The general and comparative biology of gonadotropin-inhibitory hormone (GnIH)

The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor responsible for the hypothalamic control of gonadotropin secretion. Gonadal sex steroids and inhibin inhibit gonadotropin secretion via feedback from the gonads, but a neuropeptide inhibitor of gonadotropin secretion was, until recently, unknown in vertebrates. In 2000, we identified a novel hypothalamic dodecapeptide that inhibits gonadotropin release in cultured quail pituitaries and termed it gonadotropin-inhibitory hormone (GnIH). To elucidate the mode of action of GnIH, we then identified a novel G protein-coupled receptor for GnIH in quail. The GnIH receptor possesses seven transmembrane domains and specifically binds to GnIH. The GnIH receptor is expressed in the pituitary and several brain regions including the hypothalamus. These results indicate that GnIH acts directly on the pituitary via GnIH receptor to inhibit gonadotropin release. GnIH may also act on the hypothalamus to inhibit GnRH release. To demonstrate the functional significance of GnIH and its potential role as a key regulatory neuropeptide in avian reproduction, we investigated GnIH actions on gonadal development and maintenance in quail. Chronic treatment with GnIH inhibited gonadal development and maintenance by decreasing gonadotropin synthesis and release. GnIH was also found in the hypothalamus of other avian species including sparrows and chickens and also inhibited gonadotropin synthesis and release. The pineal hormone melatonin may be a key factor controlling GnIH neural function, since quail GnIH neurons express melatonin receptor and melatonin treatment stimulates the expression of GnIH mRNA and mature GnIH peptide. Thus, GnIH is capable of transducing photoperiodic information via changes in the melatonin signal, thereby influencing the reproductive axis. It is concluded that GnIH, a newly discovered hypothalamic neuropeptide, is a key factor controlling avian reproduction. The discovery of avian GnIH opens a new research field in reproductive neuroendocrinology.     

Gonadotropin-inhibitory hormone inhibits gonadal development and maintenance by decreasing gonadotropin synthesis and release in male quail

Until recently, any neuropeptide that directly inhibits gonadotropin secretion had not been identified. We recently identified a novel hypothalamic dodecapeptide that directly inhibits gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). The action of GnIH on the inhibition of gonadotropin release is mediated by a novel G protein-coupled receptor in the quail pituitary. This new gonadotropin inhibitory system is considered to be a widespread property of birds and provides us with an unprecedented opportunity to study the regulation of avian reproduction from an entirely novel standpoint. To understand the physiological role(s) of GnIH in avian reproduction, we investigated GnIH actions on gonadal development and maintenance in male quail. Continuous administration of GnIH to mature birds via osmotic pumps for 2 wk decreased the expressions of gonadotropin common alpha and LHbeta subunit mRNAs in a dose-dependent manner. Plasma LH and testosterone concentrations were also decreased dose dependently. Furthermore, administration of GnIH to mature birds induced testicular apoptosis and decreased spermatogenic activity in the testis. In immature birds, daily administration of GnIH for 2 wk suppressed normal testicular growth and rise in plasma testosterone concentrations. An inhibition of juvenile molt also occurred after GnIH administration. These results indicate that GnIH inhibits gonadal development and maintenance through the decrease in gonadotropin synthesis and release. GnIH may explain the phenomenon of photoperiod-induced gonadal regression before an observable decline in hypothalamic GnRH in quail. To our knowledge, GnIH is the first identified hypothalamic neuropeptide inhibiting reproductive function in any vertebrate class.     

Evolutionary origin and divergence of GnIH and its homologous peptides

Probing undiscovered hypothalamic neuropeptides that play important roles in the regulation of pituitary function in vertebrates is essential for the progress of neuroendocrinology. In 2000, we discovered a novel hypothalamic dodecapeptide inhibiting gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). GnIH acts on the pituitary and gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus via a novel G protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotropin release and synthesis. Similar findings were observed in other avian species. Thus, GnIH is a key factor controlling avian reproduction. To give our findings a broader perspective, we also found GnIH homologous peptides in the hypothalamus of other vertebrates, such as mammals, reptiles, amphibians and teleosts. GnIH and its homologs share a common C-terminal LPXRFamide (X = L or Q) motif. A mammalian GnIH homolog also inhibited gonadotropin release in mammals like the GnIH action in birds. In contrast to higher vertebrates, hypophysiotropic activities of GnIH homologs were different in lower vertebrates. To clarify the evolutionary origin of GnIH and its homologs, we further sought to identify novel LPXRFamide peptides from the brain of sea lamprey and hagfish, two extant groups of the oldest lineage of vertebrates, Agnatha. In these agnathans, LPXRFamide peptide and its cDNA were identified only from the brain of hagfish. Based on these findings over the past decade, this paper summarizes the evolutionary origin and divergence of GnIH and its homologous peptides.     

Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals

Successful reproduction requires maintenance of the reproductive axis within fine operating limits through negative feedback actions of sex steroids. Despite the importance of this homeostatic process, our understanding of the neural loci, pathways, and neurochemicals responsible remain incomplete. Here, we reveal a neuropeptidergic pathway that directly links gonadal steroid actions to regulation of the reproductive system. An RFamide (Arg-Phe-NH2) peptide that inhibits gonadotropin release from quail pituitary was recently identified and named gonadotropin-inhibitory hormone (GnIH). Birds are known to have specialized adaptations associated with gonadotropin-releasing hormone (GnRH) regulation to optimize reproduction (e.g., encephalic photoreceptors), and the existence of a hypothalamic peptide inhibiting gonadotropins may or may not be another such specialization. To determine whether GnIH serves as a signaling pathway for sex steroid regulation of the reproductive axis, we used immunohistochemistry and in situ hybridization to characterize the distribution and functional role of this peptide in hamsters, rats, and mice. GnIH-immunoreactive (GnIH-ir) cell bodies are clustered in the mediobasal hypothalamus with pronounced projections and terminals throughout the CNS. In vivo GnIH administration rapidly inhibits luteinizing hormone secretion. Additionally, GnIH-ir neurons form close appositions with GnRH cells, suggesting a direct means of GnRH modulation. Finally, GnIH-ir cells express estrogen receptor-alpha and exhibit robust immediate early gene expression after gonadal hormone stimulation. Taken together, the distribution of GnIH efferents to neural sites regulating reproductive behavior and neuroendocrine secretions, expression of steroid receptors in GnIH-ir nuclei, and GnIH inhibition of luteinizing hormone secretion indicate the discovery of a system regulating the mammalian reproductive axis.     

Gonadotropin-inhibitory hormone neurons interact directly with gonadotropin-releasing hormone-I and -II neurons in European starling brain

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic dodecapeptide (SIKPSAYLPLRF-NH(2)) that directly inhibits gonadotropin synthesis and release from quail pituitary. The action of GnIH is mediated by a novel G-protein coupled receptor. This gonadotropin-inhibitory system may be widespread in vertebrates, at least birds and mammals. In these higher vertebrates, histological evidence suggests contact of GnIH immunoreactive axon terminals with GnRH neurons, thus indicating direct regulation of GnRH neuronal activity by GnIH. In this study we investigated the interaction of GnIH and GnRH-I and -II neurons in European starling (Sturnus vulgaris) brain. Cloned starling GnIH precursor cDNA encoded three peptides that possess characteristic LPXRF-amide (X = L or Q) motifs at the C termini. Starling GnIH was further identified as SIKPFANLPLRF-NH(2) by mass spectrometry combined with immunoaffinity purification. GnIH neurons, identified by in situ hybridization and immunocytochemistry (ICC), were clustered in the hypothalamic paraventricular nucleus. GnIH immunoreactive fiber terminals were present in the external layer of the median eminence in addition to the preoptic area and midbrain, where GnRH-I and GnRH-II neuronal cell bodies exist, respectively. GnIH axon terminals on GnRH-I and -II neurons were shown by GnIH and GnRH double-label ICC. Furthermore, the expression of starling GnIH receptor mRNA was identified in both GnRH-I and GnRH-II neurons by in situ hybridization combined with GnRH ICC. The cellular localization of GnIH receptor has not previously been identified in any vertebrate brain. Thus, GnIH may regulate reproduction of vertebrates by directly modulating GnRH-I and GnRH-II neuronal activity, in addition to influencing the pituitary gland.     

Effects of melatonin on peripheral reproductive function: regulation of testicular GnIH and testosterone

Study of seasonal reproduction has focused on the brain. Here, we show that the inhibition of sex steroid secretion can be seasonally mediated at the level of the gonad. We investigate the direct effects of melatonin on sex steroid secretion and gonadal neuropeptide expression in European starlings (Sturnus vulgaris). PCR reveals starling gonads express mRNA for gonadotropin inhibitory hormone (GnIH) and its receptor (GnIHR) and melatonin receptors 1B (Mel 1B) and 1C (Mel 1C). We demonstrate that the gonadal GnIH system is regulated seasonally, possibly via a mechanism involving melatonin. GnIH/ GnIHR expression in the testes is relatively low during breeding compared with outside the breeding season. The expression patterns of Mel 1B and Mel 1C are correlated with this expression, and melatonin up-regulates the expression of GnIH mRNA in starling gonads before breeding. In vitro, GnIH and melatonin significantly decrease testosterone secretion from LH/FSH-stimulated testes before, but not during, breeding. Thus local inhibition of sex steroid secretion appears to be regulated seasonally at the level of the gonad, by a mechanism involving melatonin and the gonadal GnIH system.     

Two new forms of gonadotropin-releasing hormone in a protochordate and the evolutionary implications.

The neuropeptide gonadotropin-releasing hormone (GnRH) is the major regulator of reproduction in vertebrates. Our goal was to determine whether GnRH could be isolated and identified by primary structure in a protochordate and to examine its location by immunocytochemistry. The primary structure of two novel decapeptides from the tunicate Chelyosoma productum (class Ascidiacea) was determined. Both show significant identity with vertebrate GnRH. Tunicate GnRH-I (pGlu-His-Trp-Ser-Asp-Tyr-Phe-Lys-Pro-Gly-NH2) has 60% of its residues conserved, compared with mammalian GnRH, whereas tunicate GnRH-II (pGlu-His-Trp-Ser-Leu-Cys-His-Ala-Pro-Gly-NH2) is unusual in that it was isolated as a disulfide-linked dimer. Numerous immunoreactive GnRH neurons lie within blood sinuses close to the gonoducts and gonads in both juveniles and adults, implying that the neuropeptide is released into the bloodstream. It is suggested that in ancestral chordates, before the evolution of the pituitary, the hormone was released into the bloodstream and acted directly on the gonads.     

Identification, expression, and physiological functions of Siberian hamster gonadotropin-inhibitory hormone

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. To further understand its physiological roles in mammalian reproduction, we identified its precursor cDNA and endogenous mature peptides in the Siberian hamster brain. The Siberian hamster GnIH precursor cDNA encoded two RFamide-related peptide (RFRP) sequences. SPAPANKVPHSAANLPLRF-NH(2) (Siberian hamster RFRP-1) and TLSRVPSLPQRF-NH(2) (Siberian hamster RFRP-3) were confirmed as mature endogenous peptides by mass spectrometry from brain samples purified by immunoaffinity chromatography. GnIH mRNA expression was higher in long days (LD) compared with short days (SD). GnIH mRNA was also highly expressed in SD plus pinealectomized animals, whereas expression was suppressed by melatonin, a nocturnal pineal hormone, administration. GnIH-immunoreactive (-ir) neurons were localized to the dorsomedial region of the hypothalamus, and GnIH-ir fibers projected to hypothalamic and limbic structures. The density of GnIH-ir perikarya and fibers were higher in LD and SD plus pinealectomized hamsters than in LD plus melatonin or SD animals. The percentage of GnRH neurons receiving close appositions from GnIH-ir fiber terminals was also higher in LD than SD, and GnIH receptor was expressed in GnRH-ir neurons. Finally, central administration of hamster RFRP-1 or RFRP-3 inhibited LH release 5 and 30 min after administration in LD. In sharp contrast, both peptides stimulated LH release 30 min after administration in SD. These results suggest that GnIH peptides fine tune LH levels via its receptor expressed in GnRH-ir neurons in an opposing fashion across the seasons in Siberian hamsters.     

Gonadotropin-inhibitory hormone and its receptor in the avian reproductive system

Many hormones that are classified as neuropeptides are synthesized in vertebrate gonads in addition to the brain. Receptors for these hormones are also expressed in gonadal tissue; thus there is potential for a highly localized autocrine or paracrine effect of these hormones on a variety of gonadal functions. In the present study we focused on gonadotropin-inhibitory hormone (GnIH), a neuropeptide that was first discovered in the hypothalamus of birds. We present different lines of evidence for the synthesis of GnIH and its receptor in the avian reproductive system including gonads and accessory reproductive organs by studies on two orders of birds: Passeriformes and Galliformes. Binding sites for GnIH were initially identified via in vivo and in vitro receptor fluorography, and were localized in ovarian granulosa cells along with the interstitial layer and seminiferous tubules of the testis. Furthermore, species-specific primers produced clear PCR products of GnIH and GnIH receptor (GnIH-R) in songbird and quail gonadal and other reproductive tissues, such as oviduct, epididymis and vas deferens. Sequencing of the PCR products confirmed their identities. Immunocytochemistry detected GnIH peptide in ovarian thecal and granulosa cells, testicular interstitial cells and germ cells and pseudostratified columnar epithelial cells in the epididymis. In situ hybridization of GnIH-R mRNA in testes produced a strong reaction product which was localized to the germ cells and interstitium. In the epididymis, the product was also localized in the pseudostratified columnar epithelial cells. In sum, these results indicate that the avian reproductive system has the capability to synthesize and bind GnIH in several tissues. The distribution of GnIH and its receptor suggest a potential for autocrine/paracrine regulation of gonadal steroid production and germ cell differentiation and maturation.     

Melatonin induces the expression of gonadotropin-inhibitory hormone in the avian brain

We recently identified a novel hypothalamic neuropeptide inhibiting gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). Cell bodies and terminals containing the dodecapeptide GnIH are localized in the paraventricular nucleus (PVN) and median eminence, respectively. To understand the physiological role of GnIH, we investigated the mechanisms that regulate GnIH expression. In this study, we show that melatonin originating from the pineal gland and eyes induces GnIH expression in the quail brain. Pinealectomy (Px) combined with orbital enucleation (Ex) (Px plus Ex) decreased the expression of GnIH precursor mRNA and content of mature GnIH peptide in the diencephalon, which includes the PVN and median eminence. Melatonin administration to Px plus Ex birds caused a dose-dependent increase in expression of GnIH precursor mRNA and production of mature peptide. The expression of GnIH was photoperiodically controlled and increased under short-day photoperiods, when the duration of melatonin secretion increases. To identify the mode of melatonin action on GnIH induction, we investigated the expression of Mel(1c), a melatonin receptor subtype, in GnIH neurons. In situ hybridization of Mel(1c) mRNA combined with immunocytochemistry for GnIH revealed that Mel(1c) mRNA was expressed in GnIH-immunoreactive neurons in the PVN. Melatonin receptor autoradiography further revealed specific binding of melatonin in the PVN. These results indicate that melatonin is a key factor for GnIH induction. Melatonin appears to act directly on GnIH neurons through its receptor to induce GnIH expression. This is the first demonstration, to our knowledge, of a direct action of melatonin on neuropeptide induction in any vertebrate class.     

Primary structure of a novel gonadotropin-releasing hormone in the brain of a teleost, Pejerrey

The neuropeptide GnRH is the major regulator of reproduction in vertebrates acting as a first signal from the hypothalamus to pituitary gonadotropes. Three GnRH molecular variants were detected in the brain of a fish, pejerrey (Odontesthes bonariensis), using chromatographic and immunological methods. The present study shows that one form is identical to chicken GnRH-II (sequence analysis and mass spectrometry) and the second one is immunologically and chromatographically similar to salmon GnRH. The third form was proven to be a novel form of GnRH by isolating the peptide from the brain and determining its primary structure by chemical sequencing and mass spectrometry. The sequence of the novel pejerrey GnRH is pGlu-His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly-NH(2), which is different from the known forms of the vertebrate and protochordate GnRH family. The new form of GnRH is biologically active in releasing gonadotropin and GH from pituitary cells in an in vitro assay.     

Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats

Gonadotropin-inhibitory hormone (GnIH), a newly discovered hypothalamic RFamide peptide, inhibits reproductive activity by decreasing gonadotropin synthesis and release in birds. The gene of the mammalian RFamide-related peptides (RFRP) is orthologous to the GnIH gene. This Rfrp gene gives rise to the two biologically active peptides RFRP-1 (NPSF) and RFRP-3 (NPVF), and i.c.v. injections of RFRP-3 suppress LH secretion in several mammalian species. In this study, we show whether RFRP-3 affects LH secretion at the pituitary level and/or via the release of GnRH at the hypothalamus in mammals. To investigate the suppressive effects of RFRP-3 on the mean level of LH secretion and the frequency of pulsatile LH secretion in vivo, ovariectomized (OVX) mature rats were administered RFRP-3 using either i.c.v. or i.v. injections. Furthermore, the effect of RFRP-3 on LH secretion was also investigated using cultured female rat pituitary cells. With i.v. administrations, RFRP-3 significantly reduced plasma LH concentrations when compared with the physiological saline group. However, after i.c.v. RFRP-3 injections, neither the mean level of LH concentrations nor the frequency of the pulsatile LH secretion was affected. When using cultured pituitary cells, in the absence of GnRH, the suppressive effect of RFRP-3 on LH secretion was not clear, but when GnRH was present, RFRP-3 significantly suppressed LH secretion. These results suggest that RFRP-3 does not affect LH secretion via the release of GnRH, and that RFRP-3 directly acts upon the pituitary to suppress GnRH-stimulated LH secretion in female rats.     

Three gonadotropin-releasing hormone genes in one organism suggest novel roles for an ancient peptide.

Gonadotropin-releasing hormone (GnRH) is known and named for its essential role in vertebrate reproduction. Release of this decapeptide from neurons in the hypothalamus controls pituitary gonadotropin levels which, in turn, regulate gonadal state. The importance of GnRH is underscored by its widespread expression and conservation across vertebrate taxa: five amino acids are invariant in all nine known forms, whereas two others show only conservative changes. In most eutherian mammals, only one form, expressed in the hypothalamus, is thought to exist, although in a recent report, antibody staining in developing primates suggests an additional form. In contrast, multiple GnRH forms and expression loci have been reported in many non-mammalian vertebrates. However, evidence based on immunological discrimination does not always agree with analysis of gene expression, since GnRH forms encoded by different genes may not be reliably distinguished by antibodies. Here we report the expression of three distinct GnRH genes in a teleost fish brain, including the sequence encoding a novel GnRH preprohormone. Using in situ hybridization, we show that this form is found only in neurons that project to the pituitary and exhibit changes in soma size depending on social and reproductive state. The other two GnRH genes are expressed in other, distinct cell populations. All three genes share the motif of encoding a polypeptide consisting of GnRH and a GnRH-associated peptide. Whereas the GnRH moiety is highly conserved, the GnRH-associated peptides are not, reflecting differential selective pressure on different parts of the gene. GnRH forms expressed in nonhypothalamic regions may serve to coordinate reproductive activities of the animal.     

Seasonal changes of gonadotropin-releasing hormone in the Atlantic hagfish Myxine glutinosa

To investigate seasonal reproduction in Myxine glutinosa, we measured total brain gonadotropin-releasing hormone (GnRH) and determined gonadal stages of hagfish collected from the Gulf of Maine once a month for 12 months. Thirty hagfish from each of three different size classes of small (20-35 cm), medium (35-45 cm), and large (50-60+ cm) were sampled for brains and gonads. In the medium and large class hagfish there was an increase in GnRH concentrations during April and May that correlated with male and female gonadal maturity. Also in these size classes of female hagfish, there was a similar rise in GnRH in November and then again in January that preceded the highest incidence of large eggs (stage 7). The elevated GnRH may be influencing the onset of ovarian recrudescence which has been shown in other vertebrates. These data suggest an association of the concentration of brain GnRH with gonadal maturity and provide supportive evidence of a possible seasonal reproductive cycle in M. glutinosa shown in recent studies of [J. Exp. Zool. 301A (2004) 352], correlating steroid production with gonadal maturation.     

Activation of the chicken gonadotropin-inhibitory hormone receptor reduces gonadotropin releasing hormone receptor signaling

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic peptide from the RFamide peptide family that has been identified in multiple avian species. Although GnIH has clearly been shown to reduce LH release from the anterior pituitary gland, its mechanism of action remains to be determined. The overall objectives of this study were (1) to characterize the GnIH receptor (GnIH-R) signaling pathway, (2) to evaluate potential interactions with gonadotropin releasing hormone type III receptor (GnRH-R-III) signaling, and (3) to determine the molecular mechanisms by which GnIH and GnRH regulate pituitary gonadotrope function during a reproductive cycle in the chicken. Using real-time PCR, we showed that in the chicken pituitary gland, GnIH-R mRNA levels fluctuate in an opposite manner to GnRH-R-III, with higher and lower levels observed during inactive and active reproductive stages, respectively. We demonstrated that the chicken GnIH-R signals by inhibiting adenylyl cyclase cAMP production, most likely by coupling to G_αi. We also showed that this inhibition is sufficient to significantly reduce GnRH-induced cAMP responsive element (CRE) activation in a dose-dependent manner, and that the ratio of GnRH/GnIH receptors is a significant factor. We propose that in avian species, sexual maturation is characterized by a change in GnIH/GnRH receptor ratio, resulting in a switch in pituitary sensitivity from inhibitory (involving GnIH) to stimulatory (involving GnRH). In turn, decreasing GnIH-R signaling, combined with increasing GnRH-R-III signaling, results in significant increases in CRE activation, possibly initiating gonadotropin synthesis.     

Gonadotropin-inhibitory hormone in Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii): cDNA identification, transcript localization and functional effects in laboratory and field experiments

The neuropeptide control of gonadotropin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, GnRH. We recently identified a novel hypothalamic dodecapeptide with a C-terminal LeuPro-Leu-Arg-Phe-NH2 sequence in the domestic bird, Japanese quail (Coturnix japonica). This novel peptide inhibited gonadotropin release in vitro from the quail anterior pituitary; thus it was named gonadotropin-inhibitory hormone (GnIH). GnIH may be an important factor regulating reproductive activity not only in domesticated birds but also in wild, seasonally breeding birds. Thus, we tested synthetic quail GnIH in seasonally breeding wild bird species. In an in vivo experiment, chicken gonadotropin-releasing hormone-I (cGnRH-I) alone or a cGnRH-I/quail GnIH cocktail was injected i.v. into non-breeding song sparrows (Melospiza melodia). Quail GnIH rapidly (within 2 min) attenuated the GnRH-induced rise in plasma LH. Furthermore, we tested the effects of quail GnIH in castrated, photostimulated Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), using quail GnIH or saline for injection. Again, quail GnIH rapidly reduced plasma LH (within 3 min) compared with controls. To characterize fully the action of GnIH in wild birds, the identification of their endogenous GnIH is essential. Therefore, in the present study a cDNA encoding GnIH in the brain of Gambel's white-crowned sparrow was cloned by a combination of 3' and 5' rapid amplification of cDNA ends and compared with the quail GnIH cDNA previously identified. The deduced sparrow GnIH precursor consisted of 173 amino acid residues, encoding one sparrow GnIH and two sparrow GnIH-related peptides (sparrow GnIH-RP-1 and GnIH-RP-2) that included Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Although the homology of sparrow and quail GnIH precursors was approximately 66%, the C-terminal structures of GnIH, GnIH-RP-1 and GnIH-RP-2 were all identical in two species. In situ hybridization revealed the cellular localization of sparrow GnIH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. Immunohistochemical analysis also showed that sparrow GnIH-like immunoreactive cell bodies and terminals were localized in the PVN and median eminence respectively. Thus, only the sparrow PVN expresses GnIH, which appears to be a hypothalamic inhibitory factor for LH release, as evident from our field injections of GnIH into free-living breeding white-crowned sparrows. Sparrow GnIH rapidly (within 2 min) reduced plasma LH when injected into free-living Gambel's white-crowned sparrows on their breeding grounds in northern Alaska. Taken together, our results indicate that, despite amino acid sequence differences, quail GnIH and sparrow GnIH have similar inhibitory effects on the reproductive axis in wild sparrow species. Thus, GnIH appears to be a modulator of gonadotropin release.     

Non-breeding gonadal testosterone production of male and female northern cardinals (Cardinalis cardinalis) following GnRH challenge

Yearly, testosterone (T) levels fluctuate as many vertebrates cycle through reproductive and non-reproductive periods. Among many temperate birds, it is well established that levels of T peak as gonads recrudesce for breeding and then fall as gonads regress prior to the non-breeding season. While the tissues producing breeding season T are well studied, the tissues responsible for non-breeding T have received less investigative attention. We examined the ability of male and female Northern Cardinals (Cardinalis cardinalis) to elevate gonadal T following standardized injections of gonadotropin-releasing hormone (GnRH) across three non-breeding seasons. Males and females were capable of significantly elevating gonadal T production following GnRH injections during periods of reproductive quiescence. The magnitude of T elevation varied across the non-breeding season, but not between sexes. To our knowledge, this is the first report of a significant increase in gonadal T production following GnRH injections administered in the non-breeding season.