| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N5
b The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-subunit for LH and thyroid-stimulating hormone in dispersed fish pituitary cells. Our data support the hypothesis that the ancestral salmonid had a third GnRH form when the genome doubled (tetraploidization), but the third form was lost later in some salmonids due to chromosomal rearrangements. We suggest that the salmon GnRH form compensated for the loss of the third form.
central nervous system, gonadotropin-releasing hormone, neuroendocrinology, neuropeptides, pituitary hormones
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The GnRH decapeptide was first isolated from pig and sheep as mammalian GnRH (mGnRH) [1, 2], and novel forms have been subsequently isolated from brain of other species, mainly vertebrates. The number of distinct forms now totals 15 after the recent identification of herring (hr)GnRH [3] and medaka/pejerrey GnRH [4, 5]. All forms of GnRH identified to date are 10 amino acids with a pyroglutamic acid N terminus and an amidated glycine C terminus. Amino acids 1 through 4 are tightly conserved except for position 2 in guinea pig (gp)GnRH and position 3 in lamprey (l)GnRH-I; the conservation may reflect the importance of residues 1 through 3 for the functional release of LH and FSH. Variation in GnRH peptide structure is typically the result of different amino acids in positions 5 to 8.
At least 2 forms of GnRH coexist in the brain of most vertebrate species. Chicken (c)GnRH-II is present in all jawed vertebrates and mGnRH appears in early bony fish, which suggests that these 2 forms are ancient and may have given rise to other variants that have been identified in more recently evolved species. Humans are known to have genes for both mGnRH [6] and cGnRH-II [7].
Fish provide a unique opportunity to study the evolution of GnRH in vertebrates. It is now established that at least 3 GnRH forms are expressed in many teleost fish. Three forms of GnRH probably first appeared in the herring lineage [3]. The next order of fish to evolve is thought to be salmonids and, at about the same time, several orders of fresh-water fish, including catfish and goldfish/carps; in each of these groups of fish, only 2 forms of GnRH have been identified [8]. However, some fish that evolved after herring (pejerrey, seabream, pacu, tilapia) have 3 forms of GnRH [5, 9–11]. Each form of GnRH within a species is encoded on a separate gene. It is not clear whether all teleosts that evolved after herring had 3 forms of GnRH initially. One option is that some fish inherited the genes for 3 GnRHs but lost or ceased to express the third form. The other option is that some fish evolved from an ancestor with 2 GnRHs and the others from an ancestor with 3 GnRHs. A further complication is that tetraploid fish, which includes salmon, catfish, and goldfish/carp, appear to have only 2 forms of GnRH (cGnRH-II and salmon (s)GnRH), whereas one might predict up to 6 GnRHs.
Our objective was to determine whether a representative of the earliest subfamily of salmonids (Coregoninae) had more than 2 forms of GnRH in the brain. The presence of a third form implies that the loss of 1 form of GnRH occurred much later than the initial tetraploidization of the ancestral salmonid. We selected lake whitefish (Coregonus clupeaformis) as the representative because they are available in sufficient numbers for peptide purification. Our other objective was to identify the primary structure of the third form of GnRH and determine whether the peptide was biologically active.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A total of 1159 whole brains, some with pituitaries, were collected from both sexes of lake whitefish (Coregonus clupeaformis) during the December fishery in Lesser Slave Lake, Alberta, Canada. These fish were in a nonreproductive state because spawning normally occurs in late September and October. The dissected tissue was frozen on dry ice and stored at -80°C.
GnRH Extraction
The frozen tissue (894.3 g) was pulverized in a Waring blender with liquid nitrogen. The powder was extracted in 1 N HCl:acetone solution (3:100 vol:vol) in which the tissue:liquid ratio was 1 g tissue:5 ml liquid. The extract was stirred for 3 h while being chilled with dry ice that surrounded the container. The solution was filtered through Whatman #1 filter paper and solids were reextracted in 0.01 N HCl:acetone (1:4 vol:vol) for 5 min and then refiltered. To remove hydrophobic substances from the filtrate, 5 petroleum ether (bp 30–60°C) extractions (at 20% of the extract volume) in series were used. The final peptide extract was reduced to 700 ml on a vacuum centrifuge to remove the acetone.
Initial HPLC Analysis of 12 Brains
A small batch of 12 brains was extracted as above except that the final volume for injection onto the HPLC was 2 ml. Just before injection, the extract was filtered through a 0.45-µm filter (µStar LB, Costar 8112; Corning, NY). The extract was injected onto a Supelco C-18 column that was separate from the one used for large-scale purification. Elution was identical to that described in step 2 (Table 1). Standards of synthetic cGnRH-II and sGnRH (200 ng each in 600 µl water) were injected only onto the column used for the 12-brain extract for comparison with the immunoreactive peaks.
|
HPLC Purification
The large-scale extract was filtered using Whatman #1 filter paper. Then the extract was pumped onto a column made by connecting 10 Sep-Pak cartridges (Waters Corp., Milford, MA) in series. Purification consisted of 7 additional HPLC runs using different columns and/or mobile phases and a Beckman model 166 Liquid Chromatograph (Fullerton, CA). The elution programs are described in Table 1. The HPLC injections consisted of fractions that had been pooled from the immunoreactive GnRH peaks of the previous HPLC run (Table 1), then reduced and filtered through a 0.45-µm filter (Corning). Sixty fractions (1 ml/min) from each run were collected for RIA analysis. The elution profile was monitored continuously at 280 nm, except in step 5, which was monitored at 210 nm. To avoid contamination, synthetic standards were never applied to the columns used for purification. Blanks were run between each purification step and fractions were assayed.
RIA Analysis
A 5-µl sample was collected from each fraction of each HPLC run to assay for GnRH. Each assay tube contained 5 µl of the HPLC fraction, 300 µl PBS, 100 µl GF-6 antiserum, and 100 µl of iodinated trace mGnRH. Tubes were incubated overnight at 4°C. The following day, 1 ml of charcoal-dextran in PBS was added, tubes were centrifuged, and the supernatant radioactivity was measured in a gamma counter.
The standard curve was prepared with synthetic mGnRH and ranged from 1 pg/tube to 250 pg/tube; the tracer was 125I-mGnRH. Antiserum GF-6 was raised against sGnRH but detects a number of GnRH forms [12]; GF-6 was used at a final dilution of 1:25 000. The detection level of GF-6 was 7.3 pg measured at 80% maximum binding (B/BO).
Peptide Sequencing
Four peaks of immunoreactive (ir)GnRH eluted in step 2 (Table 1) from the C-18 column. The early-eluting peak, which was the unknown peak of prime interest, was further purified as outlined in Table 1 and sequenced. The irGnRH peak that eluted in the second position was identified by comparing its elution position with the cGnRH-II standard on a separate column and by its immunoreactive profile; this peak was not purified further as we have identified and sequenced this peak from a number of other species. The irGnRH peak that eluted in the third position was purified by the same procedure shown in Table 1, but was of insufficient quantity for sequencing or identification. The 4th irGnRH peak was initially identified as sGnRH by its elution position, but was purified and sequenced because the elution position of late-eluting material is more variable.
An aliquot of the peptide purified by RP-HPLC on a diphenyl column was injected into a microbore C-18 column using 0.05% TFA and acetonitrile for elution (Table 1, step 8). Fractions were collected and analyzed with mass spectrometry as described in the next section. Sequencing was initially attempted on 10% of each sample. Failure of this sequencing indicated a blocked N terminus. Subsequent sequencing was carried out on the remaining material after treatment with pyroglutamyl aminopeptidase and microbore HPLC purification [13]. This was followed by separation and sequence analysis by automated Edman degradation on a PE-ABI Procise 494 protein sequencer (Foster City, CA).
Mass Spectroscopy
Mass spectra were measured on a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) instrument (ABI Perceptive DE-STR). Samples were mixed with a saturated solution of a-cyano-4-hydroxycinnamic acid and allowed to dry. Measurements were carried out in the delayed extraction and reflector mode with an acceleration voltage of 20 kV. Data represent averages of 100 laser shots recorded employing a 2-GHz digitizer at a 20-Hz firing rate. All measurements were externally calibrated.
Electrospray mass spectroscopy was performed on a Bruker Esquire ion-trap instrument (Framingham, MA). Samples were diluted 1:1 with 1% acetic acid in methanol and infused at a flow rate of 50 µl/h. Data are averages of 8 spectra and were recorded in the positive ion mode.
Synthesis of Whitefish GnRH Peptide
The peptides for whitefish GnRH (wfGnRH) and cGnRH-II were synthesized using a solid-phase method on a methylbenzhydrylamine resin as described [14]. Boc strategy was used with the following protecting groups: pyro-Glu(carbobenzoxy), Boc-His(tosyl), Boc-Ser(benzyl), Boc-Asn(xanthyl), and Boc-Tyr(2-bromocarbobenzoxy). The GnRHs were deprotected and cleaved from the solid support with hydrofluoric acid. After purification with reverse-phase HPLC in 2 solvent systems [15], the purity was determined by HPLC and capillary zone electrophoresis (>95%), and the composition was confirmed by mass spectral analysis.
Bioactivity of wfGnRH Peptide
Synthetic wfGnRH was tested for its effect on pituitary hormone expression in dispersed rainbow trout (Oncorhyncus mykiss) pituitary cells. Procedures were approved by the University of Victoria Animal Care Committee. The cGnRH-II was used for a comparison. The pituitary hormones for whitefish have not been isolated as proteins or cDNA. Instead, we developed an assay for the rainbow trout, a closely related salmonid, based on modification of the procedures in goldfish [16, 17]. Briefly, wfGnRH and cGnRH-II were solubilized in water and stored at -20°C. Rainbow trout growth hormone (GH) and the -subunit that serves for both gonadotropins and thyroid-stimulating hormone (GtH/TSH) were cloned in our laboratory using rainbow trout pituitary total RNA and primers based on reported sequences [18, 19]. Primers used to clone GH were 5'-AACGGCTCTTCAACATCG-3' and 5'-GACGGTCAGGTAGGTCTCG-3', forward and reverse, respectively. A nested primer strategy was used to generate the partial glycoprotein -subunit cDNA. First round products were generated by the forward 5'-CAACTGGACTATTCCTCATCC-3' and reverse 5'-GCCCATACAGACAGTTTAT-3' primers. The nested primers were forward 5‘GTCCGCACTTCTAGTCAT-3' and reverse 5'-AATAGCAGGTGCTGCAGT-3'. Rainbow trout were anesthetized in 50 mg/L Eugenol and killed by decapitation. Pituitaries were removed, washed, and treated using a combination of physical fragmentation and a trypsin/DNase treatment. Fragments were dispersed in calcium-free media (Dulbecco PBS, 25 mM Hepes, 2.2 g/L sodium bicarbonate, 0.3% bovine serum albumin, 100 000 U/L penicillin, and 100 mg/L streptomycin, pH 7.2). Cells were harvested, and cell yield and viability were determined. Cells were resuspended in culturing medium (Medium 199 with Earle salts, 25 mM Hepes, 2.2 g/L NaHCO3, 100 000 U/L penicillin, and 100 mg/L streptomycin, pH 7.2; Life Technologies, Burlington, ON, Canada) and 140 000 to 180 000 cells per well were added to a 96-well plate for 2 h at 18°C, 5% CO2, and saturated humidity. Then horse serum (Life Technologies) was added to each well to a final concentration of 1% and incubated overnight at 18°C, 5% CO2, and saturated humidity. The next morning, cells were treated with 10-9 or 10-7 M wfGnRH or 10-7 M cGnRH-II for 12 h. After the incubation, cells were harvested and total RNA was extracted.
Determination of mRNA Levels
Total RNA was extracted using TRIzol (Life Technologies) based on the guanidium thiocyanate-phenol-chloroform method of extraction [20]. The ratio of the absorbance wavelength (nm) at 260/280 for the samples ranged between 1.7 and 2.0. Subsequently, total RNA (approximately 5 µg) was loaded onto a 1.5% formaldehyde/agarose gel at 30 volts for 4–5 h. The RNA was transferred using the capillary transfer method with 0.1 N NaOH to a GeneScreen plus nylon membrane (New England Nuclear Life Science Products, Boston, MA), rinsed, and fixed on the membrane by baking for 1 h. Purified cDNA fragments for rainbow trout GH and GtH/TSH -subunit were labeled using the Random Primers DNA Labeling System (Life Technologies) with [-32P]deoxycytidine 5'-triphosphate (dCTP) 3000 Ci/mmol (New England Nuclear). A probe of 18S rRNA was made using an end-labeling kit (Amersham Pharmacia Biotech, Baie d'Urfé, QC, Canada) incorporating [
-32P]deoxycytidine 5'-triphosphate (dATP) 3000 Ci/mmol (New England Nuclear). The membrane was prehybridized for 1.5–2 h at 55°C in 30 ml of ULTRAhyb (Ambion Inc., Austin, TX). The specific probe of interest was added and left to hybridize overnight at 55°C. We used the 18S rRNA probe to control for possible variation in loading amounts. The membrane was washed following hybridization with a series of washes: 2 x 15 min with 2% SSC and 0.2% SDS; 1 x 60 min with 0.2% SSC and 0.2% SDS at 55°C. The membrane was exposed for 24–48 h in a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). Signals were analyzed with ImageQuant software (Molecular Dynamics). Between hybridizations, the membrane was stripped of probe with 2 x 10-min washes containing 0.2% SSC and 0.2% SDS. The mRNA levels were expressed with respect to the 18S rRNA signal obtained for each lane and are reported as a percentage with respect to the control (where control is 0%). Statistical analyses were performed using ANOVA followed by a Student t-test. Differences between groups were considered significant when P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the initial study with 12 brains, the HPLC-RIA of lake whitefish brains revealed 4 peaks at fractions 19–20, 27, 40, and 50–52 (Fig. 1). The first peak did not correspond to any known standard; the second peak at fraction 27 corresponded to the cGnRH-II standard; the third peak did not elute with a known standard; and the peak at fractions 50–52 eluted with the sGnRH standard.
|
In the large-scale purification, the irGnRH peaks eluted as shown in Figure 2a; the same conditions were used (Table 1, step 2) as for the small initial study. In both the initial and large-scale studies, 4 peaks eluted in the same positions. Peaks 1, 3, and 4 were separately purified as outlined in Table 1 in steps 3–8. The full purification of peak 1 is shown in Figure 2, a–f. The purification of peaks 3 and 4 followed a similar pattern. Peak 2 was identified as cGnRH-II by its elution position and cross-reactivity.
|
Sequence and Mass Spectral Analysis
Chemical sequence analysis, carried out after enzymatic removal of N-terminal pyroglutamic acid residues, yielded the following sequences:
We have designated the former novel structure as wfGnRH in accordance with the convention of naming GnRH molecules after the organism from which the primary structure is first determined. The accession numbers in SWISS PROT are P83192, for lake whitefish sGnRH, and P83193, for lake whitefish wfGnRH. Accurate mass determination by mass spectroscopy was used to confirm these sequences and to determine C-terminal amidation. Peak 1 material was analyzed by electrospray mass spectroscopy. The measured m/z = 1158.2 was in agreement with the calculated monoisotopic [M+H]+ of 1158.479 for pGlu-His-Trp-Ser-Tyr-Gly-Met-Asn-Pro-Gly-NH2. Peak 1 material (wfGnRH) was further analyzed by MALDI-MS after pyroglutamate aminopeptidase treatment. The measured m/z = 1047.43 was in excellent agreement with the calculated [M+H]+ of 1047.447 for the monoisotopic mass of H-His-Trp-Ser-Tyr-Gly-Met-Asn-Pro-Gly-NH2. The mass of the peak 4 material (sGnRH) was determined by MALDI-MS to be m/z = 1212.50. The calculated monoisotopic [M+H]+ for sGnRH is 1212.559.
Physiological Effects
Both wfGnRH and cGnRH-II peptides resulted in a significant increase of GH mRNA expression in dispersed rainbow trout pituitary cells as determined by Northern analysis by 42% and 80%, respectively (Fig. 3). The wfGnRH stimulated GtH/TSH- subunit mRNA expression 90% (a significant increase) compared with controls in dispersed rainbow trout pituitary cells. However, the 30% increase by cGnRH-II was not statistically significant.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
subunit and GH in dispersed fish pituitary cells. In addition, we identified 2 other forms of GnRH in whitefish as sGnRH and cGnRH-II; a 4th GnRH was purified by HPLC-RIA but was insufficient in amount for sequencing. Previous research detected only 2 forms of GnRH in the more recently evolved salmonids. The present evidence is the first to show that there are at least 3 forms of GnRH peptide present in the brain of a basal salmonid species, whitefish. It is possible that a tetraploid species such as whitefish has more than 3 forms of GnRH. The proof of 3 GnRH forms provides a mechanism in which the tetraploid ancestor of salmon retained all 3 forms (presumably with duplicate copies) but later-evolving salmon had chromosomal rearrangements that resulted in a loss of the third GnRH form.
Novel Structure of wfGnRH
The specialized nature and importance of GnRH in vertebrates may be inferred from the presence of the peptide in extant protochordate tunicate species that diverged as early as 600 million years ago from the ancestral line that led to vertebrates [21]. The novel wfGnRH has the basic structure that is common to most GnRH peptides isolated to date: a length of 10 amino acids and conserved amino acids in positions 1–4, 9, and 10 (Table 2). The substitutions in wfGnRH involve the part of the GnRH peptide that is believed to be important for receptor binding but not for any known functional effects. This is the first report of a methionine amino acid present in any of the GnRH forms identified to date.
|
Biological Activity of wfGnRH
We prepared synthetic wfGnRH and found that a concentration of 10-7 M stimulated a significant increase in mRNA expression of GtH/TSH- subunit and GH in dispersed rainbow trout pituitary cells. The wfGnRH had a greater effect on GtH/TSH- subunit and a similar effect on GH compared with cGnRH-II. Proof of biological activity is important because characterization of a GnRH form is determined in part by its function, mainly pituitary activity involving gonadotropic hormone (GtH) and, in fish, growth hormone (GH). Past reports show that the endogenous forms of GnRH in different fish species can release GtH and GH from pituitary cells in vitro. Each of sGnRH, cGnRH-II, hrGnRH, and pjGnRH is able to act in vitro in goldfish pituitary cells to release GtH and GH [3, 5, 32, 33]. GnRH increases the gene expression of the GtHs, FSH and LH, in mammals [34]. In fish, sGnRH elevated mRNA expression of all 3 GtH subunits (, ß1, ß2) in cultured hybrid tilapia pituitary cells [35], and sGnRH and cGnRH-II each increased mRNA expression of all 3 GtH subunits and GH in goldfish pituitary cells in vitro [17].
Origin of wfGnRH
The origin of wfGnRH could be due to substitutions in one of the GnRH genes, possibly a duplicated gene, known to exist in early teleosts before whitefish evolved (Fig. 4). Early teleost GnRH forms include mGnRH, cGnRH-II, sGnRH, and hrGnRH [3, 36, 37]. The wfGnRH differs from sGnRH and mGnRH by 2 amino acids but differs from cGnRH-II and hrGnRH by 3 amino acids (Table 2). Another clue to the origin of wfGnRH may lie in the distinct embryological origins of GnRH neurons. The cGnRH-II neuroblasts originate in the midbrain in contrast with all other GnRH neuroblasts, which are born in the olfactory placode outside the brain and then migrate into the forebrain and diencephalon (see [8]). The cGnRH-II can probably be eliminated as the source of wfGnRH because no immunoreactive wfGnRH neurons were found in the midbrain (unpublished data). Another possibility is that wfGnRH resulted from nucleotide substitutions in either herring GnRH or another ancestral GnRH, possibly mGnRH or sGnRH. The cloning of GnRH cDNAs in early teleosts and the bony fish that preceded them should give more information about the GnRH lineage.
|
Whitefish GnRH as the Missing Link in Salmonids
The present evidence of a third form of GnRH in a basal salmonid is crucial in understanding the evolution of multiple forms of GnRH within individuals. Two forms of GnRH are present in tunicates, which evolved before the vertebrates, and 2 forms are characteristic of jawless fish (lamprey), cartilaginous fish (dogfish), and early bony fish (sturgeon and others). However, early in the evolution of teleosts, 3 forms became a characteristic pattern. In the earliest teleosts (bony tongues and eels), only 2 forms are detected, mGnRH and cGnRH-II or sGnRH and cGnRH-II [36–38]. Then, at least as early as herring, 3 forms of GnRH appeared: sGnRH, cGnRH-II, and hrGnRH [3]. Thereafter, representative fish in the major orders of teleosts have 3 forms, with the exception of species that are tetraploid (salmonids, catfish, goldfish, carp), which have only 2 detectable forms of GnRH (sGnRH and cGnRH-II or catfish [cf]GnRH and cGnRH-II). The question is whether events after tetraploidization led to loss of chromosomal material or silencing of the third GnRH gene. To date, salmonid GnRH studies have been restricted to the most recently evolved subfamily, Salmoninae, and have not included the other 2 subfamilies, Coregoninae or Thymallinae (Fig. 5). The fishes of the subfamily Coregoninae are the most basal of the salmonids [39]. Therefore, we selected whitefish (Coregonus clupeaformis) as a representative of the basal Coregoninae. The presence of 3 forms of GnRH suggests that the tetraploid ancestor of salmonids inherited 2 copies of each of 3 forms of GnRH, resulting in 6 GnRH genes. Here we identify 3 distinct GnRH peptides. To date, 1 set of duplicate GnRH genes has been identified in sockeye salmon [40] and in rainbow trout [41]. The 2 cDNAs are from different genes but continue to encode the identical peptide, sGnRH. Duplicate cDNAs encoding cGnRH-II have not been identified in salmon but have been sequenced from goldfish [42]. The cDNAs for wfGnRH remain to be isolated.
|
Mechanism for Salmonid Reduction in GnRH Peptides
The diploid ancestor of salmonids is thought to have had a karyotype with 48 chromosomes that doubled to 96 with tetraploidization [39]. At present, the chromosomes of Coregoninae and Salmoninae have evolved by centric fusions, decreasing the chromosome numbers (2n) to 80 for whitefish and 57–64 for the sockeye and rainbow trout. It is not clear whether DNA has been lost or just rearranged. However, whitefish continue to express 3 functional GnRH peptides, whereas the sockeye and rainbow trout produce only 2.
The finding of a third peptide in a basal salmonid provides a missing link in our understanding of the evolution of multiple GnRH forms in vertebrates. The loss of a third GnRH form due to gene deletion or chromosomal rearrangement means that the remaining form of GnRH in the anterior brain has to compensate for the lost form, which is thought to control the pituitary. The loss may be compensated by 1 of the duplicate copies of the GnRH genes. Whereas all fish express cGnRH-II in the midbrain, late-evolving salmon express sGnRH in both the anterior brain and the preoptic region that controls the pituitary [43]. However, we do not know yet whether sGnRH in the 2 locations is the product of a single gene or the duplicate genes. In either case, the regulation of the sGnRH gene has changed compared with other teleosts that have 3 distinct GnRH peptides in the anterior, preoptic, and midbrain regions.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was funded by grants from the Canadian Institutes of Health Research, the Natural Science and Engineering Research Council, NIH (grant HD 13527 and a Shared Equipment Grant 1 S10 RR15843-01), and the Foundation for Medical Research. 
2 Correspondence. FAX: 250 721 7120; nsherwoo{at}uvic.ca
Accepted: February 6, 2002.
Received: December 10, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. Klausen, T. Tsuchiya, J. P. Chang, and H. R. Habibi PKC and ERK are differentially involved in gonadotropin-releasing hormone-induced growth hormone gene expression in the goldfish pituitary Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1625 - R1633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Klausen, D. L. Severson, J. P. Chang, and H. R. Habibi Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1634 - R1643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. D. Vickers, F. Laberge, B. A. Adams, T. J. Hara, and N. M. Sherwood Cloning and Localization of Three Forms of Gonadotropin-Releasing Hormone, Including the Novel Whitefish Form, in a Salmonid, Coregonus clupeaformis Biol Reprod, April 1, 2004; 70(4): 1136 - 1146. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Wang, O. Yun, K. Maiti, D. Y. Oh, K. K. Kim, C. H. Chae, C. J. Lee, J. Y. Seong, and H. B. Kwon Position of Pro and Ser near Glu7.32 in the Extracellular Loop 3 of Mammalian and Nonmammalian Gonadotropin-Releasing Hormone (GnRH) Receptors Is a Critical Determinant for Differential Ligand Selectivity for Mammalian GnRH and Chicken GnRH-II Mol. Endocrinol., January 1, 2004; 18(1): 105 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Adams, J. A. Tello, J. Erchegyi, C. Warby, D. J. Hong, K. O. Akinsanya, G. O. Mackie, W. Vale, J. E. Rivier, and N. M. Sherwood Six Novel Gonadotropin-Releasing Hormones Are Encoded as Triplets on Each of Two Genes in the Protochordate, Ciona intestinalis Endocrinology, May 1, 2003; 144(5): 1907 - 1919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Terasawa Gonadotropin-Releasing Hormone II: Is this Neuropeptide Important for Mammalian Reproduction? Endocrinology, January 1, 2003; 144(1): 3 - 4. [Full Text] [PDF]
|
|
|
|
|
|
S. L. Gray, B. A. Adams, C. M. Warby, K. R. von Schalburg, and N. M. Sherwood Transcription and Translation of the Salmon Gonadotropin-Releasing Hormone Genes in Brain and Gonads of Sexually Maturing Rainbow Trout (Oncorhynchus mykiss) Biol Reprod, November 1, 2002; 67(5): 1621 - 1627. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
