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BOR - Papers in Press, published online ahead of print December 10, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.023846
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BIOLOGY OF REPRODUCTION 70, 1136–1146 (2004)
DOI: 10.1095/biolreprod.103.023846
© 2004 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Cloning and Localization of Three Forms of Gonadotropin-Releasing Hormone, Including the Novel Whitefish Form, in a Salmonid, Coregonus clupeaformis1

Elaine D. Vickers3, Frederic Laberge4, Bruce A. Adams3, Toshiaki J. Hara4,5, and Nancy M. Sherwood2,3

Department of Biology,3 University of Victoria, Victoria, British Columbia, Canada V8W 3N5 Department of Zoology,4 University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 Freshwater Institute,5 Fisheries and Oceans Canada, Winnipeg, Manitoba, Canada R3T 2N6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells containing different GnRH peptides currently are thought to have distinct locations and functions in the brain. Lake whitefish is the first salmonid species to have three forms of GnRH peptide in contrast to later-evolving salmonids (salmon and trout) in which only two forms have been identified. Our objective was to isolate the cDNAs that code for these transcripts and to localize the transcripts for the three forms of GnRH in adult lake whitefish brain. Also, we provide phylogenetic analysis of these three whitefish genes based on their preprohormone sequence. From whitefish we isolated cDNAs encoding chicken (c)GnRH-II, salmon (s)GnRH, and the novel whitefish (wf)GnRH. The three cDNAs each encode only one GnRH and are placed in separate groups with phylogenetic analysis. A combination of in situ hybridization and immunocytochemistry with two antisera revealed neurons that expressed protein and/or mRNA for cGnRH-II in the midbrain and hindbrain; sGnRH in the olfactory nerve and bulb, ventral telencephalon, and preoptic area; and wfGnRH in the same latter two brain regions and the hypothalamus. Thus, in the anterior brain, cells containing sGnRH and wfGnRH were in the same brain areas but not at identical locations in the ventral telencephalon and preoptic area. Based on our results, we speculate that both sGnRH and wfGnRH have gonadotropin-releasing roles in the lake whitefish brain.

central nervous system, gonadotropin-releasing hormone, neuroendocrinology, neuropeptides


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GnRH is synthesized in nerve cells in the brain and acts on the anterior pituitary, causing the synthesis and release of gonadotropins (LH and FSH) into the blood. In turn, the gonadotropins act on the gonads to stimulate gametogenesis and steroid synthesis. Additionally, GnRH may act as a neuromodulator [1–4], pheromone [5], and regulator of reproductive behavior [6–8]. In fish, GnRH can stimulate the release of other pituitary hormones, including growth hormone [9], prolactin [10], and possibly somatolactin [11].

To date, 14 distinct GnRH peptides have been identified in vertebrates and named after the species from which they were first isolated [12]. In salmonids [13], which includes whitefish, salmon, and trout (Fig. 1), GnRH peptides have been identified in nine species by primary structure, cDNA, or gene sequence [12, 14–22]. In later-evolving salmonids (salmon and trout), only two forms of GnRH have been identified: salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II; Table 1). Based on HPLC-RIA studies using antisera GF-4 or GF-6, which detect whitefish and several other GnRH forms, none of the seven salmon or trout species that were tested contained a third form of GnRH [23, 24]. However a third form may be present, but was not detected with these antisera. In contrast, we identified three forms of GnRH in an ancient salmonid, lake whitefish. Two of the three GnRH forms were identical to those of salmon and trout, whereas a third form, whitefish GnRH (wfGnRH), was novel [12]. This was the first report of a third form of GnRH in a salmonid fish.



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  		FIG. 1. A phylogenetic diagram showing the evolutionary relationship of whitefish to other salmonids in the order Salmoniformes. A complete duplication of the genome is thought to have occurred at the beginning of the Salmoniformes order with subsequent loss, rearrangement, or mutations in many duplicate genes or chromosomes. Whitefish are the most ancient lineage, followed by grayling and then the trout and salmon species. To date, two forms of GnRH have been identified in the trout or salmon listed. GnRH has not been reported for grayling. Whitefish have retained three distinct forms of GnRH


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  		TABLE 1. Gonadotropin-releasing hormones

A third GnRH gene in other nonsalmonid fish species is found to be expressed in the brain areas associated with control of gonadotropins from the pituitary. In contrast, sGnRH neurons in teleosts are usually associated with the olfactory-terminal nerve region, and cGnRH-II neurons are present in the midbrain. The third form of GnRH is usually in the neurons of the ventral telencephalon and preoptic region. One theory suggests that in a teleost brain, each form of GnRH is in a separate population of neurons with a distinct location and function [25–29]. In salmonids, we are interested in considering the distribution of GnRH neurons when three forms are present. A number of possibilities for the expression of a third form include unique localization of the cells containing the third form in separate brain areas compared with other GnRH cells, partial localization in the same areas with neurons containing another GnRH form, or localization in the same areas with cells of another GnRH form. Another possibility is that a third form of GnRH in salmon and trout is undetected. Also, salmon and trout are not alone in having only two forms of GnRH in the brain. The other fish species with two GnRH forms are like salmonids in that a complete duplication of the genome occurred (tetraploidy); these species include catfish [30, 31], goldfish [32, 33], roach [34], and zebrafish [35–37].

In the present study we first determined the cDNA structure of each form of GnRH in lake whitefish, including the cDNA for wfGnRH; this is the first time a third cDNA has been isolated from a tetraploid species. Second, we localized cells with GnRH cRNA by in situ hybridization and compared these data to cells with protein localization using immunocytochemistry. Finally, phylogenetic analysis is used to understand the relationship of the three whitefish cDNAs to those from other vertebrates.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The University of Victoria Animal Care Committee approved all animal procedures in this study. Lake whitefish (Coregonus clupeaformis) were obtained from two sources. Twelve fish (six males, six females) were netted from the Freshwater Research Institute, Winnipeg, Manitoba. Eleven wild lake whitefish were collected by gill netting at Exeter Lake in 100 Mile House, British Columbia, Canada. Fish were deeply anesthetized with MS-222 (0.1 mg/ml) and killed. For isolation of mRNA, brains were dissected with pituitaries attached, if possible, and frozen in liquid nitrogen or stored in RNAlater (Ambion Inc., Austin, TX). All brains were transported to the University of Victoria where the tissues were frozen at -80°C.

RNA Isolation

Brain tissue was ground to a powder in liquid nitrogen using a cold mortar and pestle. Total RNA was isolated using TRIzol RNA isolation reagent (Invitrogen, Burlington, ON, Canada), based on an acid guanidinium-thiocyanate extraction method [38]. The mRNA was extracted from total RNA using the Micro Poly(A)+ Pure kit (Ambion) as per manufacturer's directions. The concentration of mRNA was quantified by a spot test on an ethidium bromide agar plate. The mRNA was stored at -80°C.

GnRH cDNA Synthesis

For sGnRH and cGnRH-II cDNA isolation, both 5' and 3' rapid amplification of cDNA ends (RACE) were done using the SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA) by following the manufacturer's instructions. For wfGnRH cDNA isolation, 5' and 3' RACE products were made using the First Choice RNA Ligase Mediated (RLM)-RACE kit (Ambion) as per the manufacturer's directions. The 3'RACE was performed using two rounds of polymerase chain reaction (PCR). The first round used forward (F) primers for salmon (s)F1, chicken (c)F1, or whitefish (w)F1 (Table 2) with reagents provided in the kit, including first-round anchor primer, and DNA Taq Polymerase (Invitrogen). The second round used 1 µl of first-round PCR product and forward primers sF2, cF2, or wF2 with the nested adapter primer. Also, 5'RACE reactions involved two rounds of PCR. The first round used reverse (R) primers sR1, cR1, or wR1 with the appropriate first-round primer, and the second round used 1 µl of first-round product and the reverse primers sR2, cR2, or wR2 with the appropriate nested adapter primer. All cDNAs were amplified at annealing temperatures indicated in Table 2 for 35 cycles with a final 7-minute extension at 72°C. All second-round PCR products were separated on a 1.5% agarose gel using electrophoresis and visualized by ethidium bromide stain on the Eagle-Eye II still video system (Stratagene, La Jolla, CA).


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  		TABLE 2. 5' and 3' PCR primer sequences for amplifying sGnRH, wfGnRH, and cGnRH-II from lake whitefish

PCR products that were considered to be candidate GnRH cDNAs (200–400 base pairs [bp]) were ligated into the pGEM T-Vector plasmid (Promega, Madison, WI). Plasmid DNA was incorporated into E. coli XL-1 blue competent cells (Stratagene, Cedar Creek, TX) by electrotransformation. Colonies were grown on plates treated with 100 mg/ml ampicillin, 0.1 M isopropyl-beta-D-thiogalactopyranoside, and 20 mg/ml X-Gal. White colonies, assumed to be recombinant colonies, were picked and grown overnight in 3 ml LB broth supplemented with 100 mg/ml ampicillin. All plasmid DNA was isolated using the QIAprep Spin Miniprep kit (Qiagen, Mississauga, ON, Canada) following the manufacturer's instructions. A 3-µl sample of DNA was digested using the restriction enzymes PstI and SphI (New England Biolabs, Mississauga, ON, Canada), followed by separation on a 1.5% agarose gel. Gels were stained in ethidium bromide, and plasmids containing candidate GnRH cDNA inserts were sequenced in both directions.

Phylogenetic Analysis

Gene and cDNA sequences encoding 71 different GnRH precursors were obtained from Genbank (http://www.ncbi.nlm.nih.gov) and the literature. The GnRH precursors (signal peptide, GnRH, processing site, and GnRH-associated peptide [GAP]) were entered into a common file using BioEdit Sequence Alignment Editor version 5.0.9 [39]. Phylogenetic analyses were carried out with Clustal X using the Neighbor-Joining method. The data were resampled by 1000 bootstrap replicates, and the proportion of Neighbor-Joining trees possessing each particular internal branch was indicated to express its level of support. Phylogenetic trees were generated using the TreeView software package, version 1.5.2 for Microsoft Windows 32 bit (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) and formatted using Microsoft Word (Microsoft Corporation, Redmond, WA). Full species names and GenBank accession numbers for the sequences of the GnRH precursors used are listed in the legend of the tree. Only sequences that included a full coding DNA sequence were used in analyses.

In Situ Hybridization Probe Preparation

Probes were prepared for in situ hybridization using the following sets of primers (Table 2): cF3 and cR3 amplified a 256 bp product within the 5' untranslated region (UTR) of the cGnRH-II cDNA; sF3 and sR3 amplified a 253 bp product within the GAP and 3'UTR regions of the sGnRH cDNA; and wF3 and wR3 amplified a 269 bp product that amplified all of GAP and part of the 3'UTR of the wfGnRH cDNA. PCR was carried out under the following conditions: 3-min denaturation at 94°C followed by 35 cycles at 94°C, 55°C, and 72°C for 30 sec each and a final extension round of 7 min at 72°C. Amplified DNA was then separated by electrophoresis gel, ligated, digested, and sequenced in both directions as above. Probe DNA (20 µg) was linearized with PstI or SphI restriction enzymes to make antisense and sense strands, respectively. Linear DNA was transcribed and labeled using a dioxigenin (DIG) kit (Roche Diagnostics, Laval, PQ, Canada) following the manufacture's instructions. Probe concentration was determined using a spot assay, where serial dilutions of each probe were compared with an RNA standard. In the assay, spots were baked on a nylon membrane; washed (tris-buffered saline [TBS], 0.2% Tween-20); and incubated in blocking buffer (TBS, 0.2% Tween-20, 10% serum) with antibody (anti-DIG-alkaline phosphatase 1:5000). The membrane was washed again before an overnight incubation of color reactions (nitro-blue tetrazolium; 75 mg/ml in 70% dimethylformamide and X-phosphate 4-toluidine salt; 50 mg/ml in 100% dimethylformamide) in detection buffer (100 mM Tris-HCl pH 9.5, 100 mM NaCl). Based on the spot assay, 100 ng of probe/200 µl hybridization buffer was used for in situ hybridization.

Tissue Preparation for In Situ Hybridization

For mRNA expression studies, fish were anesthetized and perfused intracardially with phosphate-buffered fish saline (PBFS; 0.1 M phosphate buffer; 0.725% NaCl; pH 7.4), followed by 4% paraformaldehyde in PBFS. Brains and pituitaries were removed and stored in a 30% sucrose solution. The brains were cryosectioned sagittally into 20-µm sections, collected on microscope slides, and stored at -80°C.

In Situ Hybridization

Slides were removed from -80°C and dried 3 h at 37°C. Sections were postfixed in 4% buffered paraformaldehyde. Slides were washed in 1x PBS before treatment with 0.1 µg/ml proteinase K in Tris-EDTA (TE) buffer (100 mM Tris-HCl, 50 mM EDTA, pH 8.0). Next, slides were washed in 0.1 M TE-acetic anhydride (TEA) buffer, pH 8.0, with acetic anhydride (2.5 µl/ml buffer). Sections were washed twice in 2x SSC (sodium chloride, sodium citrate); dehydrated in graded ethanol (50%, 60%, 70%, 80%, and 90%), and air-dried. Sections were prehybridized in hybridization buffer (50% formamide, 5x SSC, 50 µg/ml heparin, 500 µg/ml tRNA, 0.1% Tween-20, and 0.1 M citric acid brought up to 50 ml in sterile water) before each probe was added. Sections were hybridized (100 ng probe/200 µl buffer) at 48°C overnight. Slides were washed in 2x SSC before being treated with RNase A (40 µg/ml in TBS) at 37°C. Slides underwent a series of washes in decreasing concentrations of SSC (2x SSC, 1x SSC, 0.5x SSC, and 0.1x SSC) and two washes in TBS. Blocking buffer (TBS, 0.1% Tween-20, 10% rabbit serum) was added to sections on the slides. Antibody (anti-DIG-alkaline phosphatase) was added to fresh blocking buffer (1:5000), placed over sections with cover slips, and incubated at room temperature for 2 h. Slides were washed twice in TBS before incubating in coloration buffer (100 mM Tris-HCl pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20, brought up to 50 ml in sterile water). The color reaction was continued overnight as in the spot assay and stopped by washing slides in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Sections were dehydrated in graded ethanol as before, air-dried, and cover-slipped with an aqueous mounting medium (8 g gelatin, 60% water, 40% glycerin, and 0.1 g phenol as a preservative).

Immunocytochemistry

The antisera GF-6 and 7CR-10 were raised in rabbits in our laboratory against sGnRH or dogfish GnRH, respectively. Cross-reactivity studies showed that GF-6 strongly cross-reacted with wfGnRH and sGnRH, whereas 7CR-10 cross-reacted only with cGnRH-II and sGnRH (Table 1). Cross-reactivity was determined as described earlier [40].

Four female and seven male fish were deeply anesthetized with MS-222, then perfused through the heart with 30 ml of PBFS followed by 250 ml of 4% paraformaldehyde in PBFS. The brain was removed from the skull, left in the fixative solution overnight, and then cryoprotected in 30% sucrose PBFS for a day. Sagittal sections 40-µm thick were cut on a cryostat (American Optical Corp., Southbridge, MA). Consecutive sections were distributed among the different reactions (antibody or antibody plus blocking peptide). Immunocytochemistry was performed on free-floating sections.

The sections were incubated in PBFS containing 4% normal donkey serum, 0.4% TRITON-X, and 1% BSA for 20-min (blocking step), followed by incubation with the first antiserum (rabbit anti-GnRH) diluted 1:2500 or 1:5000 in PBFS with 0.4% TRITON-X, 1% normal serum, and 1% BSA at 4°C overnight. The next day the sections were rinsed 10 times for 10 min in PBFS with 0.02% TRITON-X and 0.25% BSA, followed by incubation with the secondary antibody (biotinylated donkey anti-rabbit) diluted 1:1000 in PBFS with 0.02% TRITON-X and 1% BSA for 60 min. The antigen of the secondary antibody was whole rabbit immunoglobulin G. The sections were rinsed again two times for 15 min in PBFS with 0.25% BSA, followed by incubation with avidin-biotin: peroxidase with 1% BSA and 0.4% TRITON-X for 120 min. After three 10-min rinses in PBFS, the Chromagen solution (100 mM NiSO4, 125 mM acetate, 10 mM imidazole, 0.03% diaminobenzidine, 0.003% H2O2) was applied to the sections for 2–15 min. The sections were finally mounted on gelatinized glass slides, dried, dehydrated in graded alcohols, and cleared in toluene. Coverslips were applied. The secondary antibodies and normal serum were from Jackson ImmunoResearch Laboratories (West Grove, PA), and the ABC kit was from Vector Laboratories (Burlingame, CA). Additional chemical supplies were from Sigma Chemical Co. (St. Louis, MO).

Controls were performed by adding 100 µg of wfGnRH or sGnRH peptide to the primary antisera solutions 30 min prior to application to the sections.

Statistical Analysis

A two-way ANOVA with unweighted means was used to determine if the number of neurons labeled by the two antisera was different in the four brain regions that showed labeling with both antisera.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Complementary DNA Sequences

We isolated three cDNA sequences that encode sGnRH, wfGnRH, and cGnRH-II separately from lake whitefish by overlapping the 5' and 3' ends. The lake whitefish sGnRH cDNA is 491 bp in length and consists of a 44-bp 5'UTR, a 249-bp open reading frame, a stop codon (TAA), and a 195-bp 3'UTR (Fig. 2). The cDNA for wfGnRH is 511 bp long, which includes a 67-bp 5'UTR; a 279-bp open reading frame (encoding the signal peptide, wfGnRH, cut site, and GAP); a stop codon (TAA); and a 162-bp 3'UTR (Fig. 3). The lake whitefish cGnRH-II cDNA is 636 bp long, which includes a 202-bp 5'UTR; a 258-bp open reading frame (encoding the signal peptide, cGnRH-II, cut site, and GAP); a stop codon (TAA); and a 173-bp 3'UTR (Fig. 4).



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  		FIG. 2. Lake whitefish sGnRH cDNA. The nucleotide and derived amino acid sequence that encodes the sGnRH precursor of lake whitefish is shown. Nucleotides are numbered 5' to 3' and amino acids N-terminal to C-terminal. Signal peptide, cut site, and GnRH-associated peptide are all underlined. Salmon GnRH and the nucleotides corresponding to the polyadenylation signal (aataaa) are in bold. The dash (-) indicates the stop codon



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  		FIG. 3. Lake whitefish wfGnRH cDNA and derived amino acids. Signal peptide, cut site, and GnRH-associated peptide are underlined. Whitefish GnRH and the nucleotides corresponding to the polyadenylation signal (aataaa) are in bold. The dash (-) indicates the stop codon



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  		FIG. 4. Lake whitefish cGnRH-II cDNA and derived amino acids. Signal peptide, cut site, and GnRH-associated peptide are underlined. Chicken GnRH-II and the nucleotides corresponding to the polyadenylation signal (aataa) are in bold. The dash (-) indicates the stop codon

Phylogenetic Relationships

An unrooted phylogenetic tree based on the amino acids of the GnRH precursors revealed three major groupings of vertebrate GnRH (GnRH I, GnRH II, and GnRH III; Fig. 5). The lake whitefish wfGnRH precursor fits with the GnRH I group; whitefish cGnRH-II and sGnRH each formed a group, GnRH II and GnRH III, with the same molecules from other species.



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  		FIG. 5. Phylogenetic relationship of precursors derived from known DNA sequences encoding gonadotropin-releasing hormone (GnRH). A) The relationship was generated with CLUSTAL W, and the unrooted tree was generated using Treeview version 1.5.2. The scale bar represents the estimated evolutionary distance as 0.1 amino acid substitutions per site. In alphabetical order the precursors, species, and accession numbers are listed (B).



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  		FIG. 5. Continued.

GnRH I includes the precursors of 10 vertebrate GnRH forms that have been sequenced and are expressed mainly in the preoptic-hypothalamic areas, the main location for neurons that control reproduction. However, within GnRH I, fish precursors group together, whereas tetrapod GnRH I precursors form a separate group of two main branches: one branch includes three amphibian precursors and the second includes precursors found in birds and mammals. The precursors for a specific form, for example mammalian (m)GnRH, do not group based on the GnRH peptide produced, but group instead with precursors from organisms that are more closely related phylogenetically. For example, the mGnRH precursor from Japanese eel is more closely related to other fish GnRH precursors that code for different forms of GnRH than it is to mGnRH-encoding precursors from tetrapods. The second group, GnRH II, is composed solely of cGnRH-II-encoding precursors. These precursors also group according to our current understanding of phylogenetic relationships for these species. The third group, GnRH III, is composed solely of precursors encoding sGnRH, which are expressed in neurons in the olfactory and other regions. The GnRH III precursors are the most different from those in GnRH I and GnRH II.

In Situ Hybridization

In situ hybridization was done with three different probes, each recognizing one of the three GnRH forms in lake whitefish. Antisense riboprobes made against the wfGnRH in the GAP-3'UTR region hybridized to cells in the ventral telencephalon, preoptic area, and hypothalamus (Fig. 6A). There were also a few cells stained in the olfactory bulb, but none in the midbrain or hindbrain. Clusters of cells appeared darkly stained. There was some background staining in all brain regions with the sense probe, but no staining with the negative control.



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  		FIG. 6. In situ hybridization showing GnRH location. The brain location is shown for each GnRH form found in lake whitefish. A) Whitefish GnRH–labeled cells in the hypothalamus. B) Salmon GnRH–labeled cells located in the preoptic area. C) Chicken GnRH-II–labeled cells found in the midbrain. Scale bars represent 50 µm in each picture.  FIG. 7. GnRH-positive neuron populations found in the lake whitefish brain. A drawing of a complete medial parasagittal section of the lake whitefish brain is shown to help localize the micrographs (rostral to the right and dorsal on top). A) olfactory nerve–olfactory bulb junction (antiserum GF-6); (B) olfactory bulb–telencephalon transition region (GF-6); (C) ventral telencephalon (antiserum 7CR-10); (D) preoptic area (GF-6); and (E) midbrain tegmentum (7CR-10). Note that the micrographs shown in (B) and (E) are at a level more medial than that illustrated by the drawing. Scale bars are 100 µm

Antisense riboprobes made against sGnRH in the GAP-3'UTR region hybridized to cells within the olfactory bulb, ventral telencephalon, and preoptic area (Fig. 6B). Stained cells were roundish and occasionally clustered. There was no staining in the midbrain or hindbrain. In controls, there was little or no staining with the sGnRH sense probe and no staining with the negative control, which lacked a probe.

Antisense riboprobes made against cGnRH-II in the 5'UTR region detected clustered cells in the midbrain and hindbrain (Fig. 6C). There was no cGnRH-II staining in the forebrain or hypothalamus. Also, there was no staining with either the cGnRH-II sense probe or the negative control.

The wfGnRH probe was tested against the three GnRH (wfGnRH, sGnRH, cGnRH-II) clones and shown to hybridize only to the wfGnRH clone. In addition, the nucleotide sequences of the three probes were compared by a pairwise blast in BlastN 2.2.4 (NCBI) using default parameters, and no significant similarity was found.

Immunocytochemistry

Immunocytochemistry revealed the existence of neurons positive for GnRH in several regions of the lake whitefish brain. Figure 7 shows representative examples of GnRH-positive neurons in the olfactory nerve, olfactory bulb, ventral telencephalon, preoptic area, and midbrain. Both 7CR-10 and GF-6 antisera labeled a similar number of neurons in anterior brain regions (P = 0.608) except the midbrain, where only 7CR-10 labeled neurons (Table 3). In the forebrain, 7CR-10 antiserum shows the distribution of sGnRH neurons, as it does not cross-react with wfGnRH. The neurons labeled with 7CR-10 were present from the olfactory nerve-bulb junction to the preoptic area with the highest number of neurons in the ventral telencephalon (Table 3).


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  		TABLE 3. Number of labeled neurons in different brain regions in lake whitefish.*

The GnRH neurons of lake whitefish were of three types: 1) large, darkly stained neurons with extensive arborization in the ventral telencephalon and preoptic area; 2) small fusiform neurons found around the olfactory bulb and sometimes in the preoptic area; and 3) large, lightly stained neurons with a limited arborization in the midbrain. Some GnRH-positive neurons in the ventral telencephalon appeared to be in contact with each other as shown in the inset in Figure 7C.

The distribution of labeled fibers was similar with both antisera. The fibers were widely distributed throughout the brain, although few were in the cerebellum and spinal cord. However, fibers stained with GF-6 were less abundant around the midbrain. Few pituitaries were attached to the brain in the present study, preventing the investigation of GnRH neuron projections to the pituitary.

The addition of sGnRH peptide to the 7CR-10 primary antibody solution abolished all cell labeling, whereas the addition of wfGnRH peptide had no effect. This confirms the specificity of sGnRH for antiserum 7CR-10. The addition of wfGnRH peptide to the GF-6 primary antibody solution abolished all cell labeling, as expected.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
GnRH was originally thought to be present in the brain as a single form that controlled both FSH and LH. It is now clear that most, if not all, vertebrates have at least two forms of GnRH in the brain, and most advanced bony fish (teleosts) have three forms of GnRH. However, only the GnRH that is primarily in neurons of the preoptic area of the brain (GnRH I group; Fig. 5) regulates the synthesis and release of LH and FSH. Neither the function of the GnRH in the midbrain neurons (GnRH II) nor the GnRH in cells of the terminal nerve or the olfactory bulb (GnRH III) is clearly known, but the location of their axons suggests that very little GnRH is delivered to the pituitary [4, 42]. The highly conserved structure of the GnRH family of peptides (i.e., human, sturgeon, and eel have identical forms of both GnRH I and GnRH II) means that many forms of GnRH are effective in releasing pituitary hormones, but their location in the brain prevents them from reaching the pituitary.

The presence of a third form of GnRH is restricted to teleost fish, but its distribution within fish is puzzling. In orders of fish that are thought to have had a complete duplication of the genome, including orders that contain salmon, trout, zebrafish, goldfish, or catfish, there are only two forms of GnRH detected in the brain. One hypothesis is that one form of GnRH is no longer expressed or is lost due to rearrangements within the chromosome or deletions/mutations within the gene or its promoter after the duplication of the genome [43]. The other hypothesis is that the third form has not yet been detected. Our evidence is the first to show that at least one species of fish with a doubled genome still retains three forms of GnRH, each encoded by a separate gene. Our localization of both GnRH I and GnRH III in the forebrain neurons or in neurons near the preoptic area suggests that other tetraploid fish could have an undetected form of GnRH, or if GnRH I is lost, they could use GnRH III instead to regulate FSH and LH, as is the case for zebrafish, goldfish, and several tetraploid species.

We determined the sequence of three full-length cDNAs from the brains of lake whitefish. All three cDNAs have a similar structural organization to other vertebrate GnRH cDNA sequences in vertebrates. Each sequence consists of 5'UTR, signal peptide, GnRH, GnRH-associated peptide, and 3'UTR; the region that is most conserved encodes the GnRH peptide with its 3' cut site. Our data support the idea that each GnRH form is encoded by a different gene in vertebrates.

The peptide structure deduced from the cDNAs for the three forms of GnRH in the whitefish matched our earlier results from peptide isolation and showed that the GnRH II form (cGnRH-II) and the GnRH III form (sGnRH) are identical to those in other vertebrates [12]. The cGnRH-II form has been identified from sharks through humans in all jawed vertebrates. The sGnRH form is present in almost all teleost fish. In contrast, the GnRH I form (wfGnRH) is a novel form and a new member of the GnRH peptide family. Comparing the whitefish and other GnRH peptides over 10 amino acid differences limits any deductions about their evolutionary relationship.

The full-length cDNA sequences for all three GnRH forms found in lake whitefish are useful for understanding the evolution of the peptide by phylogenetic analysis. Grober et al. [44] first constructed a phylogenetic tree based on 18 prepro-GnRH cDNA sequences from vertebrate species. Later, other trees were constructed based on prepro-GnRH amino acid sequences [26, 45]. The phylogenetic analyses from these earlier studies showed three major precursor groups: group one (GnRH I), which contained forms of GnRH that were located in neurons of the preoptic region; group two (GnRH II), which were mainly in neurons of the midbrain; and group three (GnRH III), which were located in neurons of the olfactory region and ventral telencephalon. The new tree that we have generated is based on 71 vertebrate GnRH precursors. Lake whitefish wfGnRH fits with the GnRH I group that includes 10 vertebrate GnRH forms (Table 1, Fig. 5). The phylogenetic analysis of the precursors in this group suggests they shared a common ancestral gene that underwent changes, including mutations in the GnRH-coding part of the gene. In analysis of whitefish cGnRH-II and sGnRH precursors, each grouped with the same molecules from other species. The precursors for the GnRH II group all share the same peptide structure (cGnRH-II), which suggests a common ancestral gene, one common to both the fish and tetrapod lineages, and also a gene that has been tightly conserved. Also, neurons containing cGnRH-II occupy a unique location in the brain compared with neurons producing other GnRH forms, suggesting a specialized and conserved function for this gene. The presence of group III (sGnRH) only in fish species suggests that this GnRH form may be derived from gene duplication early in the bony fish lineage. In teleosts, the sGnRH coding region has resisted mutation pressure.

In Situ Hybridization and Immunocytochemistry

Localization of GnRH in brain neurons is important in understanding function. Many forms of GnRH can activate release of LH and FSH if injected into the animal. However, in the brain only GnRH in preoptic neurons is delivered to the pituitary in substantial amounts. GnRH cell bodies at other locations have axons that synapse within the brain and do not terminate in the pituitary (fish) [4, 42].

Very few antisera are specific for only one form of GnRH because the peptide is only 10 amino acids and varies almost exclusively in positions 5–9. Therefore, we used two antisera, one of which (GF-6) cross-reacts with both the whitefish and salmon GnRH forms; the other antiserum (7CR-10) reacts with the salmon and chicken-II forms. If cGnRH-II is present, then only 7CR-10 would detect the peptide in cells. If wfGnRH is present, only GF-6 would detect the cells. If sGnRH is present, both antisera would detect the peptide in cells. We showed unequivocally that cGnRH-II was only in the midbrain and hindbrain neurons. This is in agreement with the literature, as the cGnRH-II form is restricted to cells in the posterior brain in jawed vertebrates [1, 4, 11, 40]. The only exception is goldfish where the chicken-II form is also expressed in some neurons in the forebrain [33, 46], although in situ hybridization has not been done. Our evidence suggests that only sGnRH is in neurons in the far anterior brain (terminal nerve region) because there was no increase in the number of GnRH neurons detected by the antiserum that cross-reacts with both wfGnRH and sGnRH compared with the antiserum that only cross-reacts with sGnRH. However, in situ hybridization, which used different probes that are each specific to only one form of the three GnRHs, showed that cells expressing sGnRH or wfGnRH are both in the forebrain area. Recent studies of the European sea bass (Dicentrarchus labrax) suggest that the distribution of cells expressing salmon or seabream GnRH are both in the olfactory bulb, ventral telencephalon, and preoptic area [47, 48]. The situation is similar in lake whitefish where both sGnRH- and wfGnRH-containing neurons are present in or near the olfactory bulb, in the ventral telencephalon, and in the preoptic area. Although we did not have sections with attached pituitaries to show termination of sGnRH-containing axons, there was a heavy abundance of sGnRH fibers (antibody 7CR-10) in the basal hypothalamic region.

It is known that sGnRH can trigger ovulation or release gonadotropins if delivered to the pituitary by a systemic injection or by application to cultured pituitary cells [14, 49–53]. Adams et al. [12] reported that wfGnRH caused a significant increase in gonadotropin mRNA expression from dispersed rainbow trout pituitary cells. Based on our results, it is possible that both sGnRH and wfGnRH have gonadotropin-releasing roles in lake whitefish brain.


   ACKNOWLEDGMENTS
 
We thank Wayne Gray, Jack Leggett, Darin Sollit, and Don Savard for assistance in obtaining the lake whitefish and gaining access to Exeter Lake; Carol Warby for testing the cross-reactivity of GF-6 and 7CR-10 with wfGnRH; Drs. Sandra Krueckl and Michael Cox for assistance and use of their microscope for in situ imaging; and Dr. Jean Rivier for peptides used to test preabsorption of the antisera.


   FOOTNOTES
 
1 Supported by the Canadian Institutes of Health Research grant (N.M.S.) and fellowship (B.A.A.); Natural Sciences and Engineering Research Council of Canada grant OGP0007576 (T.J.H.); Fonds pour la formation de Chercheurs et l'Aide à la Recherche (Québec); and the University of Manitoba Graduate Fellowship (F.L.). Back

2 Correspondence: Nancy M. Sherwood, Department of Biology, 3800 Finnerty Road, University of Victoria, Victoria, BC, Canada, V8W 3N5. FAX: 250 721 7120; nsherwoo{at}uvic.ca Back

Received: 1 October 2003.

First decision: 15 October 2003.

Accepted: 25 November 2003.


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 MATERIALS AND METHODS
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 DISCUSSION
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