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Laboratory of Reproductive Neuroendocrinology (T.U., S.K., Y.-c.H., J.R., J.J., G.E.B.), Department of Integrative Biology and Helen Wills Neuroscience Institute, University of California at Berkeley, Berkeley, California 94720-3140; and Laboratory of Integrative Brain Sciences (T.O., V.S.C., K.T.), Department of Biology, Waseda University, Shinjuku-ku, Tokyo 169-8050, Japan
Address all correspondence and requests for reprints to: Takayoshi Ubuka, Ph.D., Laboratory of Reproductive Neuroendocrinology, Department of Integrative Biology, University of California at Berkeley, 3060 Valley Life Sciences Building No. 3140, Berkeley, California 94720-3140. E-mail: ubukat{at}berkeley.edu.
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Introduction |
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GnIH homologs are present in the brains of other vertebrates, such as mammals, amphibians, and fish (for review, see Refs. 14 and 15). These peptides, categorized as RFamide-related peptides (RFRPs) (16), possess a characteristic LPXRF-amide (X = L or Q) motif at their C termini in all vertebrates tested to date (14, 15). The receptors for GnIH homologs (RFRP-Rs) have also been characterized in vertebrates (14, 15, 16). To elucidate the mode of GnIH action, we identified a novel G-protein coupled receptor for GnIH in quail (17). The identified GnIH receptor (GnIH-R) was expressed in the pituitary and specifically bound GnIH in a concentration-dependent manner. Interestingly, GnIH-R mRNA was abundantly expressed in various parts of the brain as well as in the pituitary, suggesting GnIH action not only in the pituitary, but also in the brain (17). In quail and songbirds, GnIH immunoreactive fiber terminals were observed in multiple brain areas in addition to the external layer of the ME (7, 18). Immunohistochemical studies using light and confocal microscopy indicate that GnIH immunoreactive axon terminals are in probable contact with GnRH neurons in birds (18) and mammals (19). Thus, there is potential for the direct regulation of GnRH neuronal activity by GnIH neurons (18, 19). Central administration of GnIH or GnIH homologs inhibits gonadotropin release in white-crowned sparrow (20), Syrian hamsters (19), and rats (21) in a manner similar to peripheral administration of GnIH (10, 12, 19). Accordingly, GnIH may inhibit gonadotropin secretion by decreasing GnRH neuronal activity in addition to regulating pituitary gonadotropin release directly.
In this study we investigated the interactions of GnIH neurons and the GnRH neuronal system in a seasonally breeding songbird species, European starling. Starlings are obligate seasonal breeders, and show robust, highly predictable repeated changes in reproductive physiology and behavior in response to the annual cycle of changing photoperiod. A great deal is known about the neuroendocrine control of the starling reproductive system (for review, see Refs. 22, 23, 24). Therefore, it is an ideal model system for studies on neuroendocrine control of seasonality in particular and on reproduction in general. Unlike many strains of quail, wild-caught starlings have not undergone artificial selection, thus, any data gathered provide us with an additional insight as to neuroendocrine control of reproduction in the wild. Furthermore, starlings are non-native and considered a pest species in the United States. Thus, understanding physiological mechanisms controlling their reproduction is of potential ecological and agricultural importance. The focus of the present study is the neuroanatomical interaction of the GnIH/GnRH systems; therefore, we do not attempt to address potential GnIH effects upon pituitary hormone release. Rather, our aim was to identify the potential for direct regulation of the GnRH system by GnIH. We first cloned GnIH precursor cDNA from the starling hypothalamus by a combination of 3' and 5' rapid amplification of cDNA ends (RACE). We further identified the mature starling GnIH peptide by immunoaffinity purification from starling brain, followed by mass spectrometry. The histological localization of GnIH neurons was analyzed by in situ hybridization (ISH) of GnIH precursor mRNA and immunocytochemistry (ICC) of GnIH peptide. It is generally accepted that birds possess two forms of GnRH in their brain. One form is GnRH-I, which is thought to be released at the ME to stimulate gonadotropin secretion from the anterior pituitary gland (3, 4, 25). The second form of GnRH is GnRH-II (5, 26), which is thought to influence reproductive behaviors in mammals (26, 27, 28) and birds (29). To visualize the distribution of GnIH immunoreactive fibers relative to GnRH-I and -II neurons, we performed double-label ICC using GnIH and GnRH antisera. The expression of GnIH-R on GnRH neurons is critical for the functional interaction of GnIH peptide with GnRH neurons. Accordingly, we cloned the partial cDNA of starling GnIH-R and performed ISH of starling GnIH-R mRNA combined with GnRH ICC. GnIH-R mRNA was expressed in GnRH-I and -II neurons. Our results suggest that GnIH controls reproductive physiology and behavior of vertebrates not only by inhibiting gonadotropin secretion directly at the pituitary, as has already been demonstrated in birds and mammals (6, 10, 11, 12, 19), but also by influencing GnRH neuronal activity within the brain.
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Materials and Methods |
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Identification of a cDNA encoding starling GnIH precursor
Three hypothalami from adult starlings were used for the identification of a cDNA encoding starling GnIH precursor. Total RNA, including rRNA and mRNA, was isolated by the Sepasol extraction method (Sepasol-RNA I Super; Nacalai Tesque, Kyoto, Japan). Total RNA was reverse transcribed using oligo(deoxythymidine)15 primer (Promega, Madison, WI) and reverse transcriptase (M-MLV Reverse Transcriptase; Invitrogen, Carlsbad, CA). Partial starling GnIH precursor cDNA was amplified by PCR using various primers based on quail and white-crowned sparrow GnIH precursor cDNA sequences (9, 10). All PCR amplifications were performed in a reaction mixture containing Taq polymerase (TaKaRa Ex Taq; Takara Bio Inc., Shiga, Japan). PCR products were subcloned into a pGEM-T Easy vector (Promega), and the DNA inserts of the positive clones were amplified by PCR with universal M13 primers. Amplified DNA was sequenced at the University of California at Berkeley DNA sequencing facility (Berkeley, CA) using the 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA), and a starling GnIH precursor cDNA fragment of approximately 0.3 kb was determined.
To identify the 3' end of the starling GnIH precursor cDNA, first-strand cDNA was reverse transcribed with the oligo(dT)-anchor primer (5'/3' RACE Kit, 2nd Generation; Roche Diagnostics, Mannheim, Germany) and poly(A)+ RNA, which was isolated by mRNA isolation kit (Roche Diagnostics). Gene-specific forward primers and PCR anchor primer (Roche Diagnostics) were used to amplify the 3' end of the starling GnIH precursor cDNA. The PCR products were subcloned and sequenced as described previously.
To identify the 5' end of the starling GnIH precursor cDNA, the template cDNA was reverse transcribed with gene-specific reverse primers, followed by poly(A) tailing of the cDNA with dATP and terminal transferase (Roche Diagnostics). The tailed cDNA was amplified with the oligo(dT)-anchor primer and nested gene-specific reverse primer. A second PCR was performed using PCR anchor primer and further nested gene-specific reverse primer. The second PCR products were subcloned and sequenced as described previously.
Immunoaffinity purification and mass spectrometry of starling GnIH peptide
To identify the mature starling GnIH peptide in the starling brain, we first collected GnIH immunoreactive material from the brain extract using an antiserum raised against quail GnIH (6). There were 10 starling brains boiled and homogenized in 5% acetic acid, as described previously (6, 30, 31). The homogenate was centrifuged at 10,000 g for 30 min at 4 C, and the supernatant was collected. The collected supernatant was passed through a disposable C-18 cartridge column (Mega Bond-Elut; Varian, Harbor, CA). The retained material eluted with 60% methanol was concentrated using an evaporator and loaded onto an immunoaffinity column. The affinity chromatography was performed as described previously (31, 32, 33, 34). The antiserum against GnIH was conjugated to cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) as an affinity ligand. The concentrated material was applied to the immunoaffinity column at 4 C, and the adsorbed materials were eluted with 0.3 M acetic acid containing 0.1% 2-mercaptoethanol. The eluted fractions were again concentrated and subjected to a reversed-phase HPLC column (ODS-80TM; Tosoh, Tokyo, Japan) with a linear gradient of 10–50% acetonitrile containing 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min, and fractionated every 2 min for 100 min. The immunoreactive fraction was assayed by a dot immunoblot assay, and the molecular mass of the material was analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) (AXIMA-CFR plus; Shimadzu, Kyoto, Japan). The predicted starling GnIH (SIKPFANLPLRF-NH2) was synthesized by peptide synthesizer (PSSM-8; Shimadzu), and molecular behavior of the synthetic and native peptides was further compared by MALDI-TOF MS.
ISH of starling GnIH precursor mRNA and ICC of GnIH
Adult starlings were deeply anesthetized before transcardial perfusion with 0.1 M PBS (pH 7.4), followed by fixative solution (4% paraformaldehyde in 0.1 M PBS). Brains were soaked in a refrigerated sucrose solution (30% sucrose in PBS) until they sank. Coronal sections at 20-µm thickness were collected on a cryostat at –20 C. ISH was performed according to our previous method (10, 13) using a digoxigenin (DIG)-labeled antisense RNA probe. The DIG-labeled antisense RNA probe was produced using a standard RNA labeling kit (Roche Diagnostics) using partial starling GnIH precursor cDNA (complementary to nt 198–454 in Fig. 1
) as a template. After hybridization, the sections were incubated with alkaline phosphatase-labeled sheep anti-DIG antibody (Roche Diagnostics), and the immunoreactive product was visualized by immersing the sections in a substrate solution (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate stock solution; Roche Diagnostics). Control for specificity of ISH was performed using a DIG-labeled sense RNA probe, the sequence of which was complementary to that of the antisense probe.
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GnIH ICC was also conducted on some of the sections directly without ISH. Sections were incubated in 0.3% H2O2 for 30 min, and nonspecific binding of the antibody was blocked using 2% normal goat serum in 0.2% PBS + Triton X-100 (PBS-T), and then incubated in primary antibody (rabbit anti-GnIH) at a concentration of 1:1000 in 0.4% PBS-T. Three subsequent washes in 0.2% PBS-T were followed by incubation for 1 h in biotinylated goat antirabbit IgG (1:1000 in 0.2% PBS-T). Sections were then incubated for 1 h in avidin-biotin complex (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA). The resulting complex was visualized using 0.03% 3,3 diaminobenzidine (DAB).
Double-labeling ICC of GnIH and GnRH
To determine the relative neuroanatomical distribution of GnIH and GnRH peptides, starlings were perfused, and brains were extracted and cryoprotected as already described. Coronal sections (40 µm) were taken throughout the whole brain and collected into PBS. Double-label ICC for GnRH and GnIH was performed as described by Bentley et al. (18). The primary antibody used was rabbit-anti GnRH (HU60H; kindly donated by Dr. H. Urbanski, Oregon National Primate Research Center) at a concentration of 1:10,000 in 0.2% PBS-T. Although the antibody we used does not distinguish between the two forms of GnRH, we can identify them based on their separate locations in the brain (see Fig. 3, A and D) and from their distinctive appearance: GnRH-II neurons are stubby with few fibers emanating from them (18, 35, 36). GnRH-I and -II were labeled with DAB (brown) and GnIH was labeled with Vector VIP (purple). Thus, the GnIH neurons and fibers were identified by their purple color in contrast to brown GnRH-immunoreactive neurons labeled with DAB.
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0.4 kb) was sequenced at the University of California at Berkeley DNA sequencing facility to assess the homology to quail GnIH-R cDNA. ISH was performed using a DIG-labeled antisense RNA probe that was produced from the cloned starling partial GnIH-R cDNA as a template. After hybridization, the sections were incubated with alkaline phosphatase-labeled sheep anti-DIG antibody, and immunoreactive product was detected by the substrate solution. Control for specificity of ISH of GnIH-R mRNA was performed using a DIG-labeled sense RNA probe, the sequence of which was complementary to the antisense probe sequence.
Immunocytochemical analysis of GnRH was further conducted on the same sections previously labeled for GnIH-R mRNA by ISH. After immersing the sections in rabbit-anti GnRH (HU60H), which is the same antibody used for double labeling of GnIH and GnRH peptides, they were incubated with goat antirabbit secondary antibody conjugated to the fluorophore Alexafluor 488 or 568 (Molecular Probes, Inc., Eugene, OR).
The ratio of GnRH immunoreactive cells that also express GnIH-R mRNA was further calculated as follows. First, pictures of brain sections of four different birds double stained by ICC for GnRH and ISH for GnIH-R mRNA were taken. Ten to 20 cells that apparently express GnRH-I and another 10–20 cells that apparently express GnRH-II were randomly chosen from each bird. The ratio of the cells that also expresses GnIH-R mRNA was calculated for each bird, and the mean ratio of four birds was further calculated. The cells where the staining was subtle in its density, as shown by gray arrows in Fig. 7E or 8D, were treated as not expressing GnIH-R mRNA.
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Results |
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shows that starling GnIH precursor cDNA (GenBank EF486798) is composed of 912 nucleotides, containing a short 5' untranslated sequence of 40 bp, a single open reading frame of 519 bp, and a 3' untranslated sequence of 353 bp with the addition of various lengths of poly(A) tail. The open reading frame begins with the putative start codon ATG at position 41 according to the Kozak rule (37) and terminates with a stop codon TGA at position 560. A single polyadenylation signal (AATAAA) was found in the 3' untranslated region at position 895. The open reading frame region encoded a 173-residue polypeptide. We predict that the GnIH transcript is translated with Met1 as an initiation methionine, because Met1 was followed by hydrophobic moiety, which is typical in a signal peptide region. There was 91% nucleotide homology between starling and white-crowned sparrow GnIH precursor polypeptide cDNA (GenBank AB128164), and 75% homology with quail GnIH precursor cDNA (GenBank AB039815).
The GnIH precursor polypeptide included three LPXRF (X = L or Q) sequences with Gly residue as a C-terminal amidation signal followed by monobasic endoproteolytic residues Arg or Lys (Fig. 1, underlined) (14, 15, 16). SIKPFANLPLRF-NH2 was predicted as the mature starling GnIH peptide (processed form of the double underlined amino acid sequence in Fig. 1) because of its homology with quail GnIH (SIKPSAYLPLRF-NH2) (6, 9).
Identification of mature starling GnIH peptide
We first used immunoaffinity purification using the antiserum against quail GnIH, and the immunoreactive material was subjected to the mass spectrometric analysis (MALDI-TOF MS). The observed mass value of the molecular ion peak in the spectrum was 1401.89 m/z ([(M + H]+) (Fig. 2 and Table 1); this value was almost identical to the calculated mass of the predicted starling GnIH (SIKPFANLPLRF-NH2) [1401.84 m/z ([M + H]+)] (Table 1). The predicted starling GnIH was then synthesized, and its molecular behavior was also analyzed by MALDI-TOF MS, which produced a molecular ion peak of 1401.75 m/z ([M + H]+) (Fig. 2, inset, and Table 1). Although the GnIH immunoreactive material was not sequenced, it is highly probable that the predicted peptide is produced from the precursor as an endogenous ligand in a manner similar to quail GnIH (6, 9).
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), and the product was used for ISH. An intense expression of starling GnIH precursor mRNA was distributed in the PVN (Fig. 3B) of the hypothalamus (Fig. 4B). ICC for GnIH peptide was further conducted on the same sections. As shown in Fig. 4A, intense immunoreactivity with GnIH peptide was also found in the PVN. There was a clear cellular colocalization of GnIH precursor mRNA and GnIH peptide (Fig. 4C). Colocalization of GnIH precursor mRNA and GnIH peptide also demonstrated the specificity of each localization method. Dense populations of GnIH immunoreactive beaded fibers were observed emanating from GnIH neuronal cell bodies, presumably transporting GnIH (Fig. 4, A and C).
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) because hypothalamic hormones that regulate anterior pituitary function are generally thought to be released at the ME. ICC indicates GnIH immunoreactive fibers in the external layer of the ME (Fig. 5, A and B), which is consistent with a role for GnIH in pituitary hormone regulation.
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) and GnRH-II neurons in the midbrain (Fig. 3D). Dense populations of GnRH-I neurons (Fig. 6A, stained brown) and GnRH-II neurons (Fig. 6B, stained brown) were clustered in the preoptic area and midbrain, respectively. Dense populations of GnIH immunoreactive fibers were also observed in both of these regions (Fig. 6, A and B, fibers labeled in purple). Furthermore, GnIH immunoreactive axon terminal-like structures were observed in close proximity to both GnRH-I (Fig. 6, A and C) and GnRH-II (Fig. 6, B and D) neuronal dendrites or cell bodies.
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Expression of starling GnIH-R mRNA in GnRH-I neurons
The close proximity of GnIH axon terminals with GnRH-I neurons predicted a direct action of GnIH on GnRH-I neurons. To verify this prediction, we localized the expression of GnIH-R mRNA in GnRH-I neurons by ISH of GnIH-R mRNA combined with GnRH ICC on the same brain sections. ISH revealed expression of starling GnIH-R mRNA in the preoptic area of the hypothalamus (Figs. 3A, and 7, A, B, and E). The cells expressing GnIH-R mRNA were clustered in the medial portion of the preoptic area (Fig. 7A). Most of the cells that expressed GnIH-R mRNA showed putative neuronal morphology identified from their neuron-shaped cell bodies (Fig. 7B). Subsequent ICC for GnRH peptide on the same sections used for ISH of GnIH-R mRNA revealed a discrete bilateral population of GnRH-I immunoreactive cell bodies in the preoptic area (Fig. 7C). Closer observation of the labeled section revealed the coexpression of GnIH-R mRNA in GnRH-I neurons (Fig. 7, D and E). Although most of the GnRH-I neurons observed coexpressed GnIH-R mRNA (80.5%), the expression of GnIH-R mRNA was not detectable in some GnRH-I neurons. GnIH-R mRNA was also expressed in cells that do not express GnRH-I in the preoptic area (Fig. 7, D and E). In sharp contrast to the preoptic area, populations of cells that express GnIH-R mRNA were not observed in the telencephalic region where GnIH immunoreactive fibers are sparse (Fig. 7F), nor were they observed in regions lateral to the preoptic area (Fig. 7A).
Expression of starling GnIH-R mRNA in GnRH-II neurons
The close proximity of GnIH axon terminals with GnRH-II neurons also predicted a direct action of GnIH on GnRH-II neurons. In the same manner as for GnRH-I neurons, we investigated the expression of GnIH-R mRNA in GnRH-II neurons by GnIH-R mRNA ISH combined with GnRH ICC on the same brain sections. Populations of cells that expressed starling GnIH-R mRNA were observed in the medial portion of the midbrain (Figs. 3D and 8A). Most of the cells that expressed GnIH-R mRNA showed an apparent neuronal structure, as identified from their distinctive morphology (Fig. 8, A and B). The staining of GnIH-R mRNA in the cells of the midbrain tended to be stronger (Fig. 8, A and B) than that of the preoptic area (Fig. 7, A and B), suggesting active production of GnIH-R mRNA in this area. ICC for GnRH peptide was further conducted on the same sections used for ISH of GnIH-R mRNA. As shown in Fig. 8C, a dense population of GnRH-II immunoreactive cell bodies was clustered in a discrete region of the midbrain. Clear cellular colocalization of GnRH-II and GnIH-R mRNA was observed (89.4%), although the expression of GnIH-R mRNA was not detectable in some of the GnRH-II neurons (Fig. 8, C and D).
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Discussion |
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We first cloned starling GnIH precursor cDNA from the hypothalamus, and found that starling GnIH precursor mRNA encoded three peptides that possess characteristic LPXRF-amide (X = L or Q) motifs at the C termini, as in quail (9) and white-crowned sparrow (10). Mature starling GnIH peptide was further isolated by immunoaffinity purification, and its structure was identified as SIKPFANLPLRF-NH2 by mass spectrometric analyses. Although we did not isolate the other two LPXRF-amide (X = L or Q) peptides encoded in the precursor gene in this study, we cannot exclude that they are also produced as endogenous ligands.
The cloned partial starling GnIH-R cDNA, which was used for ISH in this study, was highly homologous (92%) to quail GnIH-R cDNA, which encodes a functional receptor that binds GnIH in a concentration-dependent manner (17). Two distinct types of receptors for RFRPs have been cloned in the chicken, which are chicken NPFF-R1 (designated as RFRPR in Ref. 38) and chicken NPFF-R2 (designated as NPFFR in Ref. 38). Functional studies measuring ligand-induced modulation of G
i2 mRNA expression in COS-7 cells transfected with RFRPR or NPFFR cDNA have shown that RFRP has higher affinity to RFRPR (NPFF-R1) than NPFFR (NPFF-R2). The homology of cDNA sequences of the partial starling GnIH-R cloned in this study and RFRPR (NPFF-R1) was 93%, whereas NPFFR (NPFF-R2) was 67% (supplemental Fig. 1). Accordingly, we consider that the cloned partial starling GnIH-R cDNA encodes a functional receptor (RFRPR) that binds GnIH at higher affinities than NPFFR.
ISH of GnIH precursor mRNA and ICC of GnIH peptide revealed GnIH neurons clustered in the PVN of starling hypothalamus. GnIH immunoreactive fibers also extended to the external layer of the ME. These findings are consistent with studies in quail and white-crowned sparrows (6, 7, 8, 10), suggesting direct regulation of pituitary function in birds. Dense populations of GnIH immunoreactive fibers were also found in the preoptic area, where the bilateral population of GnRH-I neuronal cell bodies exist. Furthermore, double-label ICC using GnIH and GnRH antiserum revealed GnIH immunoreactive axon terminals in putative contact with GnRH-I neurons, as has been shown in other avian and mammalian species. In addition, GnIH-R mRNA was apparently coexpressed in most of the GnRH-I neurons (80.5%), providing strong evidence for the regulation of GnRH-I neurons by GnIH. In birds, GnRH-I is thought to be released at the ME to stimulate gonadotropin secretion from the pituitary (3, 4, 25). Accordingly, it is possible that GnIH may also control gonadotropin secretion by decreasing GnRH-I neuronal activity. Because GnIH-R mRNA was also expressed in cells that do not contain GnRH-I, it is also possible that GnIH may control GnRH-I neurons indirectly via other neurons.
The mechanism of action of GnIH has not yet been elucidated. GnIH decreases LH release from cultured quail (6) and chicken (11) pituitary, and GnIH-R is expressed in the pituitary (17). Thus, it appears that GnIH has a direct inhibitory effect on the pituitary. However, because of the relatively long pituitary incubation periods used in these in vitro experiments [100 min (6) and 120 min (11)], it is still unclear if the inhibitory effect of GnIH on LH release results from the inhibition of gonadotropin synthesis (11, 12), or from the inhibition of exocytosis of gonadotropin containing vesicles in gonadotropes, or both. The present study further strengthens the idea that GnIH directly acts on GnRH-I neurons to influence gonadotropin secretion. Clearly, further study is required to investigate the possible modes of action of GnIH upon the hypothalamo and pituitary components of the hypothalamo-pituitary-gonadal axis.
In starlings, GnIH immunoreactive fibers were located in the midbrain, where GnRH-II neuronal cell bodies are clustered. Double-label ICC using GnIH and GnRH antiserum also revealed GnIH immunoreactive axon terminals in putative contact with GnRH-II neurons. ISH of GnIH-R mRNA followed by GnRH ICC further showed coexpression of GnIH-R mRNA in GnRH-II neurons (89.4%). Again, this is strong evidence that GnIH acts directly on the GnRH system. GnRH-II (5, 26) is thought to control reproductive behaviors in mammals (26, 27, 28) and birds (29). Intracerebroventricular administration of GnIH to female white-crowned sparrows inhibits copulation solicitation, without affecting locomotor activity (20). Furthermore, rhodaminated GnIH binds in vivo to the midbrain area where GnRH-II neurons exist (20). Accordingly, it is possible that GnIH inhibits the reproductive behavior of birds by inhibiting GnRH-II neuronal activity. The mammalian homolog of GnIH also inhibits reproductive behaviors in male rats (21). Based on our findings and on the widespread distribution of GnIH immunoreactive fibers in birds (7, 18) and mammals (19), direct regulation of reproductive behaviors by GnIH is also a distinct possibility in other vertebrates.
The mammalian homolog of GnIH-R is GPR147 (OT7T022, NPFF-1, RFRP-R) (15, 16). RF-amide related peptide, RFRP, which is the mammalian GnIH homolog, suppressed the production of cAMP in Chinese hamster ovary cells transfected with GPR147 (16, 39). RFRP mRNA is expressed in the hypothalamus, and RFRP immunoreactive neuronal fibers are widely distributed in the rat brain (16). GPR147 mRNA is also widely distributed in rat brain. Thus, the RFRP-GPR147 system may function as an inhibitory mechanism in the mammalian brain. The molecular mechanisms underlying the GnIH-GnIH-R system have not been studied in birds, but we predict that the inhibitory action of GnIH in birds may be achieved by decreasing cAMP production in the target cell, as it is in mammals. Whatever the cellular mechanisms might be, our data are the first to provide strong evidence for a direct action of GnIH on the GnRH system.
All the evidence collected to date suggests that GnIH acts as a physiological inhibitor of the vertebrate reproductive system. This evidence comes from anatomical studies on birds and rodents (6, 7, 8, 18, 19). Other investigations have examined effects of GnIH on reproductive behavior (20, 21), gonadotropin synthesis and release (10, 12, 19, 21), and gonadal development (12) in vivo. In vitro studies found direct inhibitory effects of GnIH on the anterior pituitary gland synthesis and release of gonadotropin (6, 11). To demonstrate that GnIH is a physiologically important regulator of the reproductive system in intact, unmanipulated animals, a causal physiological relationship between GnIH and the entire reproductive system, including the brain GnRH system, pituitary gland, and gonads, should be established. This will at the very least involve demonstrating changes in the activity of the entire reproductive system as a function of season (18), age (8), ontogenic stage (8), etc. in intact organisms are correlated with changes in GnIH synthesis and release, GnIH-R, and neuron connectivity. Our data demonstrating the presence of neuroarchitecture by which GnIH might influence GnRH-I and -II neurons in the avian brain are a critical step in this direction.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online September 27, 2007
Abbreviations: DAB, Diaminobenzidine; DIG, digoxigenin; GnIH, gonadotropin-inhibitory hormone; GnIH-R, gonadotropin-inhibitory hormone receptor; ICC, immunocytochemistry; ISH, in situ hybridization; MALDI-TOF MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; ME, median eminence; NPFF-R, neuropeptide FF receptor; PBS-T, PBS + Triton X-100; PVN, paraventricular nucleus; RACE, rapid amplification of cDNA end; RFRP, RFamide-related peptide; RFRP-R, RFamide-related peptide receptor.
Received July 17, 2007.
Accepted for publication September 20, 2007.
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and follicle-stimulating hormone β subunit expression in the pituitary of the domestic chicken. J Neuroendocrinol 16:999–1006[CrossRef][Medline]This article has been cited by other articles:
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M. Murakami, T. Matsuzaki, T. Iwasa, T. Yasui, M. Irahara, T. Osugi, and K. Tsutsui Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats J. Endocrinol., October 1, 2008; 199(1): 105 - 112. [Abstract] [Full Text] [PDF]
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E. M. Gibson, S. A. Humber, S. Jain, W. P. Williams III, S. Zhao, G. E. Bentley, K. Tsutsui, and L. J. Kriegsfeld Alterations in RFamide-Related Peptide Expression Are Coordinated with the Preovulatory Luteinizing Hormone Surge Endocrinology, October 1, 2008; 149(10): 4958 - 4969. [Abstract] [Full Text] [PDF]
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 T. J Greives, L. J Kriegsfeld, G. E Bentley, K. Tsutsui, and G. E Demas Recent advances in reproductive neuroendocrinology: a role for RFamide peptides in seasonal reproduction? Proc R Soc B, September 7, 2008; 275(1646): 1943 - 1951. [Abstract] [Full Text] [PDF]
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