Potential roles for GNIH and GNRH-II in reproductive axis regulation of an opportunistically breeding songbird

General and Comparative Endocrinology 173 (2011) 20–26

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General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen

Potential roles for GNIH and GNRH-II in reproductive axis regulation of an opportunistically breeding songbird Nicole Perfito a,⇑, Richard Zann b, Takayoshi Ubuka a, George Bentley a,c, Michaela Hau d a

Dept. of Integrative Biology, University of California, Berkeley, CA, USA La Trobe University, Melbourne, Victoria, Australia c Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA d Max-Planck Institute for Ornithology, Radolfzell, Germany b

a r t i c l e

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Article history: Received 18 October 2010 Revised 2 March 2011 Accepted 18 April 2011 Available online 23 April 2011 Keywords: Opportunistic breeding HPG axis Timing of reproduction Zebra finch GnRH GnIH Neuroendocrine

a b s t r a c t The ability to breed at any time of year enables opportunistically breeding species to respond to good conditions whenever they occur. We investigate the neuroendocrine basis for this relatively unusual reproductive pattern in the avian world. One proposed mechanism for year-round breeding ability is tonic activation of gonadotropin-releasing hormone-I (GnRH-I) production that is flexibly modified by gonadotropin-inhibitory hormone (GnIH) production during unfavorable conditions. GnIH could inhibit GnRH secretion from the hypothalamus and/or inhibit GnRH action on the anterior pituitary gland. We studied neuroendocrine patterns in wild Australian zebra finches (Taeniopygia guttata) sampled during a breeding period in Southern Australia, a non-breeding period in central Australia, and in juvenile males in the latter location. We asked whether patterns in immunoreactivity of three neuropeptides important for reproductive axis regulation, GnRH-I, GnRH-II and GnIH, during periods of breeding and non-breeding reflect this flexibility. We found that the numbers and sizes of immunoreactive (-ir) GnRH-I cells did not vary between breeding stages and ages. Contrary to our predictions, irGnIH cell number and size, as well as the synthesis of GnIH mRNA were similar in breeding and non-breeding conditions. However, breeding males had more and larger irGnRH-II cells in the midbrain compared to non-breeding males. Hence, while changes in irGnIH cells are not associated with fluctuations in gonadotropin secretion or gonad volume, the regulation of irGnRH-II cells might represent a previously-unidentified mechanism by which reproductive flexibility can be achieved; namely via behavioral neurotransmitter actions of GnRH-II rather than through the typical sensory-CNS integration-GnRH-I route. Published by Elsevier Inc.

1. Introduction Opportunistic breeders live in environments with unpredictable variations in resource availability. If food resources for successfully raising young are transient and vary unpredictably in time and space, individuals should maintain the ability to reproduce yearround when favorable conditions present themselves [15,16,46]. The ways in which the hypothalamo–pituitary–gonadal (HPG) axis, the neuroendocrine cascade that regulates reproduction, is organized to support this breeding pattern are not well understood. In seasonally breeding birds, which undergo regular seasonal changes in reproductive competence and quiescence, the main hypothalamic peptide involved in reproductive activation is avian gonadotropin-releasing hormone-I (hereafter called GnRH-I; [2,13,50,51]). ⇑ Corresponding author. Address: Dept. of Integrative Biology, University of California Berkeley, 3060 Valley Life Sciences Bldg #3140, Berkeley, CA 94720-3140, USA. Fax: +1 510 643 6264. E-mail address: nperfi[email protected] (N. Perfito). 0016-6480/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.ygcen.2011.04.016

GnRH-I immunoreactivity (-ir) increases when days get longer, reaches a peak during the breeding season and spontaneously decreases while day lengths are still long and reaches a minimum after the onset of photorefractoriness in many species [10,18,48]. GnRH-I secretion is reduced first, followed eventually by a reduction in its synthesis [10,11,14,18,19,44,48,50]. During the photorefractory period the reproductive axis is quiescent (i.e., GnRH-I synthesis is halted, circulating gonadotropin and sex steroid concentrations are low and testes are regressed), individuals usually undergo molt, and photosensitivity is regained (with an accompanying increase of GnRH-I-ir) only after experiencing short days [12,13]. A second form of GnRH, GnRH-II, is highly conserved in its presence across vertebrates [26,36]. In mammals and birds, GnRH-II cell bodies are found in the midbrain and exogenous GnRH-II stimulates female sexual behavior when infused centrally [23–25,30,45]. Because GnRH-II does not play a major role in pituitary regulation [34,35,55], only a few studies have investigated seasonal regulation of GnRH-II in birds [33,47]. For opportunistic

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species such as zebra finches (Taeniopygia guttata), that show aseasonal cycles in reproductive activation, sexual interactions between mates could play an important role in synchronization of breeding readiness. Sexual behavior might be a particularly powerful stimulatory cue because zebra finches are monogamous, breed in large colonies and remain with the same partner during breeding and non-breeding periods [59]. The opportunistically breeding bird species studied to date show irGnRH-I fluctuations in the preoptic area that are much less dramatic than seasonal species [29,39,40]. However circulating concentrations of luteinizing hormone (LH) and the volumes of the testes can vary predictably with bouts of breeding in avian opportunists [21,42]. What, then, controls the decrease in the activity of such peripheral reproductive components during unfavorable conditions? One possibility are the actions of a new hypothalamic peptide, gonadotropin-inhibitory hormone (GnIH), discovered in quail and shown to inhibit synthesis and secretion of gonadotropins by the anterior pituitary gland in birds [38,49,54] and mammals [22,27]. In vivo and in vitro experiments indicate inhibitory actions of GnIH on the anterior pituitary gland, the GnRH neurons themselves, and on the gonad directly [1,6,27,31,32,49,52,53,58]. One proposed explanation for maintenance of the HPG axis year-round is that GnRH-I synthesis remains tonically active [16,20]. If GnIH synthesis and secretion increase during unfavorable conditions, it could act to inhibit secretion of GnRH and/or to inhibit GnRH action on the anterior pituitary gland. Gonadotropin secretion, testis function and reproductive behavior would thus be reduced causing a hiatus in breeding. GnIH also appears to bind to GnRH-II neurons via its cognate receptor and causes inhibition of sexual behavior in estradiol-primed female white-crowned sparrows (Zonotrichia leucophrys; [3,4,52]). Taken together, these studies suggest that flexible breeding could occur: (1) simply through GnRH-I activation that is uncoupled from photoperiodic information and GnIH inhibition would act to fine-tune final onset of reproductive readiness, (2) tonic availability of GnRH-I and -II that are only transiently inhibited by GnIH activation during poor conditions, or (3) tonic activation of both GnRH-I and GnIH that are balanced to keep the reproductive system in a semi-activated state and GnRH-II activating sexual behavior during good conditions to stimulate complete reproductive readiness (Table 1). In scenario 1, we expect GnRH-I and GnIH activity to be high during breeding and low during nonbreeding. In scenario 2, we expect GnRH-I and -II to be tonically available, and GnIH activity to be low during breeding and high during non-breeding. In scenario 3, we expect GnRH-I and GnIH to tonically available and GnRH-II activity to be high during breeding and low during non-breeding (Table 1). We compared immunoreactivity of GnRH-I, GnRH-II and GnIH in brain tissue collected just days apart from free-living adult zebra finches beginning a breeding bout in southern Australia and from non-breeding birds experiencing drought conditions in central Australia. We expected that if GnIH was critical for maintaining the non-breeding state, immunoreactivity of the peptide and/or production of its mRNA would be elevated in non-breeding birds as measured with immunohistochemistry (IHC) and in situ hybridization (ISH). In order to label GnIH mRNA we cloned a partial sequence of GnIH. Lastly, since the actions of GnIH could result in an

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anterior pituitary gland less sensitive to GnRH stimulation, we injected exogenous GnRH and measured the responsiveness of anterior pituitary via plasma LH concentrations in breeding and non-breeding birds. With these measurements we discuss which components of this (neuro-) hormonal cascade are different between breeding and non-breeding states. 2. Methods 2.1. Animals and tissue collection Seven adult male zebra finches were captured in breeding condition in northern Victoria and six adults in non-breeding condition three days later in Alice Springs, Australia (see [42] for study site descriptions). We also captured four juvenile males (<90 days post-hatch) in Alice Springs during the same time. Birds were killed by rapid decapitation after deep anesthesia with Isoflourane (Phoenix Pharmaceutical, Inc.). Brains were removed and immediately flash frozen on dry ice. Brain tissue was stored on dry ice until transport to University of California, Berkeley and then stored at 80 °C. Brain tissue was cut serially into 20 lm sections in the coronal plane using a cryostat and mounted directly onto slides. Volume of the testis was calculated from the formula for an ovoid sphere: V = 4/3Pa2b, where V is volume, a is the radius of the testis at its widest point and b is half the long axis. 2.2. DNA sequencing of the partial zebra finch cDNA fragment Brains from 3 adult male captive-reared zebra finches were used for sequencing and the production of the mRNA probe. Total RNA of the diencephalon was isolated by the Trizol extraction method (Invitrogen, Carlsbad, CA). Three forward (50 -AGGCTGCAGAGCAGA GAAGA-30 , 50 -GAAAAGCAGAGGAGTCT-30 , and 50 -TGCCAAATTCAG TTGCT-30 ) and three reverse (50 -CTGAGATTTGGAAGA GCTTTTG-30 , 50 -CACAGAGATTTGGGAAGTCA-30 , and 50 -GAACCAGGGATATGAA TTCTAACA-30 ) primers were synthesized (Sigma Genosys) based on the known sequence for the white-crowned sparrow (Z. leucophrys) precursor GnIH cDNA (GenBank accession #AB128164). Firststrand cDNA was reverse transcribed (M-MLV Reverse transcriptase; Invitrogen) using the manufacturer’s instructions, using nine different primer combinations. The strongest band using gel electrophoresis resulted from the combination of forward primer 50 AGGCTGCAGAGCAGAGAAGA-30 and reverse primer 50 -CACAGAGA TTTGGGAAGTCA-30 . The cDNA PCR product was purified with Amersham Pharmacia MicroSpin columns using the manufacturer’s instructions, and the products were subcloned into a pGEM-T Easy vector (Promega) and DNA inserts of positive clones were amplified using universal M13 primers. Amplified DNA was sequenced at the University of California Berkeley DNA sequencing facility. GenBank accession #EF212881. 2.3. In situ hybridization of GnIH mRNA Localization of GnIH mRNA expression was identified by in situ hybridization using the digoxigenin (DIG)-labeled antisense RNA probe (Roche Diagnostics). Control for the specificity of the

Table 1 Potential scenarios in which a flexible breeding schedule could be regulated by GnRH-I, -II and GnIH with predictions for the relative immunoreactivity that would be expected for each of the three peptides. Immunoreactivity for:

1. GnRH-I responds to local predictive cues (GnIH fine tunes)

2. GnRH-I and –II are always available (GnIH inhibits actions)

3. GnRH-I and GnIH maintain partial activation (GnRHII completes activation)

GnRH-I GnRH-II GnIH

Breeding > non-breeding No prediction Breeding > non-breeding

Non-breeding Breeding = non-breeding Breeding < non-breeding

Breeding = non-breeding Breeding > non-breeding Breeding = non-breeding

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in situ hybridization of GnIH mRNA was performed using the complementary DIG-labeled sense RNA probe. Briefly, sections were post-fixed in 4% paraformaldehyde (PFA) for 10 min, washed three times in PBS (10 nM phosphate buffer), and incubated in proteinase K (10 lg/ml) at 37 °C for 30 min. Slides were rinsed with RNase-free water, treated with 0.2 N HCl for 10 min, and rinsed again. Hybridization was carried out at 50 °C for 15–17 h with 500 ng/ml DIG-oligonucleotide probe mixture dissolved in 50% hybridization medium, 40% formamide, 10% water and 0.1% SDS. After washing three times with 40% formamide in 5 SSC (Roche Diagnostics), sections were washed two times each for 15 min in 50% formamide in 2 SSC and in 50% formamide in 1 SSC at 50 °C, then washed with PBS. The sections were then treated for 30 min with 1.5% blocking reagent (Roche Diagnostics) in PBS and incubated with alkaline phosphatase-labeled sheep anti-DIG antibody (1:1000 dilution in blocking solution) for one hour. Sections were washed three times for 15 min with 0.05% Tween in PBS, followed by a PBS rinse, then incubated in alkaline buffer for 5 min. Immunoreactive products were visualized by immersing sections for 24 h in a substrate solution (nitro blue tetrazolium and 5-bromo-4chloro-3indolyl phosphate in alkaline buffer). The reaction was stopped after two PBS rinses using 20 mM EDTA in PBS. Sections were rinsed with PBS and coverslipped. 2.4. Immunocytochemistry of GnIH and GnRH In order to balance any possible variation in labeling across ICC runs, 2–3 brains from each group were processed together in each run. GnRH and GnIH were labeled on alternate sections. Sections were post-fixed with 4% paraformaldehyde in 0.1 M phosphate buffered saline for 20 min and washed three times in PBS. To remove endogenous peroxidase activity, sections were incubated in 1% hydrogen peroxide in absolute methanol for 10 min and then washed three times with PBS. Non-specific immunoreactivity was blocked by incubation in 2% normal goat serum in PBS containing 0.1% Triton X-100 (PBS-T) for one hour at room temperature. Sections were then incubated in primary antibody (diluted 1:5000 for GnIH and GnRH) for 72 h at 4 °C, and washed in 0.1% PBS-T. The primary antibody for GnRH was donated by H. Urbanski and was a polyclonal rabbit anti-GnRH antiserum that has been validated for use in birds [10,29]. The primary antibody for GnIH was polyclonal rabbit anti-white-crowned sparrow GnIH that has been used in several bird and mammalian species [7,27,38,52]. Sections were then incubated in biotinylated secondary antibody (goat anti-rabbit IgG diluted 1:250 in 0.1% PBS-T) for one hour, and washed in 0.1% PBS-T. The antibody-antigen complex was localized using the Vectastain ABC Elite Kit; 1:200 in 0.1% PBS-T, followed by two washes in 0.1% PBS-T and one wash in 0.1 M PBS. Sections were visualized with diaminobenzidine tetra-hydrochloride (DAB, Sigma). The reaction was terminated with 5 washes in 0.1 M PBS and left to dry overnight. Slides were then dehydrated and cover slipped.

to the treatment group of all images. To calculate optical density, the average value for all pixels in the outlined area was taken as the mean intensity of staining relative to background on unlabeled portions of the same tissue section and is expressed as a percent above background density. 2.6. GnRH injections To measure the ability of the anterior pituitary to secrete LH, birds were injected with exogenous GnRH. Different birds captured were captured from the two populations described in Section 2.1 for GnRH injections, and additionally from the Victoria population during a period of non-breeding 15 months earlier (described in [42]). Males and females were caught passively in mist nets or baited seed traps and given jugular injections of either chicken gonadotropin-releasing hormone 1 (c-GnRH-1; Sigma, 500 ng in 10lL lactated saline) or saline alone [5,57]. We collected 100 ll of whole blood before injections and 10 min after injections into micro-capillary tubes after puncturing the alar wing vein with a 26-gauge needle. Plasma was kept on ice until separated by centrifugation, collected with a Hamilton syringe and stored at 20 °C until assay. 2.7. LH radioimmunoassay Plasma LH was measured in the laboratory of John Wingfield at the University of Washington using a micromodification of the radioimmunoassay (RIA) originally devised by Follett and colleagues [17]. Plasma samples were measured in two assays in 15 ll duplicates. Inter-assay variation was 9.13%, intra-assay variation was 4.36%, and the detection limit was 0.04 ng/ml. 2.8. Statistical analyses Testis volume, ir-cell numbers, cell area and optical density were compared using an analysis of variance (ANOVA) with least significant difference (LSD) post hoc pairwise comparisons. To test for differences in the GnRH injection study, LH concentrations were log-transformed to satisfy equal variance assumptions. We used a two-way ANOVA with experimental treatment (GnRH or saline) and breeding condition group (breeding Victoria, nonbreeding Alice Springs, non-breeding Victoria) as main effects. We used least significant difference (LSD) post hoc tests to identify differences if main effects or interactions were significant. All data are presented as means ± SEM. 3. Results 3.1. Reproductive morphology Testes were significantly larger in breeding adults compared to non-breeding adults or juveniles (F(2, 16) = 10.71, p = 0.002; LSD p < 0.004; Fig. 1).

2.5. Cell counts, area measurements and optical density 3.2. ICC and ISH The neuroanatomical distribution of GnRH containing neurons is largely conserved across vertebrate groups [35,37]. Cell bodies of GnRH-I and GnRH-II are found in discrete areas; therefore, we used location and morphology to distinguish these immunoreactive cells. GnRH-I cell bodies were found in the preoptic area and GnRH-II in the midbrain. Slides were examined using bright-field light microscopy and digital images of immunoreactive cells were captured using a Zeiss Axioscope. Cell bodies were counted, outlined and a two-dimensional area was calculated in every fifth section from the tractus septomesencephalicus (TSM) caudally through the midbrain using NIH Image 1.61. The counter was blind

GnRH-I-ir was similar regardless of breeding state (cell number, p = 0.35; cell area, p = 0.27; optical density, p = 0.83; Figs. 2a and 3 top row). However, non-breeding adult males had significantly fewer GnRH-II-ir neurons than did breeding adult males (F(2, 14) = 3.87, p = 0.05; LSD p = 0.02) and juvenile males had an intermediate number. GnRH-II cell size and optical density were similar among groups (cell area, p = 0.42; optical density, p = 0.51; Figs. 2b and 3 middle row). GnIH immunoreactivity was similar regardless of breeding state (cell number, p = 0.99; cell area, p = 0.30; optical density, p = 0.30; Figs. 2c and 3 bottom

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Fig. 1. Testis volume of adult male zebra finches captured during a non-breeding period in Alice Springs (open bars), during a breeding period in Northern Victoria (light gray bars), and in non-breeding juvenile males (NB Juveniles, dark gray bars) in Alice Springs. Data are mean ± 1SEM.

row); however, breeding adult males had larger cell area than non-breeding juveniles when GnIH mRNA was measured (F(2, 14) = 7.08, p = 0.009; LSD p = 0.003; Fig. 2d). Groups had similar numbers and optical density of cells containing GnIH mRNA (cell number, p = 0.77; optical density, p = 0.61). 3.3. GnRH injections In males, GnRH injections caused a significant increase in plasma LH concentrations compared to saline (treatment F(1, 44) = 13.76, p = 0.001; Fig. 4, top panel), and LH concentration increased in a similar direction across sampling times (interaction

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treatment  breeding condition F(2, 44) = 2.37, p = 0.12). The effect of GnRH injections in males resulted mainly from significantly increased LH secretion during the non-breeding Victoria sample (LSD p < 0.001), since injections in either of the other sampling points did not cause a significant rise in LH (LSD p > 0.05). The absolute concentrations of plasma LH after GnRH injection were similar across sampling points (p > 0.05). Non-breeding males in Victoria had significantly lower LH concentrations after saline injections than saline injected birds during either the non-breeding Alice Springs or breeding Victoria sampling points (LSD p < 0.01). In females, GnRH injections also caused a significant increase in plasma LH concentrations compared to saline (treatment F(1, 37) = 15.14, p < 0.001; Fig. 4, bottom panel), but this was dependent on which breeding state and location birds were sampled (interaction treatment  breeding condition F(2, 37) = 3.84, p = 0.03). When females were in non-breeding condition, either in Victoria or Alice Springs, exogenous GnRH caused increased LH secretion by the pituitary gland (LSD p < 0.02), but when females were breeding it did not (LSD p = 0.86). The absolute concentrations of plasma LH after GnRH injection were similar across sampling points (LSD p > 0.05). After saline injections, plasma LH was lower in nonbreeding Victoria females than breeding Victoria females, and non-breeding Alice Springs females had LH that was intermediate between the two.

4. Discussion As in other flexible breeders, in wild zebra finch males GnRH-Iir does not change significantly between periods of breeding and non-breeding [29,39,40]. However, contrary to our prediction, GnIH, as measured via ICC and ISH, was not associated with nonbreeding down-regulation of the reproductive axis. Even though testis volume is considerably smaller and LH secretion is reduced, neither the numbers nor sizes of GnRH-I-ir and GnIH-ir cells change during non-breeding periods. It is important to remember

Fig. 2. Immunoreactive cell number (total cell numbers), area (relative units) and optical density (% above background) of GnRH-I (A), GnRH-II (B) and GnIH (C) and (D) GnIH mRNA in free-living adult male zebra finches captured during a non-breeding period in Alice Springs (open bars), during a breeding period in Northern Victoria (light gray bars) and in free-living non-breeding juvenile males (NB Juveniles, dark gray bars). Data are mean ± 1SEM. Sample sizes are given at the bottom of bars. ⁄p < 0.05.

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Fig. 3. Representative sections for GnRH-I (top row), GnRH-II (middle row) and GnIH (bottom row) immunoreactivity in male zebra finches during a period of non-breeding (left column), breeding (middle column) and from a small sample of non-breeding juveniles (right column). All images were taken at the same magnification, and 50 lm scale bars in the right column correspond to images in their respective rows.

Fig. 4. Plasma LH concentration 10 min following either saline (open bars) or GnRH (filled bars) injections in free-living adult male (top panel) and female (bottom panel) zebra finches. LH secretion increased in both males and females during nonbreeding in Victoria, but during a non-breeding bout during drought conditions (Alice Sp). LH increased only in females. GnRH injections did not alter LH secretion in males or females above breeding levels in Victoria.

that ICC cannot tell us about changes in release, and we were unable to measure GnIH in the pituitary portal system at these time-points, and therefore cannot gain information on the release of GnIH to the pituitary. At a minimum, these data suggest that

there is no increase in GnIH synthesis during the non-breeding period. By contrast, GnRH-II peptide is markedly reduced (both numbers of GnRH-II-ir neurons and to a lesser degree cell size) during non-breeding periods. This exciting finding may point to an alternate mechanism by which flexible breeders might regulate their reproductive axis [41]. To the extent that GnRH-II is involved in sexual behavior in birds and mammals [23–25,30,45], up-regulation of the synthesis and secretion this peptide in zebra finches might facilitate social interactions between mates as conditions improve for breeding. How environmental conditions might be transduced to affect GnRH-II neurons is unknown, but evidence in rodents suggests that energetic status might be important [24]. We know that performing sexual behaviors (seeing and/or hearing conspecific song, as well as responding behaviorally with courtship behaviors) can itself have effects on the reproductive axis in birds (i.e., increased secretion of sex steroids and LH; [8,9,28]). Tonic availability of GnRH-I might allow for behavioral effects of GnRH-II to feedback positively on the reproductive axis to stimulate LH secretion fully and thus complete gonadal development and gamete maturation. While testes can regress in non-breeding zebra finches, some spermatogenesis continues in the gonad even when testis volume is reduced [40,43,56]. Both of these characteristics, GnRH-II availability and incomplete gonadal inactivation, might allow individuals to switch on reproductive readiness regardless of the time of year. It should be noted that our small sample of non-breeding juvenile males had GnRH-II numbers that were similar to breeding adult males. This suggests that either GnRH-II down-regulation is not required for non-breeding or that during the first transition from non-breeding to breeding in juveniles is regulated differently with respect to GnRH-II. Our GnRH injections give an index of the responsive ability of the anterior pituitary to elevated GnRH. Both males and females that were in non-breeding condition (Victoria) were responsive to exogenous GnRH and increased their output of LH into circulation. During this time period, males had very small testes and females had fully regressed ovarian follicles (Fig. 3, [42]). During breeding, when baseline plasma LH was already elevated [42]

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and when saline injected birds had higher LH concentrations compared to non-breeding birds at the same location (Fig. 4), the pituitary did not increase LH secretion in response to exogenous GnRH. This does not suggest that the pituitary gland was unresponsive to GnRH, but perhaps that a higher dose of GnRH is required to demonstrate any further capacity of the pituitary in the zebra finch to secrete LH. Alternatively, the limit for secretion might have already been reached, so that injections did not cause a further rise in LH. During non-breeding in Alice Springs, females but not males responded to GnRH injections. Although this population had been experiencing drought conditions and had not bred for the previous 5 months, males had maintained intermediate testes volumes and LH concentration while many females (42%) had fully regressed ovarian follicles [42]. These data suggest that females might regain the ability to respond to favorable conditions more quickly than males, and could therefore drive reproductive activation through behavioral means as described above.

[9] [10]

[11]

[12]

[13] [14]

[15]

5. Conclusions The picture emerging for avian species with highly flexible breeding patterns is one of fairly large reductions of peripheral structures (e.g., testis and follicle volume) and of gonadotropin secretion by the pituitary gland during periods of non-breeding, but at the same time maintenance of readily available hypothalamic neuropeptides like GnRH-I and GnIH. Our data suggest that regulation of GnRH-II might offer a new way to think about how flexibility is regulated; namely via behavioral neurotransmitter actions of GnRH-II rather than through the typical sensory-CNS integration-GnRH route.

[16] [17]

[18]

[19]

[20]

Acknowledgments

[21]

We thank Norm Crighton, the Powney family, Anita Smyth, Mitchell Jones, Cameron Wallace, Gary Weir and the Centre for Arid Zone Research for logistical support and accommodation. We thank Jim Adelman and Katherine Beebe for field assistance and John Wingfield for the use of his laboratory to conduct the LH assay. Funding was provided to G.E.B. by NSF IOS 0641188 and 0920753. Data were collected under the Victorian Department of Sustainability and Environment Wildlife Permit No. 10003656 and the Northern Territory Parks and Wildlife Permit No. 18356.

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