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Endocrinology Vol. 139, No. 4 2015-2024
Copyright © 1998 by The Endocrine Society

ARTICLES

Presence of Salmon Gonadotropin-Releasing Hormone (GnRH) and Compounds with GnRH-Like Activity in the Ovary of Goldfish1

Debananda Pati2 and Hamid R. Habibi

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4

Address all correspondence and requests for reprints to: Dr. H. R. Habibi, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4. E-mail: habibi{at}acs.ucalgary.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The presence of ovarian GnRH and/or compounds with GnRH-like activity was investigated in the goldfish ovary. Goldfish ovary was extracted using an acetone/HCl/ether mixture and was purified by Waters C-18 Sep-Pak columns (ovarian extract). The goldfish ovarian extract was found to 1) stimulate gonadotropin release and subunit messenger RNA production in the goldfish pituitary that was inhibited by a GnRH antagonist; 2) stimulate germinal vesicle breakdown in the prophase-I arrested follicle-enclosed goldfish oocytes in vitro, which was inhibited by a GnRH antagonist; 3) immunoreact with various GnRH antisera; and 4) bind to GnRH receptors in the goldfish pituitary and ovary as well as rat pituitary. Further purification with HPLC revealed the presence of two compounds with GnRH-like activity. One with identical chromatographic characteristics, amino acid composition, and primary structure to that of the salmon GnRH (sGnRH), and the other a novel compound with GnRH-like activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS THE primary stimulator of gonadotropin release and synthesis in vertebrates. To date, the primary structures of eleven GnRH variants have been elucidated, and two or more molecular forms have been found to be expressed in species from all vertebrate classes (1, 2), including placental mammals (3, 4). The goldfish brain contains two forms of GnRH: [Trp7, Leu8]-GnRH (salmon GnRH; sGnRH) and [His5, Trp7, Tyr8]-GnRH (chicken GnRH-II; cGnRH-II) (5, 6). Functional diversity of GnRH is well documented in fish and other vertebrates, and there is evidence for the presence of both GnRH and GnRH receptors in various peripheral tissues. Studies in a number of species have provided evidence for a paracrine/autocrine regulatory role of ovarian GnRH or compounds with GnRH-like activity (7, 8, 9). The possibility of intraovarian production of GnRH and/or compounds with GnRH-like activity has been reinforced by demonstration of ovarian compounds with GnRH-like activity (10, 11, 12) and ovarian GnRH gene expression (13, 14, 15, 16). A recent study demonstrated the expression of both sGnRH and cGnRH-II precursor messenger RNA (mRNA) in the brain and ovary of goldfish (6). However, to date, GnRH peptides have not been biochemically isolated and sequenced from the vertebrate ovary. Other studies in fish also indicated the presence of ovarian compounds with GnRH-like activity, but no information is available on their structural identity (17, 18, 19). Other evidence in goldfish supporting a paracrine/autocrine role for GnRH peptides in ovary include those demonstrating the presence of ovarian GnRH receptors (20) and effects of GnRH peptides on reinitiation of oocyte meiosis and follicular steroidogenesis in the goldfish ovary (9, 21, 22).

In this study, we determined the primary structure of a GnRH peptide with identical amino acid sequence to that of sGnRH and demonstrate the presence of a novel compound with GnRH-like activity in the goldfish ovary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female goldfish, Carassius auratus, common or comet varieties (ranging from 8–10 cm in length) were purchased from Aquatic Imports (Calgary, Alberta, Canada). Fish were maintained in a 1500-liter semirecirculating aquarium (60% replacement per day) at 17 C on a 16-h light, 8-h dark photoperiod and fed a commercial fish diet.

Ovarian extraction
Goldfish ovaries were extracted based on a protocol described previously (23, 24). Briefly, frozen ovaries (20–30 g) along with dry ice were powdered with a precooled pestle and mortar held on dry ice. The powdered ovary was added to acetone: 1 N HCl (100:3, vol/vol) in a ratio of 1.0 g frozen tissue to 5.0 ml extraction fluid. The extraction mixture was stirred on ice for 3 h. Every hour, the mixture was homogenized for 1 min with a polytron. The mixture was filtered by suction through Whatman no. 1 filter paper after 3 h, and the residue was further homogenized and reextracted in acetone: 0.01 N HCl (80:20, vol/vol) in 40% of the original extraction fluid, stirred for 5 min, and again filtered. The combined filtrates were refiltered and extracted with petroleum ether to remove the hydrophobic substances; the ratio of the filtrate (aqueous phase) to the petroleum ether was maintained at 4:1 (vol/vol) for each extraction. Extraction was repeated four to five times until the volume of the aqueous phase became constant. Following the last extraction, the separation funnel was placed on ice for 30 min to allow the pigments present in the goldfish ovary to form an interphase between the ether and aqueous phases. The final aqueous phase was separated and centrifuged for 20 min at 17,000 x g (4 C), and the clear supernatant was purified using Waters C-18 Sep-Pak (Waters Associates, Milford, MA) columns (4 ml/column). The Sep-Pak columns were washed with 0.1% trifluoroacetic acid (20 ml/Sep-Pak), and the absorbed proteins/peptides eluted with acetonitrile: organopure water (60:40, vol/vol; 4 ml/Sep-Pak). The eluted material (ovarian extract) was aliquoted in 5–10 g frozen tissue equivalents per vial, lyophilized and stored at -25 C until further characterization. Once the presence of GnRH-like substance in the ovarian extract was established (see below), a bulk extraction was carried out from 975 g of goldfish ovary (obtained from approximately 2500 goldfish) using identical protocols. The method used to obtain goldfish muscle extracts (control tissue) was identical to that used for the goldfish ovaries as described above.

Reverse-phase HPLC
The HPLC analysis of the goldfish ovarian extract was carried out using a Beckman-Gold system, and the elution profile was continuously monitored at 2 wavelengths, 220 nM and 280 nM. For bulk purification, each batch of ovarian extracts (195 g ovary equivalent) was dissolved in 400 ml of organic pure water and filtered through a 0.22 mm Gelman filter. The filtrate was injected into a reverse phase preparative column (250 x 21.5 mm, particle size 10 mM with large pore C-18 matrix) (Hipore RP-318, Bio-Rad, Richmond, CA) preceded with a C-18 direct-connect cartridge guard column (10 x 4.6 mm; Alltech, Deerfield, IL) in ambient temperature. The mobile phase was acetonitrile (CH3CN) containing 0.1% trifluoroacetic acid (TFA) (solvent A) and 0.1% TFA in organic pure water (solvent B) with a flow rate of 5 ml/min. The extract was eluted initially for 20 min with 17% solution A, followed by a linear gradient from 17–35% solution A over 15 min, and from 35–90% solution A over a 90-min period. The immuno- and receptor-active fractions (1 min) from the preparative column were subjected to analytical HPLC using a Vydac C-18 reverse phase (polymer coated silica) column (0.46 x 25 cm; particle size 5 mM; pore size 300 Å) (Scientific Products and Equipment, Concord, Ontario, Canada) in ambient temperature with a solvent flow rate of 1 ml/min. The mobile phase was acetonitrile (CH3CN) containing 0.1% TFA (solvent A) and 0.1% TFA in organic pure water (solvent B), under the following elution conditions: an isocratic initial elution of 17% solution A for 35 min, followed by a linear gradient from 17–35% solution A over 15 min, and 35–80% solution A over a 5-min period. The HPLC fractions (500 ml or 250 ml) were collected over a period of 55 min and used for further analysis.

GnRH RIA
The Sep-Pak purified ovarian extract and the HPLC purified fractions were tested by RIA using various polyclonal GnRH antibodies. The GnRH RIA was carried out as described previously (25) with certain modifications. In brief, the rabbit antichicken GnRH-II (cGnRH-II) antiserum (8NW4, a gift from Dr. R. E. Peter, University of Alberta, Edmonton, Canada), antisalmon GnRH (sGnRH) antisera (S-30–3, a gift from H. J. Th. Goos, University of Utrecht, The Netherlands) and PBL no. L49 (a gift from W. W. Vale, Salk Institute, CA) were used each at final concentrations of 1:90,000. The peptides, sGnRH and cGnRH-II, were iodinated using lactoperoxidase method as described previously (20, 26). Briefly, 1 mCi Na125I (carrier free, Amersham, IMS 300) in 50 ml of 0.5 M phosphate buffer (pH 7.4) was added to a conical vial containing 5 mg GnRH peptide in 10 ml of 0.1 N acetic acid and 40 ml of 0.1 M phosphate buffer, and 15 ml of 2% lactoperoxidase (Boehringer Mannheim, Laval, Québec, Canada). The reaction was initiated by addition of 15 ml of 0.01% H2O2 in 0.1 M phosphate buffer (pH 7.4) at 4 C. Following 3 min of gentle agitation, the reaction mixture was injected to a 200 ml injector loop in a Beckman HPLC system. The monoiodinated GnRH peptide was purified by RP-HPLC using a Beckman C-18 column eluted with a linear gradient of acetonitrile and water (10–50%) in 0.1% trifluoroacetic acid (TFA). Fractions (250 ml) were collected in a automated fraction collector (Pharmacia), and aliquots were counted for radioactivity to determine the elution profile. The RIA tubes contained 100 ml of appropriate GnRH antiserum, 1% normal rabbit serum, 200 ml of reconstituted sample or standard, and 200 ml of iodinated sGnRH or cGnRH-II tracer (15,000–18,000 cpm/200 ml). The tubes were incubated at 4 C for 48 h with periodic manual shaking, followed by the addition of 200 ml of goat antirabbit gamma globulin antiserum (Calbiochem, CA, or Capricorn Supplies, Manitoba, Canada) (1 U/tube) and further incubation at 4 C for 16 h. At the end, the tubes were centrifuged for 20 min at 1100 x g in a refrigerated centrifuge, the supernatant decanted, and the radioactivity in the pallet fraction counted using a Packard Auto-multichannel {gamma}-counter. The data were processed by a computerized RIA program, and the minimum concentration of GnRH resulting in significant (P < 0.01) displacement of the specifically bound iodinated GnRH (27) (sensitivity), as well as the concentrations of GnRH peptides resulting in 20%, 50%, and 80% displacement (ED20, ED50, and ED80, respectively) of the iodinated GnRHs were estimated. The parallelism between displacement curves of the serially diluted GnRH peptides or ovarian extracts were estimated following logit-log transformation and calculation of the slopes of the regression lines using Student’s t test (28). Table 1Go summarizes the antiserum characteristics, in terms of antigen used, titer, sensitivity, and cross-reactivity of the various native GnRH peptides.


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  		Table 1. Characteristics of various polyclonal GnRH antisera and their cross-reactivity to native GnRH peptides

 
Biological activity of the ovarian extract
The effect of goldfish ovarian extract on the maturational gonadotropin (GTH-II; luteinizing hormone-like; see Ref. 29 for detail) release was determined in the superfused goldfish pituitary as described previously (30). Pituitary fragments (three pituitary equivalents per column) were treated with either 100 nM sGnRH or increasing concentrations of ovarian extract in a pulsatile fashion (3-min pulse, every 60 min). Five or 10-min fractions were collected using an automated fraction collector. Samples were frozen at -25 C until determination of GTH-II concentration by RIA as described before (30). The goldfish ovarian extract was also tested for GnRH receptor binding using goldfish ovarian and rat pituitary membranes using the techniques described previously (20, 31). The effect of ovarian extract on the reinitiation of oocyte meiosis and follicular steroidogenesis was investigated using a germinal vesicle breakdown (GVBD) assay and a testosterone RIA as previously described (21, 22). The effect of goldfish ovarian extract on GTH-II subunit ({alpha} and ß) mRNA production was investigated as described before (29). In brief, groups of mixed sex goldfish (six), predominantly at regressed stages of gonadal development, were injected ip with 4 mg of sGnRH, or 2.0 g equivalent of ovarian extract either alone or in combination with 100 nM GnRH-ANT per fish. The fish were anesthetized and killed after 12 h, pituitaries removed, and total RNA was extracted using the guanidine thiocyanate-phenol-chloroform extraction method (32). Five micrograms of total RNA from each treatment group was resolved in 1.2% agarose/formaldehyde gel and transferred onto a Hybond-N+ nylon membrane (Amersham, Arlington Heights, IL). The membrane was pre hybridized (overnight at 60 C) and hybridized (24 h at 60 C) with 32P-carp GTH-IIß and carp GTH-II{alpha} probes, labeled using random primer methods as described previously (29). The autoradiogram was scanned by a computerized denisitometer scanner and quantified using a gelscan program provided by NIH (Bethesda, MD). The intensity of the bands in ethidium bromide stained RNA gel was used as an internal marker to normalize the loading.

Structural analysis
Two HPLC purified fractions having functional GnRH activity were subjected to micro amino acid analysis and sequencing. The amino acid compositions were determined using an Applied Biosystems model 420H derivatizer-analyzer system (carried out at the Protein Microchemistry Centre, University of Victoria, Canada). In this procedure, Trp and Cys residues were not determined. The primary structure and amino acid sequence of the GnRH peptide present in fraction 2 was determined at the University of Calgary Protein Sequencing Facility by automated Edman degradation of more than 300 pmol peptide sample, using an Applied Biosystems 470A protein sequencer with off-line phenylthiohydantoin-amino acid identification under gradient elution conditions. The sequencing was carried out following incubation of the purified peptide with calf liver pyroglutamate aminopeptidease (Sigma) and further purification by reverse-phase HPLC on a Vydac C18 column.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of ovarian extract on GTH-II release and synthesis in goldfish pituitary
Pulsatile treatment of goldfish pituitary (3-min pulse) with increasing concentrations of goldfish ovarian extract stimulated the release of GTH-II in a dose-related manner (Fig. 1AGo). Addition of a GnRH antagonist([D-pGlu1, D-phe2, and D-Trp3,6]-GnRH; GnRH-ANT) totally blocked the ovarian extract-induced GTH-II release (Fig. 1BGo). Experiments were also carried out to study the effect of goldfish ovarian extract on the production of GTH-II{alpha} and GTH-IIß mRNA levels, in vivo. Injection of ovarian extract (2.0 g eqv/fish) significantly (P < 0.05) increased the pituitary GTH-II{alpha} and GTH-IIß mRNA levels, compared with controls (Fig. 2Go). Concomitant treatment with GnRH-ANT (100 nM) significantly blocked the ovarian extract-induced GTH-II subunit mRNA production. In this experiment, sGnRH was also injected as positive control as shown in Fig. 2Go.



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  		Figure 1. A, GtH-II release from superfused goldfish pituitary fragments after pulsatile treatments (3 min pulse every 60 min) with increasing concentrations of goldfish ovarian extract. At the end of the experiment, pituitary fragments were exposed to 3-min pulse of 0.1 mM calcium ionophore (A23187). Fractions were collected every 5 min, and the GtH-II concentration was measured by an RIA. Each value represents the mean ± SEM of four observations (two experiments, each carried out using two columns). mg eqv., mg ovary (wet weight) from which the extract was prepared. B, GtH-II release from superfused goldfish pituitary fragments after pulsatile treatment of increasing concentrations of ovarian extract alone or in combination with a GnRH antagonist ([D-pGlu1, D-phe2, and D-Trp3,6]-GnRH; GnRH-ANT) (100 nM). The effect of 100 nM GnRH-ANT alone was also tested. At the beginning and at the end of the experiment, pituitary fragments were exposed to 100 nM sGnRH as a positive control. Fractions were collected every 10 min and the GtH-II concentration was measured by an RIA. Each value represents the mean ± SEM of four observations (four different columns). Antag, GnRH-ANT; ext, ovarian extract.

 


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  		Figure 2. A–B, Effect of goldfish ovarian extract on the production of GtH-II subunit mRNA in the goldfish pituitary, in vivo. Six fish per group were injected with 4 mg of sGnRH, and 2.0 g equivalent of ovarian extract either alone (A) or in combination with 100 nM GnRH-ANT (B) (n = 2). Total RNA was extracted 12 h post injection. Five micrograms of RNA were loaded per lane on a formaldehyde gel for Northern analysis as previously described in detail by Khakoo et al. (29). Fish used in this study were sexually regressed. 18s RNA in the lower panel is shown to compare loading.

 
Displacement of bound 125I-GnRH analog by ovarian extract from pituitary and ovarian GnRH receptors
Goldfish ovarian extract was found to inhibit iodinated GnRH analog binding in a dose-related fashion from membrane prepared from goldfish ovary and pituitary as well as rat pituitary (Fig. 3Go, A–C). The serially diluted goldfish ovarian extract displaced 125I-sGnRH-A from both high and low affinity GnRH binding sites in the goldfish ovary (Fig. 3AGo) and pituitary (Fig. 3BGo). The goldfish ovarian extract was also found to displace 125I-[D-Lys6]-GnRH in the rat pituitary (Fig. 3CGo). In each case, the displacement curve for the extract was found to be parallel to the displacement curve for unlabeled GnRH (Fig. 3Go, A–C), indicating the binding of ovarian extract to the same classes of binding sites as that of unlabeled peptide.



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  		Figure 3. A–C, Binding of goldfish ovarian extract to goldfish ovary (A), goldfish pituitary (B), and rat pituitary (C) membrane preparations. Values (mean ± SEM) are the fraction of the bound label (percentage of total counts) after subtraction of the nonspecific binding in the presence of the excess unlabeled GnRH (10–6 M). Results were obtained in two experiments, each carried out in triplicate using different batches of ovarian extracts.

 
Effect of goldfish ovarian extract on the reinitiation of oocyte meiosis
Administration of goldfish ovarian extract to the follicle-enclosed goldfish oocytes stimulated the reinitiation of oocyte meiosis as indicated by GVBD, in vitro in a dose-related fashion (Fig. 4AGo). Concomitant treatment with GnRH-ANT inhibited the ovarian extract-induced GVBD response in a dose-related manner (Fig. 4BGo). As a positive control, we tested the effect of sGnRH alone and in combination with GnRH-ANT on GVBD response as shown in Fig. 4BGo.



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  		Figure 4. Effect of goldfish ovarian extract on reinitiation of oocyte meiosis and steroidogenesis. A–B, Dose-related effect of goldfish ovarian extract on GVBD response (A) and its inhibition by GnRH-ANT (B) in follicle-enclosed goldfish oocytes incubated in vitro. GVBD values were determined after 36 h incubation with ovarian extract alone (A), or ovarian extract in combination with graded doses of GnRH-ANT (B). Each value represents percentage GVBD determined using 160 oocytes (four fish, each contributing 40 oocytes incubated in groups of 20 oocytes per well, i.e. four different experiments). *, Significant difference with respect to appropriate controls according to a binomial test of proportions. The effect of sGnRH (100 nM) alone and in combination with increasing doses of GnRH-ANT has been included for a comparison.

 
Immunoreactivity of goldfish ovarian extract
Immunoreactivity of goldfish ovarian extract was tested in standard RIA using three GnRH antisera, S-30–3, PBL no. L49, and 8NW4. The degree of cross-reactivity of these antisera with various molecular forms of GnRH are summarized in Table 1Go. The goldfish ovarian extract was found to bind effectively with all antisera tested, and the displacement curves obtained by serially diluted samples indicated the presence of compounds structurally similar to GnRH peptides (Fig. 5BGo). The S-30–3 antiserum, which has a very high specificity for sGnRH, was found to be more sensitive in detecting the immunoreactive GnRH in the goldfish ovary.



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  		Figure 5. A–C, Immunoreactivity of goldfish ovarian extract with various polyclonal GnRH antisera, determined by a double antibody GnRH RIA. A, Displacement of 125IcGnRH-II to rabbit anti-cGnRH-II antiserum (8NW4) by cGnRH-II and crude goldfish ovarian extract. B and C, Displacement of 125IsGnRH to rabbit anti-sGnRH antisera (S-30–3 and PBL no. L49) by sGnRH and crude goldfish ovarian extract. B/Bo, Bound to maximum bound ratio. Each point represents the mean of triplicate determinations. The quantities of immunoreactive GnRH in the extract was measured by equating the amount of GnRH standard and the corresponding equivalent of ovarian extract resulting in 50% displacement in the RIA.

 
Purification and characterization of goldfish ovarian extract
Initially, we analyzed small batches (5–10 g eqv) of Sep-Pak purified extracts by reverse-phase HPLC using a Vydac C-18 column. Fractions were collected and tested for ovarian GnRH receptor binding, immunoreactivity with two GnRH antisera (8NW4 and S-30–3), and GTH-II release activity from superfused goldfish pituitary in vitro. Four HPLC fractions were found to displace 125I-sGnRH-A in the goldfish ovary as shown in Fig. 6Go; the first peak eluted at 11.6 min, which did not correspond to any known form of GnRH (unknown); the second peak coeluted with the region corresponding to mammalian GnRH at 19.5 min; the third peak coeluted with lGnRH-I at 26.2 min; and the last peak coeluted with sGnRH at 50.3 min. All these fractions were also tested for immunoreactivity and for biological activity. Using two GnRH antisera, 8NW4 and S-30–3, three fractions were found to display immunoreactivity. These included the fractions eluted at 11.6 min (unknown), 26.2 min (coeluted with lGnRH-I), and 50.3 min (coeluted with sGnRH) (Fig. 6AGo). The HPLC fractions were also tested for GTH-II release activity using superfused goldfish pituitary fragments in vitro. Fractions corresponding to the retention time of known GnRH peptides were lyophilized and reconstituted in the superfusion media, and administered in a pulsatile manner to the pituitary fragments in vitro (Fig. 6BGo). The GTH-II release was quantified for each treatment by determination of the area under the curve. Four of these fractions eluted between 4.5–6.25 min, 6.25–9.25 min, 9.25–18.0 min, and 49.5–54.25 min were found to significantly stimulate the GTH-II release. In general, these studies provided evidence for the presence of a biologically active GnRH-like substance(s) in the HPLC purified fractions. However, the quantity was not sufficient to investigate amino acid composition and biochemical structure. For this reason, we purified a larger quantity of goldfish ovarian extract using a preparative column (Hipore RP-318, Bio-Rad) as well as analytical column. In this experiment, we collected 975 g of goldfish ovary at different stages of maturity. The ovaries were removed, snap frozen in liquid nitrogen, and stored at -70 C until purification. The ovaries were extracted and Sep-Pak purified as described above. Figure 7Go shows a typical elution profile in the preparative column. In this system, GnRH standards were eluted in a narrow window of time, between 39.2 min and 46.3 min, and it was not possible to clearly separate all known GnRH forms. The ovarian extract in the preparative column was resolved into two major immunoreactive peaks (peak 1 and peak 2) detected by three GnRH antisera (S-30–3, PBL no. L49, and 8NW4) (Fig. 8Go). The antiserum PBL no. L49 was found to bind to a number of fractions not identified by other antisera used (Fig. 8CGo) presumably due to its broad specificity as indicated in Table 1Go. The fractions in and around the region that corresponds to the retention time of synthetic GnRH peptides (35–50 min) were tested for GnRH receptor binding using rat pituitary membranes (Fig. 9Go). In this context, fully mature goldfish ovary was not available at that time of the year, and rat pituitary was found previously to bind to all these forms of GnRH peptides (31) and was therefore used as a substitute. The displacement binding profile was consistent with the immunoreactive profile indicating the presence of two major peaks (peak 1 and peak 2) (Fig. 9Go). To further purify the peak 1 and peak 2, we used an analytical HPLC with a Vydac C-18 column. Fractions corresponding to peak 1 or peak 2 obtained from preparative column were further analyzed using an analytical HPLC C-18 column as described above (for elution profile of peak 1, see Fig. 10AGo; for peak 2, see Fig. 11AGo). Fractions (500 ml) were collected, and aliquots of these samples were tested for immunoreactivity. Using anti-sGnRH antiserum S-30–3, two major immunoreactive peaks were detected: one at 11.6 min, which did not correspond with the retention time of the known GnRH forms (Fig. 10BGo), and the other at 50.27 min, which coeluted exactly with the synthetic sGnRH (Fig. 11BGo). The samples were also tested, using 8NW4 antibody. Only the early eluting peak (peak 1; 11.6 min) was found to immunoreact with antiserum 8NW4 (results not shown). The two HPLC fractions were further analyzed for amino acid content, and the peak 1 fraction was found to contain excessive impurity and provided inconsistent results. However, the amino acid content of peak two was found to correspond exactly to that of the synthetic salmon GnRH with no tryptophan because the latter amino acid is entirely destroyed during acid hydrolysis (Table 2Go). The peptide in peak 2 was subjected to automated Edman degradation to obtain amino acid sequence following incubation with pyroglutamyl aminopeptidase and HPCL purification. During cycle 8 of operation, it was possible to identify the sequence of 7 amino acids but were not able to detect Trp during cycle 6 to confirm its identity in the peptide molecule (Table 3Go). In sGnRH, the second Trp residue occurs at position 7, and all other evidence obtained in this study suggest very strongly that the unidentified residue is likely to be Trp.



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  		Figure 6. Purification of Sep-Pak-purified goldfish ovarian extract (10 g eqv) by HPLC and C-18 column eluted with a stepwise solvent gradient of acetonitrile and water in 0.1% trifluoroacetic acid in water as described in detail in the Materials and Methods section. Fractions (250 ml) were collected and aliquots tested for GnRH binding in the goldfish ovary and GnRH immunoreactivity using anti-cGnRH-II (8NW4) and anti-sGnRH (S-30–3) antisera (A), and release of gonadotropin from superfused goldfish pituitary, in vitro. (B) (+) indicates positive response, and (-) indicates no response for gonadotropin release. Multiple (+) or (-) provides a relative measure of activity for gonadotropin-release response. The retention time of synthetic cfGnRH, lGnRH-III, cGnRH-I, mGnRH, lGnRH-I, cGnRH-II, and sGnRH has been indicated by arrows. Dotted lines represent the solvent gradient (see Materials and Methods for details). B, GtH-II release from superfused goldfish pituitary fragments after pulsatile treatment of various fractions of ovarian extract (A–L). Ten grams of Sep-Pak-purified goldfish extract was repurified by HPLC using a C-18 column. Fractions of 250 ml from various regions as shown in the lower panel (e.g. A = fractions from 0 to 3.0 min, B = 3.0–4.5 min and so on) were pulled, lyophilized, and reconstituted in the superfusion media and administered as 3-min pulses every 60 min. At the beginning and at the end of the experiment, pituitary fragments were exposed to a 3-min pulse of 100 nM sGnRH as a positive control. Fractions were collected every 10 min, and the GtH-II concentration was measured by an RIA. Each value represents the mean ± SEM of four observations (four different columns). The GtH-II release was quantified for each treatment by determination of the area under the curve. The arrow indicates the retention time of corresponding synthetic GnRH peptides in this system. Values with dissimilar superscripts are significantly different (P < 0.05) from others using a Student’s t test.

 


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  		Figure 7. HPLC elution profile (at an absorbance of 280 nM) of the crude goldfish ovarian extract (195 g equivalent), repurified using a reverse phase preparative column (Hipore, RP318; Bio-Rad), at a flow rate of 5 ml/min. One-minute fractions (5 ml) were collected. Dotted lines represent the solvent gradient (see Materials and Methods for solvent gradient program). The arrow indicates the retention time of corresponding synthetic GnRH peptides in this system.

 


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  		Figure 8. A–C, The immunoreactive profile of preparative HPLC purified ovarian extract samples using three different antisera raised against cGnRH-II (8NW4) and sGnRH (S-30–3 and PBL no. L49). Aliquots of 725 ml (25 g equivalent) of preparative column (Hipore, RP318; Bio-Rad) eluted samples were lyophilized, reconstituted in RIA buffer, and measured for immunoreactive GnRH using homologous GnRH RIA using appropriate antisera and 125I-sGnRH and 125I-cGnRH-II as iodinated tracer.

 


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  		Figure 9. The GnRH receptor binding of immunoreactive GnRH fractions from preparative HPLC purified samples, using rat pituitary membrane preparations. One-milliliter (32 g equivalent) of preparative column (Hipore, RP318; Bio-Rad) eluted samples with retention time from 35.5 min to 57.5 min were individually lyophilized, reconstituted in RRA buffer, and measured for GnRH binding in a rat pituitary GnRH receptor assay, using 125I-D-[Lys6]-GnRH as a labeled ligand. Details of the assay conditions have been previously described (31). Values (mean ± SEM, n = 4) represent specific binding (B/T, in %) determined by subtraction of nonsaturable binding in the presence of excess [D-Lys6]-GnRH (10–6 M). Peak-1 and Peak-2, Tubes giving maximal immunoreactive GnRH in the GnRH RIA (Fig. 8Go).

 


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  		Figure 10. A and B, Repurification of Peak-1 (preparative HPLC; Fig. 8Go) using reverse phase HPLC and a Vydac C-18 analytical column with a stepwise gradient of acetonitrile in O.1% TFA (see Materials and Methods for details). 0.5-minute fractions (500 ml) were collected and aliquots were tested for RIA with sGnRH antiserum (S-30–3) (B). The arrow indicates the elution time of lGnRH-III.

 


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  		Figure 11. A and B, Repurification of peak 2 (preparative HPLC; Fig. 8Go) using reverse phase HPLC and a Vydac C-18 analytical column with a stepwise gradient of acetonitrile in O.1% TFA (see Materials and Methods for details). 0.5-minute fractions (500 ml) were collected, and aliquots were tested for RIA with sGnRH antiserum (S-30–3) (B). The arrow indicates the elution time of sGnRH.

 

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  		Table 2. Amino acid composition of the HPLC-purified fraction coeluting with the synthetic salmon GnRH (Glu-His-Tyr-Ser-Leu-Glu-Trp-Lys-Pro-Gly-NH2)

 

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  		Table 3. Automated Edman degradation of GnRH peptide sample after digestion with pyroglutamyl aminopeptidase

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we purified and determined the primary structure of an ovarian GnRH with identical sequence to that of the native salmon GnRH in goldfish. This is the first demonstration of an ovarian GnRH compound by amino acid sequencing. These findings are based on clear evidence demonstrating GnRH-like biological activity, receptor binding activity, and immunoreactivity of the goldfish ovarian extract. The goldfish ovarian extract was found to stimulate the production of GTH-II subunit mRNA in the goldfish pituitary in vivo as well as stimulating GTH-II release from the goldfish pituitary in vitro. The release and synthesis of GTH-II in response to ovarian extract was inhibited in the presence of a GnRH antagonist, indicating specific action via GnRH receptors. In addition, goldfish ovarian extract stimulated GVBD in the prophase-I arrested follicle enclosed goldfish oocytes, which was also inhibited by a GnRH antagonist, indicating specific interaction with GnRH receptors confirming previous observations in goldfish (21, 33). Furthermore, serially diluted ovarian extract displaced 125I-sGnRH-A from both high and low affinity GnRH binding sites in the goldfish ovary and pituitary, confirming specific interaction with the GnRH receptors in the pituitary and ovary.

The HPLC analysis of the ovarian extract revealed the presence of three fractions that could bind to the goldfish ovaries, release GTH-II from cultured goldfish pituitaries, and cross-react with various GnRH antibodies. However, two of these fractions displayed high potency including an early eluting peak that did not correspond with the retention time of the known GnRH forms and a fraction that coeluted exactly with sGnRH. Using preparative and analytical HPLC, these two immunoreactive and biologically active fractions (peak 1 and peak 2) were purified in greater quantities and subjected to amino acid analysis. Although we were successful in determining the primary structure of the peak 2 as salmon GnRH, the early eluting peak was not of sufficient purity to provide meaningful amino acid profile. However, in view of the immunoreactivity data and HPLC elution profile, it is likely that peak 1 may be a novel form of GnRH peptide or a different compound with GnRH-like activity. In addition, it may be speculated that this compound contain a blocked NH2- and COOH-termini because it was recognized by the NH2 and COOH-terminus directed antisera 8NW4 and S-30–3. Recent studies in goldfish demonstrated the expression of both sGnRH and cGnRH-II in the ovary (6). However, we were unable to detect cGnRH-II peptide in the goldfish ovarian extracts used in this study, indicating that cGnRH-II may not be expressed as much as sGnRH during the time that the samples were collected. Other studies also indicated the presence of an early-eluting compound with GnRH-like activity in the ovary of African catfish (17) and seabream (18). The ovarian extract from catfish was found to have similar activities as that observed for the peak 1 in the present study.

In summary, the present study provides clear evidence for the presence of salmon GnRH as well as a novel compound with GnRH-like activity in the goldfish ovary. These findings collectively support the hypothesis that GnRH and GnRH-like peptides are produced in the fish ovary and play a role in the regulation of ovarian function.


   Acknowledgments
 
We wish to thank Biomedical Computing Technology Information Centre (Nashville, TN) for providing LIGAND and ALLFIT computer programs.


   Footnotes
 
1 This study was supported by a Natural Sciences and Engineering Research Council Grant U-0037946 (to H.R.H). D.P. was supported by an Alberta Minister of Advanced Education International Education Award. Back

2 Present address: Texas Children’s Cancer Center, Baylor College of Medicine, 6621 Fannin Street MC 3–3320, Houston, Texas 77030-2399. Back

Received September 29, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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