 3 mm for 45 s, 60 8C for 1 min – 45 cycles. in pre-pubertal and 2 mm in foetal lambs). For RNA extraction, Primer-Probe sets (Eurogentecs S.A, Liege, Belgium) for 100–200 mg of tissue were then harvested from an area close to qPCR were designed using Primer Express (Applied Biosystems) the ventricle and near the base of the hypothalamus from each software and used FAM as the 50-reporter and DDQ1 as the tissue block. Pituitary glands were cut along the mid-sagittal 30-quencher. plane (both posterior and anterior glands) and w100–200 mg tissue harvested from the mid-sagittal face for RNA extraction. Statistical analysis

RNA extraction For relative quantification of mRNA concentrations the comparative CT method was used, wherein the expression of Total RNA was extracted from the tissue using Trizol each gene of interest was quantified relative to the expression (Invitrogen) according to the manufacturer’s instructions. of the housekeeping gene (b-actin; User Bulletin no. 2, PE cDNA was obtained by reverse transcription of the resultant Biosystems, UK). Validation experiments (data not shown) mRNA using random hexamers (Promega), M-MLV reverse confirmed that the amplification efficiencies of the galaninergic transcriptase (Invitrogen) and RNasin (Promega), as described genes and b-actin were comparable, the slope of the difference previously (O’Shaughnessy & Murphy 1993). mRNA and between CT values for the standard curves for each gene were cDNA purity and quantity were assessed by spectroscopy !0.1 and within 5% of the b-actin slope. All results are G (yield averaged 1000 ng/ul and 260/280 ratio 2.0 0.2). expressed as the meanGS.E.M. Relative expression levels of galanin and the three receptor isoforms were compared between areas of the hypothalamus mRNA sequencing and quantification by ANOVA (Genstat, release 10, VSN International, Hemel Partial mRNA sequences for ovine galanin, GALP, GALR1, Hempstead, UK). As the expression of galanin and the three GALR2 and GALR3 were obtained as follows; primer receptor isoforms was not found to differ significantly between sequences for the genes of interest were designed based upon the two hypothalamic slices, hypothalamic expression was regions of high interspecies (rat,mouse,cow,human) averaged for each animal. Values were then multiplied by 1000 homology using available (GenBank) sequences. Synthesised (for ease of data handling) prior to further statistical analysis. To primers were used with samples of ovine hypothalamic and equalise variance between groups the data were log pituitary gland cDNA in standard RTPCR protocols and the transformed and, due to the non-orthogonal nature of the RTPCR products separated on a 1% agarose gel. Where bands data (largely due to the disparity in numbers between groups), of an appropriate size were visualised, the associated cDNA differences in expression between sexes and ages studied

Table 1 Primer and probe sequences used to isolate and quantify ovine galanin, GALP and galanin receptor mRNAs.

Forward 50–30 Probe Reverse 30–50 RT-PCR b-actin TCCTTCCTGGGCATGGAATC GGGCGCGATGATCTTGATCT Galanin TACCTTCTCGGACCACATGC TGCAGGAAAGTGAGAAACTC GALR1 CCTTGGCATAGCAGAAGCAG ATGTCGGTGGACGCGTACGT GALR2 CGCTCATCTTCCTCGTGGG AGCCGTCCAGGGTGTAGATG GALR3 TAGACAGCCCAGGGAGTATG GTCTTTGCCCTCATCTTCCT GALP CGAGGAGGCTGGACCCTCAA CAGGTCTAGGATCTCAAG qPCR b-actin CCCTGAGGCTCTCTTCCA CTTCCTTCCTGGGCATGGAATCC GGAATTGAAGGTAGTTTCGTGAAT Galanin GAGAGGCTGGACCCTGAACA TGCCGGCTACCTTCTCGGACCA CGTGAAATGACCTGTGGTTGTC GALR1 CACACCACGTAGGCCTTCTTG CGCTGGTTGGGCCACTGCTCC GACGTCAGCAACCAGACCTTCT GALR2 AGCCGTCCAGGGTGTAGATG CTTGGAAGGGCACGCAG CGACCTGTGTTTCATCGTGTG GALR3 ACGACGGATCTATTCATACCTCAAC CTGGCGGCAGCTGACCTCTGCT GATGGCGGCCTGGAAAG www.reproduction-online.org Reproduction (2009) 137 141–150

Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access 148 C M Whitelaw and others within each tissue type by REML (Genstat, release 10, VSN Clarke IJ, Thomas GB, Yao B & Cummins JT 1987 GnRH secretion International), using a linear mixed model analysis where the throughout the ovine estrous-cycle. Neuroendocrinology 46 82–88. fixed model was sex x age and individual animals were Crawley JN, Robinson JK, Langel U & Bartfai T 1993 Galanin -M40 and antagonist-C7 block galanin-induced feeding. Brain included as the random model term. Differences between Research 600 268–272. treatment groups were calculated using least significant Cunningham MJ, Scarlett JM & Steiner RA 2002 Cloning and distribution of differences analysis, calculated from the standard error of galanin-like peptide mRNA in the hypothalamus and pituitary of the the difference (S.E.D.) between means obtained from the macaque. Endocrinology 143 755–763. REML analysis. Delemarre-van de Waal HA, Burton KA, Kabigting EB, Steiner RA & Clifton DK 1994 Expression and sexual dimorphism of galanin messenger-ribonucleic-acid in growth hormone-releasing hormone neurons of the rat during development. Endocrinology 134 Declaration of interest 665–671. There is no conflict of interest that could be perceived as Depczynski B, Nichol K, Fathi Z, IIsmaa T, Shine J & Cunningham A 1998 Distribution and characterization of the cell types expressing GALR2 prejudicing the impartiality of the research reported in this mRNA in brain and pituitary gland. Annals of the New York Academy of manuscript. Sciences 863 120–128. Dufourny L & Skinner DC 2005 Distribution of -immunoreactive neurons in the ovine hypothalamus: colocalization Funding with GnRH. Brain Research 1054 73–81. Dufourny L, Schofield N & Skinner DC 2003 Immunoreactive galanin This work was supported by the Biotechnology and Biological expression in ovine gonadotropin-releasing hormone neurones: no Sciences Research Council (Grant number S18947). effects of gender or reproductive status. Journal of Neuroendocrinology 15 1062–1069. Elmquist JK, Fox CA, Ross LR & Jacobson CD 1992 Galanin-like immunoreactivity in the adult and developing Brazilian opossum References brain. Developmental Brain Research 67 161–179. Evans NP, Land RB, McNeilly JR & Webb R 1991 Role of gonadal negative Baranowska-Bik A, Baranowska B, Wolinska-Witort E, Chmielowska M, feedback on the gonadotropin responses to gonadotropin-releasing- Martynska L & Bik W 2005 Galanin modulates pituitary hormones hormone (GnRH) in ram lambs from two lines of sheep selected for their release. Neuroendocrinology Letters 26 468–472. luteinizing-hormone response to GnRH. Journal of Reproduction and Baratta M, Saleri R, Mascadri C, Coy DH, NegroVilar A, Tamanini C & Fertility 93 549–558. Giustina A 1997 Modulation by galanin of growth hormone and Evans NP, Dahl GE, Mauger D & Karsch FJ 1995a induces both gonadotropin secretion from perifused pituitary and median eminence qualitative and quantitative changes in the pattern of gonadotropin- of prepubertal male calves. Neuroendocrinology 66 271–277. releasing-hormone secretion during the presurge period in the ewe. Bartfai T, Langel U, Bedecs K, Andell S, Land T, Gregersen S, Ahren B, Endocrinology 136 1603–1609. Girotti P, Consolo S, Corwin R et al. 1993 Galanin-receptor ligand M40 Evans NP, Dahl GE, Mauger DT, Padmanabhan V, Thrun LA & Karsch FJ peptide distinguishes between putative galanin-receptor subtypes. PNAS 1995b Does estradiol induce the preovulatory gonadotropin-releasing- 90 11287–11291. hormone (GnRH) surge in the ewe by inducing a progressive change in the Berridge MJ & Irvine RF 1989 Inositol phosphates and cell signaling. Nature 341 197–205. mode of operation of the GnRH neurosecretory-system. Endocrinology Bhide SA & Puranik SS 2005 Galanin immunohistochemistry and electron 136 5511–5519. microscopic studies in developing human fetal mammillary bodies. Evans NP, Dahl GE, Padmanabhan V, Thrun LA & Karsch FJ 1997 Estradiol Journal of Chemical Neuroanatomy 29 289–295. requirements for induction and maintenance of the gonadotropin- Bouret S, Prevot V, Croix D, Howard A, Habert-Ortoli E, Jegou S, Vaudry H, releasing hormone surge: implications for neuroendocrine processing Beauvillain JC & Mitchell V 2000 Expression of GalR1 and GalR2 of the estradiol signal. Endocrinology 138 5408–5414. galanin receptor messenger ribonucleic acid in Faure-Virelizier C, Croix D, Bouret S, Prevot V, Reig S, Beauvillain JC & neurons of the rat arcuate nucleus: effect of testosterone. Endocrinology Mitchell V 1998 Effects of estrous cyclicity on the expression of the 141 1780–1794. galanin receptor Gal-R1 in the rat preoptic area: a comparison with the Cai A, Bowers RC, Moore JP & Hyde JF 1998 Function of galanin in the male. Endocrinology 139 4127–4139. anterior pituitary of -treated Fischer 344 rats: autocrine and Freeman M 1993 The neuroendocrine control of the ovarian cycle of the rat. paracrine regulation of prolactin secretion. Endocrinology 139 In The Physiology of Reproduction, pp 613–658. Eds E Knobil & JD Neill. 2452–2458. New York: Raven Press. Castellano JM, Navarro VM, Fernandez-Fernandez R, Roa J, Vigo E, Gabriel SM, Kaplan LM, Martin JB & Koenig JI 1989 Tissue-specific sex- Pineda R, Steiner RA, Aguilar E, Pinilla L & Tena-Sempere M 2006 Effects differences in galanin-like immunoreactivity and galanin messenger- of galanin-like peptide on luteinizing hormone secretion in the rat: RNA during development in the rat. 10 369–374. sexually dimorphic responses and enhanced sensitivity at male puberty. Hammond PJ, Khandan-Nia N, Withers DJ, Jones PM, Ghatei MA & Bloom SR American Journal of Physiology. Endocrinology and Metabolism 291 1997 Regulation of anterior pituitary galanin and vasoactive intestinal E1281–E1289. peptide by oestrogen and prolactin status. Journal of Endocrinology Chaillou E & Tillet Y 2005 Nutrition and hypothalamic in 152 211–219. sheep: histochemical studies. Histology and Histopathology 20 Herbison AE, Robinson JE & Skinner DC 1993 Distribution of estrogen 1209–1225. receptor-immunoreactive cells in the preoptic area of the ewe – Chaillou E, Tramu G & Tillet Y 1999 Distribution of galanin immunor- colocalization with glutamic-acid decarboxylase but not luteinizing- eactivity in the sheep diencephalon. Journal of Chemical Neuroanatomy hormone-releasing hormone. Neuroendocrinology 57 751–759. 17 129–146. Hohmann JG, Clifton DK & Steiner RA 1998 Galanin: analysis of its Chambers GB, Whitelaw C, Robinson JE & Evans NP 2007 Distribution of galanin receptor-2 immunoreactive neurones in the ovine hypothalamus: coexpression in gonadotropin-releasing hormone and growth hormone- no evidence for involvement in the control of gonadotrophin-releasing releasing hormone neurons. Annals of the New York Academy of hormone secretion. Journal of Neuroendocrinology 19 966–973. Sciences 863 221–235. Cheung CC, Clifton DK & Steiner RA 1996 Galanin: an unassuming Hohmann JG, Krasnow SM, Teklemichael DN, Clifton DK, Wynick D & moves to center stage in reproduction. Trends in Steiner RA 2003 Neuroendocrine profiles in galanin-overexpressing and Endocrinology and Metabolism 7 301–306. knockout mice. Neuroendocrinology 77 354–366.

Reproduction (2009) 137 141–150 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access Galaninergic mRNA in the ovine hypothalamus/pituitary 149

Hsu DW, Hooi SC, Hedley-Whyte ET, Strauss RM & Kaplan LM 1991 Murakami Y, Kato Y, Shimatsu A, Koshiyama H, Hattori N, Yanaihara N Coexpression of galanin and adrenocorticotropic hormone in human &ImuraH1989 Possible mechanisms involved in growth-hormone pituitary and pituitary adenomas. American Journal of Pathology 138 secretion induced by galanin in the rat. Endocrinology 124 897–909. 1224–1229. Hull KL & Harvey S 2002 GH as a co-gonadotropin: the relevance of Niimi M, Takahara J, Sato M & Kawanishi K 1990 Immunohistochemical correlative changes in GH secretion and reproductive state. Journal of identification of galanin and growth hormone-releasing factor- Endocrinology 172 1–19. containing neurons projecting to the median-eminence of the rat. Hyde JF, Moore JP & Cai A 1998 Galanin in normal and hyperplastic Neuroendocrinology 51 572–575. anterior pituitary cells. Annals of the New York Academy of Sciences 863 Ohtaki T, Kumano S, Ishibashi Y, Ogi K, Matsui H, Harada M, Kitada C, 48–55. Kurokawa T, Onda H & Fujino M 1999 Isolation and cDNA cloning of a Iqbal J, Manley TR, Ciofi P & Clarke IJ 2005 Reduction in adiposity affects novel galanin-like peptide (GALP) from porcine hypothalamus. Journal of the extent of afferent projections to growth hormone-releasing hormone Biological Chemistry 274 37041–37045. and somatostatin neurons and the degree of colocalization of O’Shaughnessy PJ & Murphy L 1993 Cytochrome P-450 17 alpha- neuropeptides in growth hormone-releasing hormone and somatostatin hydroxylase protein and mRNA in the testis of the testicular feminized cells of the ovine hypothalamus. Endocrinology 146 4776–4785. (Tfm) mouse. Journal of Molecular Endocrinology 11 77–82. Jureus A, Cunningham MJ, Li D, Johnson LL, Krasnow SM, Teklemichael DN, Planas BK, Easkind PE & Millar MA 1994 Activation of galanin pathways Clifton DK & Steiner RA 2001 Distribution and regulation of galanin-like across puberty in the male rat: assessment of regional densities of galanin peptide (GALP) in the hypothalamus of the mouse. Endocrinology 142 binding sites. Neuroscience 63 859–867. 5140–5144. Planas BK, Easkind PE & Millar MA 1995 Galanin-binding sites in the Krasnow SM, Hohmann JG, Gragerov A, Clifton DK & Steiner RA 2004 female rat brain are regulated across puberty yet similar to the male Analysis of the contribution of galanin receptors 1 and 2 to the central pattern in adulthood. Neuroendocrinology 61 646–654. actions of galanin-like peptide. Neuroendocrinology 79 268–277. Rajendren G & Li XP 2001 Galanin synaptic input to gonadotropin- Lang R, Berger A, Santic R, Geisberger R, Hermann A, Herzog H & Kofler B releasing hormone perikarya in juvenile and adult female mice: 2005 Pharmacological and functional characterization of galanin-like implications for sexual maturity. Developmental Brain Research 131 peptide fragments as potent galanin receptor . Neuropeptides 39 161–165. 179–184. Rossmanith WG, Marks DL, Clifton DK & Steiner RA 1994 Induction of Larm JA & Gundlach AL 2000 Galanin-like peptide (GALP) mRNA galanin gene-expression in gonadotropin-releasing-hormone neurons expression is restricted to arcuate nucleus of hypothalamus in adult with puberty in the rat. Endocrinology 135 1401–1408. male rat brain. Neuroendocrinology 72 67–71. Scarlett JM, Cunningham MJ, Clifton DK & Steiner RA 2001 Cloning and Lehman MN & Karsch FJ 1993 Do gonadotropin-releasing-hormone, mapping of the distribution of galanin-like peptide (GALP) mRNA in the tyrosine hydroxylase-immunoreactive, and beta-endorphin-immuno- hypothalamus of the monkey. FASEB Journal 15 A811–a811. reactive neurons contain estrogen-receptors – a double-label immuno- Selvais PL, Denef JF, Adam E & Maiter DM 1995 Sex-steroid control of cytochemical study in the suffolk ewe. Endocrinology 133 887–895. galanin in the rat hypothalamic–pituitary axis. Journal of Neuroendo- Leibowitz SF, Akabayashi A, Alexander JT & Wang J 1998 Gonadal steroids crinology 7 401–407. and hypothalamic galanin and : role in eating behavior Shen ES, Meade EH, Perez MC, Deecher DC, Negro-Vilar A & Lopez FJ 1998 and body weight control in female rats. Endocrinology 139 1771–1780. Expression of functional estrogen receptors and galanin messenger Lorimer DD & Benya RV 1996 Cloning and quantification of galanin-1 ribonucleic acid in immortalized luteinizing hormone-releasing expression by mucosal cells lining the human gastrointestinal neurons: estrogenic control of galanin gene expression. Endocrinology tract. Biochemical and Biophysical Research Communications 222 139 939–948. 379–385. Sizer AR, Rokaeus A & Foster GA 1990 Analysis of the ontogeny of galanin Matsumoto H, Noguchi J, Takatsu Y, Horikoshi Y, Kumano S, Ohtaki T, in the rat central nervous-system by immunohistochemistry and Kitada C, Itoh T, Onda H, Nishimura O et al. 2001 Stimulation effect of radioimmunoassay. International Journal of Developmental Neuroscience galanin-like peptide (GALP) on luteinizing hormone-releasing hormone- 8 81–97. mediated luteinizing hormone (LH) secretion in male rats. Endocrinology 142 3693–3696. Skinner DC, Evans NP, Delaleu B, Goodman RL, Bouchard P & Caraty A Melander T, Hokfelt T, Rokaeus A, Fahrenkrug J, Tatemoto K & Mutt V 1985 1998 The negative feedback actions of on gonadotropin- Distribution of galanin-like immunoreactivity in the gastrointestinal-tract releasing hormone secretion are transduced by the classical progesterone of several mammalian-species. Cell and Tissue Research 239 253–270. receptor. PNAS 95 10978–10983. Melander T, Hokfelt T & Rokaeus A 1986 Distribution of galanin-like Skinner DC, Caraty A & Allingham R 2001 Unmasking the progesterone immunoreactivity in the rat central-nervous-system. Journal of Comparative receptor in the preoptic area and hypothalamus of the ewe: no Neurology 248 475–517. colocalization with gonadotropin-releasing neurons. Endocrinology Mennicken F, Hoffert C, Pelletier M, Ahmad S & O’Donnell D 2002 142 573–579. Restricted distribution of (GalR3) mRNA in the adult Skofitsch G & Jacobowitz DM 1986 Quantitative distribution of galanin- rat central nervous system. Journal of Chemical Neuroanatomy 24 like immunoreactivity in the rat central-nervous-system. Peptides 7 257–268. 609–613. Merchenthaler I, Lopez FJ & Negrovilar A 1993 Anatomy and physiology of Smith KE, Walker MW, Artymyshyn R, Bard J, Borowsky B, Tamm JA, central galanin-containing pathways. Progress in Neurobiology 40 Yao WJ, Vaysse PJJ, Branchek TA, Gerald C & Jones KA 1998 Cloned 711–769. human and rat galanin GALR3 receptors – pharmacology and activation C Mitchell V, Bouret S, Prevot V, Jennes L & Beauvillain JC 1999 Evidence for of G-protein inwardly rectifying K channels. Journal of Biological expression of galanin receptor Gal-R1 mRNA in certain gonadotropin Chemistry 273 23321–23326. releasing hormone neurones of the rostral preoptic area. Journal of Takatsu Y, Matsumoto H, Ohtaki T, Kumano S, Kitada C, Onda H, Neuroendocrinology 11 805–812. Nishimura O & Fujino M 2001 Distribution of galanin-like peptide in the Moenter SM, Brand RC & Karsch FJ 1992 Dynamics of gonadotropin- rat brain. Endocrinology 142 1626–1634. releasing-hormone (GnRH) secretion during the GnRH surge – insights Tarasov KV, Tarasova YS, Crider DG, Anisimov SV, Wobus AM & Boheler KR into the mechanism of GnRH surge induction. Endocrinology 130 2002 Galanin and galanin receptors in embryonic stem cells: accidental 2978–2984. or essential? Neuropeptides 36 239–245. Moenter SM, Caraty A, Lehman MN & Karsch FJ 1993 Characterization and Tempel DL, Leibowitz KJ & Leibowitz SF 1988 Effects of PVN galanin on regulation of preovulatory secretion of gonadotropin-releasing-hormone. macronutrient selection. Peptides 9 309–314. Human Reproduction 8 51–56. Todman MG, Han SK & Herbison AE 2005 Profiling neurotransmitter Morris JL, Gibbins IL & Osborne PB 1989 Galanin-like immunoreactivity in receptor expression in mouse gonadotropin-releasing hormone neurons sympathetic and parasympathetic neurons of the toad Bufo marinus. using green protein-promoter transgenics and microarrays. Neuroscience Neuroscience Letters 102 142–148. 132 703–712. www.reproduction-online.org Reproduction (2009) 137 141–150

Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access 150 C M Whitelaw and others

Tourlet S, Ziyazetdinova G, Caraty A, Tramu G, Delsol G & Tillet Y 2005 Wang S, Hashemi T, Fried S, Clemmons AL & Hawes BE 1998 Differential Oestradiol effect on galanin-immunoreactive neurones in the dience- intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes. phalon of the ewe. Journal of Neuroendocrinology 17 145–151. Biochemistry 37 6711–6717. Tsaneva-Atanasova K, Sherman A, van Goor F & Stojilkovic SS 2007 Waters SM & Krause JE 2000 Distribution of galanin-1,-2 and-3 receptor Mechanism of spontaneous and receptor-controlled electrical activity in messenger RNAs in central and peripheral rat tissues. Neuroscience 95 pituitarysomatotrophs:experiments and theory. Journal of Neurophysiology 265–271. 98 131–144. Wood RI, Ebling FJ, I’Anson H, Bucholtz DC, Yellon SM & Foster DL 1991 Urbanski HF & Ojeda SR 1990 A role for N-methyl-D-aspartate (NMDA) Prenatal androgens time neuroendocrine sexual maturation. Endo- receptors in the control of LH-secretion and initiation of female puberty. crinology 128 2457–2468. Endocrinology 126 1774–1776. Wynick D, Hammond PJ, Akinsanya KO & Bloom SR 1993 Galanin Vrontakis ME, Yamamoto T, Schroedter IC, Nagy JI & Friesen HG 1989 regulates basal and oestrogen-stimulated lactotroph function. Nature Estrogen induction of galanin synthesis in the rat anterior-pituitary gland 364 529–532. demonstrated by in situ hybridization and immunohistochemistry. Neuroscience Letters 100 59–64. Wang SK, He CG, Hashemi T & Bayne M 1997 Cloning and expressional Received 13 June 2008 characterization of a novel galanin receptor – identification of different pharmacophores within galanin for the three galanin receptor subtypes. First decision 4 August 2008 Journal of Biological Chemistry 272 31949–31952. Accepted 30 September 2008

Reproduction (2009) 137 141–150 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/28/2021 04:17:06AM via free access