S09-5 Gonadotropin-inhibitory hormone in birds: possible modes of action

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1 52(Supplement): , 2006 S09-5 Gonadotropin-inhibitory hormone in birds: possible modes of action George E. BENTLEY 1*, Nicole PERFITO 1, Ignacio T. MOORE 1, Kazuyoshi UKENA 2, Kazuyoshi TSUTSUI 2, John C. WINGFIELD 1 1. Dept. of Zoology, Box , University of Washington, Seattle, Washington 98195, U.S.A; *gb7@u.washington.edu 2. Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima , and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo , Japan Abstract Gonadotropin inhibitory hormone (GnIH) inhibits gonadotropin release in vitro in a dose-dependent manner (Tsutsui et al., 2000). In the present study, we investigated the relative neural distributions of GnRH and GnIH, and in vivo activity of exogenous GnIH in song sparrows (Melospiza melodia) and house sparrows (Passer domesticus). Using immunocytochemistry (ICC), GnIH-containing neurons were localized in both species in the paraventricular nucleus, the GnIHcontaining fibers emanating from those neurons to multiple brain locations, including the median eminence and brainstem. Double-label ICC with light microscopy and fluorescent ICC with confocal microscopy indicate a high probability of colocalization of GnIH and GnRH (-I and -II) neurons and fibers within the avian brain. Thus, GnIH might be acting at the level of the hypothalamus as well as at the anterior pituitary to regulate gonadotropin release. In an in vivo experiment, cgnrh-i alone or a cgnrh-i/gnih cocktail was injected intravenously into song sparrows. Blood was collected at 2, 5 and 10 minutes post-injection. GnIH rapidly (within 2 minutes) attenuated the GnRH-induced rise in plasma luteinizing hormone. As GnRH- II is involved with regulation of reproductive behaviors, GnIH may play a role in regulating them as well. In sum, GnIH is (1) present in photoperiodic songbird species, (2) has the potential to regulate gonadotropin release at more than one level, and (3) might also be involved in the regulation of reproductive behaviors. Key words Rfamide function, GnRH, GnIH inhibition, Songbird, Photoperiodism 1 Introduction The recent discovery of a vertebrate hypothalamic peptide that, in a dose-dependent manner, directly inhibits LH release from cultured pituitary glands in vitro has provided the potential for new insights into the regulation of gonadotropin release, and could profoundly enhance our understanding of the mechanistic basis for the timing of reproduction (Tsutsui et al., 2000). This inhibitory peptide, named gonadotropin-inhibitory hormone (GnIH), is a potential direct regulator of gonadotropin release in vivo. In this paper, we focus on the distribution and gonadotropininhibiting activity of the GnIH protein, establishing its presence in the songbird brain and its distribution relative to chicken gonadotropin-releasing hormone-i and -II (cgnrh- I and -II). Photoperiodic animals rely on the annual cycle of changing photoperiod to restrict their reproductive activity to the most opportune time of year (Rowan, 1926; Nicholls et al., 1988; Wilson and Donham, 1988; Bronson, 1989; Dawson et al., 2001). In most photoperiodic bird species, changes in reproductive activity are dependent upon, but not directly proportional to, changes in photoperiod. During short photoperiods of winter and early spring, birds are photosensitive and GnRH is secreted at a low level such that the gonads remain in an immature state, or mature slowly. As photoperiod increases, photostimulation occurs, resulting in increased GnRH secretion, full gonadal maturation and breeding. Even though long days continue, however, the breeding season ends abruptly with a profound termination of GnRH release from the hypothalamus, followed by a decrease in synthesis as well (Dawson et al., 1985; Foster et al., 1987; Hahn and Ball, 1995; Reinert and Wilson, 1996; Cho et al., 1998; Deviche et al., 2000). This nonbreeding, or photorefractory, state is caused directly by long photoperiods and, in many species, is maintained indefinitely unless the birds experience a period of short days once again. Thus, long days cause not only reproductive maturation but also regression to a prepubertal state, in sequential order (Bentley et al., 1997). This pattern of puberty and reverse puberty in response to changes in photoperiod is shown in many avian species, including song sparrows, Melospiza melodia (Wingfield, 1993). Based upon research by Tsutsui and co-workers (Tsutsui et al., 2000; Satake et al., 2001), we predicted that, as is the case for GnRH, GnIH is important for the timing of reproduction in all photoperiodic bird species. Thus the aims of the present study were, first, to establish the pres- 2006

2 George E. BENTLEY et al.: Gonadotropin-inhibitory hormone in birds 179 ence of GnIH in the brains of species of two families of songbirds (song sparrows, Emberizidae, and house sparrows, Passeridae); secondly, to determine the neuroanatomical relationship of GnIH with GnRH; and, thirdly, if GnIH is present in the brains of song sparrows, to determine whether or not it had gonadotropin-inhibiting capability. The experiment by Tsutsui et al. (2000) demonstrated inhibition of baseline LH release from cultured anterior pituitary glands by GnIH. The incubations in that experiment lasted for 100 minutes before medium was collected and gonadotropins measured. If GnIH plays an active role in modulating gonadal growth, or in inhibiting gonadotropin release either during the breeding season (for temporary cessation of breeding as a result of adverse environmental cues), or at the end of the breeding season (to hasten gonadal regression), then it must inhibit LH release in vivo. In addition, it must inhibit LH release from stimulated pituitary, rather than from the baseline release in vitro. 2 Materials and methods All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and with the approval of the University of Washington Institutional Animal Care and Use Committee. 2.1 Neuroanatomical distribution of GnIH protein in song sparrows and house sparrows For immunocytochemistry (ICC), two males and two females of each of song sparrows and house sparrows (Passer domesticus) were used. Birds were terminally anesthetized with an intramuscular injection of 7.5 mg sodium pentobarbital, and perfused transcardially with 0.9% heparinized saline (150 IU/10 ml), followed by 4% paraformaldehyde in 0.1 mol/l phosphate buffer-saline, ph 7.4 (PBS). Brains were extracted, post-fixed for 2 hours in the same fixative at 4 C and cryoprotected overnight in PBS containing 30% sucrose. Sagittal or coronal sections (50 m) were taken throughout the whole brain and collected into PBS. Immunocytochemistry for GnIH was then performed. Sections were washed three times in PBS, background immunoreactivity blocked overnight using 2% normal goat serum (NGS) in 0.2% PBS-T (PBS + Triton X-100), and then incubated in primary antibody (rabbit anti-gnih) at a concentration of 1:1 000 in 0.4% PBS-T. Three subsequent washes in 0.2% PBS-T were followed by incubation for 1 hour in biotinylated goat anti-rabbit IgG (1:1 000 in 0.2% PBS-T) and another set of washes. Sections were then incubated for 1 h in avidin-biotin complex (ABC; Vectastain Elite Kit, Vector Labs). The resulting complex was visualized using 0.03% 3,3- diaminobenzidine (DAB) intensified with 0.15% nickel sulfate. Alternately-collected pre-absorption control sections from one brain were processed in the same immunocytochemistry run as the experimental sections. In the only modification to the protocol above, sections were incubated in synthetically-produced GnIH peptide at a concentration of 20 µg/ml concurrently with the primary antibody, instead of primary antibody alone. 2.2 Double-label fluorescence immunocytochemistry and confocal microscopy House sparrow brain tissue (paraformaldehyde perfused, 40 m coronal sections) was processed for doublelabel immunocytochemistry. Immunocytochemistry for GnRH was performed first. The protocol used was similar to the protocol for GnIH ICC above, with the following changes. First, sections were washed, incubated in 0.3% H 2 O 2 for 30 minutes, and washed again before background immunoreactivity was blocked with NGS. Secondly, the primary antibody used was rabbit-anti chicken GnRH (HU60H; kindly donated by Dr. H. Urbanski) at a concentration of 1: in 0.2% PBS-T. This antibody detects cgnrh-i and- II. The secondary antibody used for GnRH was goat antirabbit IgG conjugated to the fluorophore, Alexafluor 488 (Molecular Probes, Inc.), and that for GnIH was goat antirabbit IgG conjugated to Alexafluor 568. Images were collected using a Biorad MRC 600 confocal microscope, and processed using NIH Image, Image J (NIH) and Adobe Photoshop In vivo demonstration of active and rapid inhibition of LH release by exogenous GnIH Photorefractory male song sparrows (n=7 per group) were used for this experiment. Photorefractory birds were used so that we could control the amount of GnRH given to each bird, thereby eliminating any confounding effects from endogenous GnRH. The pituitary gland of photorefractory birds remains responsive to exogenous GnRH even though little or no endogenous GnRH is released in this reproductive condition (Nicholls et al., 1988; Wingfield et al., 1979). Group I (control) was given an i.v. injection (into the right jugular vein) of 10 ng GnRH in 20 µl physiological saline. Group II (experimental) was given an i.v. injection of a mixture of 10 ng GnRH plus 1000 ng GnIH in 20 µl physiological saline. Blood samples were taken from the alar vein at 2, 5 and 10 min. after injection. This protocol has been used previously to demonstrate rapid gonadotropin-releasing activity of GnRH in songbirds (e.g., Wingfield et al., 1979; Wingfield and Farner, 1993). Plasma was assayed for LH using the homologous chicken LH radioimmunoassay (RIA) (Follett et al., 1972), and validated for songbirds (Dawson and Goldsmith, 1982). Included in the RIA were samples of known LH concentration spiked with ng GnIH to check for cross-reactivity with the LH antiserum. No such crossreactivity was detected in this assay (data not shown). All samples were run in duplicate in a single assay. 2.4 Statistical analysis Assay data were analyzed by repeated measures analysis of variance (ANOVA), with injection type as a between-subjects factor and time as a within-subjects factor, followed by Fisher s PLSD for post-hoc analysis. 3 Results 3.1 Neuroanatomical distribution of GnIH protein in song sparrows and house sparrows

3 180 Dense populations of GnIH-ir neurons were found only in the paraventricular nucleus (PVN) of the hypothalamus in all subject birds, regardless of sex or species. These GnIH-ir neurons tend to be bipolar or tripolar (Fig. 1). No GnIH-ir neurons were detected elsewhere in the brain. Furthermore, pre-absorption control sections exhibited no immunoreactivity (Fig. 1F). Thus we are confident that the antibody is specific for the GnIH peptide in songbirds. In addition to dense immunoreactivity in the population of neurons within the hypothalamus, there are extensive networks of branching beaded fibers emanating from those cells, presumably transporting GnIH. Some of the fibers extend to terminals in the ME, consistent with a role for GnIH in pituitary gonadotropin regulation. In both sparrows, other fibers extend through the brain caudally at least as far as the brainstem and possibly into the spinal cord, consistent with multiple regulatory roles for GnIH. 3.2 Double-label fluorescence immunocytochemistry and confocal microscopy Confocal microscopy indicates that the cgnrh-i and GnIH proteins are in extremely close proximity to one another, and fibers of each are probably in contact, although electron microscopy and/or tract-tracing will be necessary to determine this conclusively. Close inspection of each area indicates close proximity of GnIH fibers to the cgnrh-i neurons and fibers in the pre-optic area (POA). Presumably these fibers project rostrally from the GnIH neurons in the PVN. The PVN also contains cgnrh-i fibers which pass directly through and in close proximity to the population of GnIH neurons and fibers as they project to the ME. It is apparent that GnIH-ir fibers are also in close proximity to cgnrh-ii neurons in the midbrain (data not shown). Pre- sumably these fibers also emanate from the GnIH-ir neurons in the PVN, but again, this was impossible to discern definitively from the present study. Figure 2 indicates colocalization or extremely close proximity of the cgnrh-i and GnIH peptides in fiber terminals in the ME, with both wavelengths being emitted from the same location. Note the clear separation of immunoreactivity for GnIH and cgnrh-i in fibers dorsal to the ME, where each emits in a separate wavelength. 3.3 In vivo demonstration of active and rapid inhibition of LH release by exogenous GnIH The LH data from the injection experiment are shown in Fig. 3. Analysis of variance indicated a significant interaction of time and injection (ANOVA: F= (1, 16); P= 0.006), allowing for post-hoc analysis to highlight time points at which injections had different effects. At 2 minutes postinjection, birds injected with GnRH alone had higher plasma LH at 2 minutes than at 10 minutes (Fisher s LSD: P<0.001). Birds injected with the GnRH/GnIH cocktail also had elevated plasma LH at 2 minutes post-injection (Fisher s LSD: P <0.05), but this increase was much attenuated compared to that in the control group (Fisher s LSD: P<0.05). The difference in plasma LH concentration between the two groups was no longer evident at 5 minutes and 10 minutes post-injection, by which time plasma LH had returned to baseline values in both groups. 4 Discussion The aims of the present study were to determine the presence, distribution relative to GnRH, and gonadotropininhibiting capability of GnIH in the brains of two sparrow species, both of which are highly photoperiodic. The data Fig. 1 A. coronal section from male song sparrow brain with dense bilateral populations of GnIH neurons and fibers in the PVN; B. coronal section from male Song Sparrow brain with bilateral population of GnIH neurons and fibers in the caudoventral PVN, extending towards the ME; C. sagittal section from female song sparrow brain with dense GnIH immunoreactivity in the PVN; D. higher magnification of C; E. GnIH fibers and terminal fields in the ME; F. sagittal section from female song sparrow brain with the antiserum preincubated with a saturating concentration of GnIH peptide (preabsorption control) Acronyms: Cb = cerebellum, CO = optic chiasm, CoA = anterior commissure, ME = median eminence, PVN = paraventricular nucleus. Note the total lack of immunoreactivity in subfigure F. Scale bars, 100 µm.

4 George E. BENTLEY et al.: Gonadotropin-inhibitory hormone in birds 181 Effects of GnRH and GnRH/GnIH coocktail upon plasma LH in vivo 4 Plasma LH (ng/ml) GnRh (10 ng) 10 ng GnRH ng GnIH 0.5 Fig. 2 Separated confocal images of the median eminence of a sparrow brain showing emission of the two fluorophores in different wavelengths Subfigure A shows emission from the GnIH-ir fibers, and B shows emission from GnRH-ir fibers. The median eminence is visible in both wavelengths, indicating probable co-localization of GnIH and GnRH, or at least extremely close proximity of the two peptides at the terminal fields. Arrows indicate fibers of each peptide that are not co-localized. Images depict the same 0.5 mm plane. clearly demonstrate that GnIH is present in abundance in the hypothalami of both sparrows. It appears that there are greater numbers of GnIH-immunoreactive neurons in them than in quail hypothalami, although a direct comparison was not performed (but see Tsutsui et al., 2000). It is unclear yet whether the presence of GnIH in the brainstem is unique to sparrow species or songbirds in general, or whether it occurs in non-songbirds as well. The presence of beaded fibers containing, and presumably transporting, GnIH in multiple brain areas is consistent with the likelihood that this dodecapeptide plays multiple roles in the regulation of physiology and behavior, as is the case for GnRH (Millar et al., 1987). Double-label ICC and confocal microscopy indicate that GnRH and GnIH proteins may be in contact with one another at the level of the hypothalamic neurons, as well as at the fiber level in the hypothalamus, and at the terminal fields in the ME (with the caveat that we have not shown synapses between the two types of neuron and fiber). This observation invokes the question of the mode(s) of action of GnIH. It is as yet unclear how GnIH exerts its effects in terms of receptor binding and downstream processing, or of competition with GnRH for binding sites; nor is it known exactly where GnIH is acting. It may be that GnIH only acts at the level of the anterior pituitary gland (Tsutsui et al., 2000). Alternatively, GnIH could act by inhibiting GnRH release from GnRH fiber terminals in the ME, in addition to acting at the level of the pituitary. The histological data presented here raise the possibility of inhibitory/modulatory action at multiple levels and over different time-frames. In addition, our data indicate that GnIH is likely to act upon cgnrh-ii neurons, as identified by their neuroanatomical location and typical stubby appearance. In this way, GnIH Time post-injection (min) Fig. 3 The effects of intra-jugular injection of GnIH on GnRH-elicited LH release in photorefractory male song sparrows Note that plasma LH was lower in the GnRH + GnIH group than in the control (GnRH alone) group at 2 minutes post-injection. has the potential to affect reproductive behavior in addition to gonadotropin release, as exogenous cgnrh-ii affects copulation solicitation in estrogen-primed songbirds (Maney et al., 1997). The results from the injection experiment confirm previous data that GnRH rapidly elicits LH release from the song sparrow pituitary in vivo (Wingfield et al., 1979; Wingfield and Farner, 1993). More pertinent to the present study is that the data clearly demonstrate rapid (within 2 minutes) reduction by GnIH of this GnRH-elicited LH release. It is possible that in both groups of song sparrows, plasma LH may have increased faster and further than indicated in the first 2 minutes post-injection, but such an effect was not detectable within this experimental paradigm. Even were this to be the case, we would predict that GnIH would attenuate the rise in LH equally as rapidly as GnRH elicits it. The most parsimonious explanation for the rapid attenuation of a GnRH injection-elicited LH rise by GnIH is its action upon the pituitary gland, but this does not negate the possibility of inhibitory action at multiple levels and over different time-frames (see above). Overall, these data confirm the previous in vitro demonstration of gonadotropininhibitory activity by GnIH (Tsutsui et al., 2000), extending those findings to in vivo activity. It remains to be seen what environmental factors elicit GnIH release in song sparrows and house sparrows, and what the complete range of physiological and behavioral effects of GnIH might be. Data herein indicate potential modulation of activity of the HPG axis, which is primarily under photoperiodic control in these species. The fact that GnIH can affect pituitary gonadotropin release in vitro and in vivo suggests that at least one function of GnIH might be rapid-time regulation of gonadotropin release within the broader time-frame of the breeding season. In other words, GnIH might act to fine-tune the timing of onset of breeding.

5 182 One might envisage increased GnIH release during periods of stress (induced, e.g., by inclement weather), whereby birds could temporarily cease reproductive activity without complete regression of the reproductive system. Moreover, action of GnIH in multiple areas of the brain might allow for direct regulation of reproductive behaviors in response to environmental cues/stressors. GnIH might well also act within a longer time-frame. It is possible that GnIH content and release is regulated seasonally within the photoperiodic system. If this is the case, then GnIH may play a primary role in timing the annual reproductive cycle in songbirds, specifically in terminating the breeding season (photorefractoriness). In summary, the data present the first demonstration of the presence of GnIH in the brains of two families of songbirds, and its potential for regulating GnRH at multiple levels. This finding builds upon growing evidence of the importance of RFamide peptides in regulatory functions within the vertebrate brain. Even though the discovery of a gonadotropin antagonist is in itself significant to reproductive biology (Tsutsui et al., 2000), the action of RFamide peptides appears not to be limited to neuroendocrine activity. Acknowledgements The authors would like to thank Todd Clason for his expert technical help. Todd Sperry provided useful discussion on the subject matter. The research was funded by: NIH (R01 MH to J.C.Wingfield), NSF (IBN to J.C.Wingfield), Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan ( , , and to K.Tsutsui), and a NSF Minority Postdoctoral Fellowship (to I.T. Moore). References Bentley GE, Goldsmith AR, Juss TS, Dawson A, The effects of nerve growth factor and anti-nerve growth factor antibody on the neuroendocrine reproductive system in the European starling, Sturnus vulgaris. J. Comp. 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