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Regular Article |
a Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N5
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene regulation, gonadotropin-releasing hormone, ovary, seasonal reproduction, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GnRH is expressed in the vertebrate brain in four distinct locations: the preoptic area that innervates the median eminence or pituitary (in fish); the midbrain; the terminal nerve; and sites in the forebrain (septum, stria terminalis, amygdala, and internal capsule) [4–6]. GnRH produced in the preoptic area acts as a hypophysiotropic factor controlling reproductive functions via the brain-pituitary-gonadal axis. The specific actions of the GnRH forms found in the olfactory/terminal nerve and the midbrain regions have not been elucidated but are thought to influence sexual behavior [7].
Nonneural expression of GnRH has been established in mammals and fish [8–15]. In gonads of mammals, in situ hybridization studies have localized mGnRH mRNA to granulosa cells of the rat ovary and to Sertoli cells of the rat and human testis [13, 14]. In gonadal tissue of fish, several forms of GnRH have been identified as mRNA, but the predominant form identified to date is salmon GnRH (sGnRH). The sGnRH mRNA is expressed in the ovary of midshipman [16], goldfish [17], rainbow trout [18], and sockeye salmon [8]. The sGnRH peptide, which is present only in teleost fish, has been isolated and sequenced from gonads only from ovary of goldfish [19] and identified only once by RIA in rainbow trout ovary [20]. In testis, sGnRH mRNA has been identified from midshipman [16], Haplochromis burtoni [4], rainbow trout [18], and sockeye salmon [8], yet the sGnRH peptide has not been identified in the testis of any fish species.
Two genes that encode sGnRH were identified in salmonids, which are tetraploid [18, 21–23]. The genes encoding sGnRH have four exons, encoding a 5' untranslated region, a signal peptide, GnRH, the GnRH-associated peptide (GAP), and a 3' untranslated region. The sGnRH gene-1 and -2 differ by 2 nucleotides out of 30 in the GnRH region but produce identical sGnRH peptides. The sGnRH-1 and -2 cDNAs encode signal peptides with an amino acid identity of 78% and GAPs with an identity of 70% due to a frameshift in the C-terminal region of GAP [18, 23]. Both sGnRH genes are expressed as mRNA in brain and gonads. The sGnRH gene-1 uses only one start site, whereas sGnRH gene-2 uses two different start sites and undergoes alternative splicing to produce three different transcripts in the gonads of rainbow trout and sockeye salmon [8, 18, 24].
The sGnRH gene-1 and -2 are expressed intermittently in the gonads of rainbow trout and sockeye salmon throughout the first 2 yr of life. Rainbow trout sexually mature during their third year of life. The study by von Schalburg and Sherwood [18] showed increased expression of sGnRH mRNA and peptide in precociously developing fish (fish sexually maturing at 2 yr of age), suggesting sGnRH in some circumstances plays an autocrine/paracrine role in the development of the ovary and testis.
The objective of the present work was to examine the expression pattern of sGnRH gene-1 and -2 and the sGnRH peptide in brain and gonads of rainbow trout sexually maturing in their third year of life. The present experiment was designed to correlate mRNA expression with the presence of the sGnRH peptide in salmonid reproductive tissues during maturation. The expression pattern of different transcripts of the sGnRH genes in the maturing salmonid may provide clues to the function of nonneuronally expressed GnRH in the gonads compared with that in the brain.
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MATERIALS AND METHODS |
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Two-year-old rainbow trout (Oncorhynchus mykiss) (n = 120), weighing 400–500 g, were obtained from Mountain Trout Sales (Sooke, BC, Canada) and housed outdoors at the Aquatics Facility at the University of Victoria in compliance with the regulations of the University of Victoria Animal Care Committee. On the same day of each month, nine trout were randomly removed from the tank and killed using 0.1% tricaine methanesulfonate (MS-222) (Argent Chemical Laboratories, Redmond, WA). The ratio of females to males at each sampling month is shown in Table 1. Whole brains and ovaries or testes were removed from the fish and the diameter of each ovary and testis was measured before freezing. Averages for diameter were calculated for each gonad type for each month. To assess ovulation in female fish, gentle pressure was applied to the abdomen to determine whether eggs were released. Each tissue was frozen using liquid nitrogen, pooled by tissue (brains were pooled without regard to sex) and stored at -80°C for later use.
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Isolation of RNA and Reverse Transcription-Polymerase Chain Reaction
Each month, frozen pooled brains were crushed into a fine powder using a chilled mortar and pestle. A small sample (
100 mg) of this pooled and mixed tissue was removed for total RNA extraction. For ovarian and testicular tissue, a small piece of the gonad was removed from each individual at dissection, pooled by gonad type, and frozen. For each month, the pooled ovarian or testicular tissue was ground into a fine powder as above and 100 mg was used for RNA extraction. Total RNA was isolated from the powdered tissues using TRIzol (Life Technologies, Burlington, ON, Canada) as described by the manufacturer. The isolated RNA was precipitated in isopropanol at -80°C. The RNA from each tissue (brain, ovary, and testis) at each month was treated with DNase I at 37°C for 1 h to remove genomic DNA. To further test for genomic contamination, a polymerase chain reaction (PCR) (as below) using primers S and T (as below) was performed using 1 µl (0.5 µg) of the RNA as template. RNA that was free of genomic contamination was then reverse transcribed in a 50-µl reaction that contained 1 µg of RNA, 2 mM oligo dT, 2 mM dNTPs, 1x first strand buffer, 0.01 M DTT, 5 U RNase inhibitor, and 100 U Superscript II reverse transcriptase (Life Technologies). The reaction was incubated at 42°C for 90 min and the enzyme was heat inactivated at 90°C for 10 min.
Five PCRs were performed on the cDNA from each tissue type for each month. One of the reactions was a positive control to assess the integrity of the cDNA generated from the reverse transcription reaction. This reaction contained primers designed to hybridize with salmon tubulin, using the sense primer T10 (5'-CAGGTGTCCACGGCTGTGGTG-3') and the antisense primer T11 (5'-AGGGCTCCATCGAAACGCAG-3'). Two PCR reactions contained sense and antisense primers designed to detect the two different transcripts produced from sGnRH gene-1. To detect a long sGnRH mRNA-1, the sense primer 80 (80 nucleotides upstream of primer B) (5'-GGAGAAGGGATTCTAATCC-3') was used with the antisense primer T (5'-TTGAATGCTCCATCATCGC-3'). To detect the short sGnRH mRNA-1, the sense primer S (5'-AGGAATAGACCGAACGGAC-3') was used with the antisense primer T. The two different transcripts produced from sGnRH gene-2 were detected in two additional PCR reactions. To detect the long sGnRH mRNA-2, the sense primer 80 was used with the antisense primer C (5'-TTATTTATGGGGCATCCATTTC-3'). To detect the short sGnRH mRNA-2, the sense primer B (5'- TAGGAAGGAATACACAGAACGG-3') was used with the antisense primer C.
Each 50-µl reaction contained 2.5 U Taq polymerase, 1x Taq buffer, 2.5 mM MgCl2, 0.2 mM dNTPs (Life Technologies), and 20 pmol of each 5' and 3' primer. Each PCR was carried out under the following conditions: denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, extension at 72°C for 45 sec for 32 cycles, and a 7-min extension. The PCR products were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining using an Eagle Eye II still video system (Stratagene, La Jolla, CA). Representative bands were isolated and cloned into pGEM Vector-T (Promega, Madison, WI) and sequenced to confirm their identity. PCR results were repeated for each month.
Southern Blot to Confirm PCR Results
To confirm some of the PCR results, products generated from the PCRs were run on a 1.5% agarose gel, dried, and exposed to 32P-labeled probes designed against sGnRH-1 and sGnRH-2 mRNAs. The probes were labeled using a random priming labeling kit (Life Technologies). Probes and blocking DNA (500 µl sea urchin sperm DNA at a concentration of 300 mg/ml) were added to a hybridization buffer of 6x sodium chloride sodium phosphate EDTA (SSPE), 5x Denhardt solution, 0.5% SDS, and 0.05% sodium pyrophosphate. Hybridization occurred at 55°C with agitation. Dried gels were washed with 0.1x salt sodium citrate and 0.5% SDS at 55°C 5 times for 30 min, exposed to a phosphoimaging screen overnight, and developed on the STORM phosphoimager (Amersham Pharmacia Biotech, Piscataway, NJ).
Identification of sGnRH Protein by HPLC/RIA
Extraction of peptides from tissue The quantity of pooled ovary and pooled testis for the first 5 mo (January–May) of the third year was too small to accurately assay peptide levels. Assessment of sGnRH peptide levels in individual months was performed in gonadal tissue collected in June–December. The weight of pooled tissue for peptide analysis ranged from 2.5 to 5.8 g for brains, 17.3 to 23.2 g for ovaries, and 7.2 to 36.0 g for testes.
The tissues were ground into a fine powder using a chilled mortar and pestle. Peptides were extracted with an acetone-HCl mixture, and soluble lipids were removed with petroleum ether as previously described [25]. The acetone-water-soluble mixture was reduced in a vacuum centrifuge to approximately 2 ml and filtered through a µStar LB 45-µm filter (Costar, Corning, NY).
HPLC
The filtrate was loaded onto a Supelcosil LC-18 column (25.0 cm x 4.6 mm; 5-µm particle size; Supelco Canada, Oakville, ON, Canada) attached to a guard column of the same material. Each filtered extract (2 ml) was loaded in repeated injections of 700 µl each at 0, 2.5, and 5 min onto a 1-ml loop. An isocratic program of 83% 0.25 M triethylammonium formate (pH 6.5) and 17% acetonitrile was used over a 10-min period at a flow rate of 1 ml/min. After 10 min, the percentage of acetonitrile was elevated to 24% over 7 min and maintained at 24% for 43 min. Sixty fractions of 1 ml each were collected in polyallomer tubes; 100 µl was removed from each fraction, vacuum dried, reconstituted in PBS with 0.1% gelatin, and assayed for immunoreactive GnRH (irGnRH).
A blank of 700 µl of Milli-Q water (Millipore, Bedford, MA) was injected onto the column; fractions were collected and assayed prior to each extract to ensure that the column was free of any GnRH from previous HPLC analyses. Synthetic standards were applied to an identical column and run under identical conditions. Four GnRH forms (mGnRH, cGnRH-II, dogfish GnRH, and sGnRH) were combined at 200 ng each and applied to the HPLC as above. The elution positions of the standards on the chromatograph were confirmed by absorbance peaks (A = 280 nm) and a GnRH-specific RIA [26].
RIA Aliquots of 100 µl of each fraction collected from the HPLC were taken, dried, and assayed for irGnRH by methods previously described [27]. The extracts were assayed using GF-6 antiserum at a final dilution of 1:25 000 and a tracer of 125I-labeled synthetic mGnRH. The GF-6 antiserum was raised in a rabbit in our laboratory against sGnRH. GF-6 cross-reacts with mGnRH (100% cross-reactivity with mGnRH tracer), seabream (sb) GnRH (94.1%), sGnRH (25.7%), and cGnRH-II (10.5%) [6]. The limit of detection (B/B0 = 80%) for GF-6 in the 27 radioimmunoassays performed in the present study ranged from 1 to 5 pg.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reproductive Stage of Rainbow Trout at Collection
The diameter of gonads from each fish was measured at each collection time and averaged for each month (Fig. 1). The average diameter of the ovaries and testes at each collection time shows an increase in gonad size as the year progresses. The dramatic increase in the size of the gonads over the course of the 13 mo is typical of sexually maturing salmonids. Fish became gravid in the autumn of their third year and spawned between October and December.
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Messenger RNA and Protein Expression of sGnRH in Brain
In rainbow trout brain, the sGnRH genes are expressed continuously throughout the third year of life (Figs. 2 and 3). Both the sGnRH-1 and -2 genes are transcribed, producing mRNAs. In brain, the sGnRH gene-1 always used a downstream promoter, producing only one sGnRH-1 mRNA transcript in all 13 mo assessed (Fig. 3). Like sGnRH gene-1, sGnRH gene-2 is expressed continuously in the sexually maturing rainbow trout brain (Fig. 3). Yet unlike sGnRH-1, we show that the sGnRH gene-2 uses alternate start sites to produce two different transcripts in brain (Fig. 3). The long sGnRH-2 transcript was produced from May to September, while the conventional transcript was expressed for all months (Fig. 3). The long sGnRH-2 transcript results from an extended 5' UTR and retention of intron 1 (Fig. 2a). Negative control PCR reactions, which contained no DNA, were run with all primer sets for each month (Fig. 2a). In addition, the long sGnRH-2 transcript was identified in brain in 2 mo (August and September) that did not show the long transcript in either the ovary or testis sample (Fig. 3), ruling out the chance of cross contamination from one sample to the next. This is the first report of an upstream start site being used for the sGnRH gene-2 in neural tissue.
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HPLC/RIA data showed that both forms of GnRH peptide found in rainbow trout, sGnRH and cGnRH-II, were expressed in rainbow trout brain throughout their third year of life (Table 1, Fig. 4). The location of cGnRH-II in the brain suggests it does not influence the release of gonadotropins from the anterior pituitary and thus is not discussed here. The levels of sGnRH in the brain of sexually maturing rainbow trout fluctuate, with the highest levels seen in May, June, and July of the third year (Table 1).
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Messenger RNA and Protein Expression of sGnRH in Ovary and Testis
Nonneural expression of sGnRH mRNAs in the gonads of sexually maturing rainbow trout is intermittent. Both sGnRH gene-1 and -2 are expressed in both ovary and testis but not continuously and not always together. In ovary, sGnRH-1 mRNA was expressed from January to November, with the exception of February (Fig. 3). In testis, sGnRH-1 mRNA was expressed in April, June, August, September, and October (Fig. 3). As in the brain, the sGnRH gene-1 did not use an upstream promoter in the gonads and only the short transcript was produced.
The sGnRH gene-2 expression was also variable (Fig. 3). In ovarian tissue, sGnRH gene-2 is expressed in 11 of the 13 mo studied (January–October). In the ovary, both the upstream and downstream promoters are detected and both the long and the conventional sGnRH-2 transcripts are produced. The upstream promoter was used on and off throughout the third year in January, in May–July, and again in October. The downstream promoter was used to produce the conventional transcript from February to April and again in August and September (Fig. 3). In testis, sGnRH-2 mRNA is expressed from May to September, with the exception of June. The upstream promoter for sGnRH-2 was used only in May; the downstream promoter was used in the remaining months (Fig. 3).
Due to the immaturity of the gonads from January to May of the third year, the sample size of the gonadal tissue was too small to accurately assay peptide levels in these tissues in each of the months. Assessment of sGnRH peptide levels in individual months was performed in gonadal tissue collected in June–December. The sGnRH peptide was not detected in any HPLC fraction from the ovary and testis from any of the months of the third year (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression pattern of sGnRH-1 and -2 transcripts in rainbow trout brain, ovary, and testis was assessed over 13 mo in their third year of life. Promoter usage by the sGnRH genes in sexually maturing rainbow trout showed that a long sGnRH-2 transcript, in addition to the conventional sGnRH-1 and -2 mRNA transcripts, was produced. The sGnRH gene-1 produced only one transcript using a downstream promoter in both brain and gonads (Fig. 3). Previous experiments on Year 1 and Year 2 trout detected only the conventional sGnRH mRNA-2 in the brain, in which sGnRH gene-2 used the downstream promoter [8, 18]. In the present experiment on Year 3 trout, GnRH gene-2 used both an upstream and downstream promoter in the brain of sexually maturing rainbow trout. This is the first report of an upstream start site being used by the sGnRH gene-2 in neural tissue. The conventional transcripts for sGnRH gene-1 and -2 are expressed every month, but the long sGnRH mRNA-2 is expressed only in May–September, during the rapid growth of the gonads before growth reaches a plateau (Fig. 1) and before spawning. Expression of this transcript coincides with maximal sGnRH peptide in the brain (May–July). The long transcript may be required to cause this increase in production of sGnRH.
Alternative splicing of the GnRH gene was originally described in human placenta, where intron 1 was retained in the mRNA for mammalian GnRH (mGnRH) [12]. Dong et al. [28] showed two start sites were present in the mGnRH gene. The downstream promoter was used for transcription of GnRH in hypothalamic (GT1-7) cells and the upstream promoter was used in nonneural placental cell lines. Dong et al. [29] also examined promoter usage of the mGnRH gene in monkeys and showed that, when mGnRH mRNA was expressed in reproductive tissues, an upstream start site was used.
In fish, the production of gonad-specific transcripts has been reported in seabream, rainbow trout, and sockeye salmon. In seabream, a mRNA for cGnRH-II and sbGnRH in ovary, but not brain, retained all three introns, creating a truncated GnRH-associated peptide (GAP protein) from a premature stop codon [30]. In rainbow trout, several sGnRH transcripts have been identified: a first conventional transcript that contains the four exons of the GnRH gene; a second transcript that retains intron 1; a third that retains a portion of intron 1 [18]; and in maturing rainbow trout, a fourth testis-specific transcript that retains all introns [24]. Both Uzbekova et al. [24] and Nabissi et al. [30] tested for genomic DNA contamination, suggesting the transcript that retained all three introns was amplified from mRNA and not from genomic DNA. In the present study, RNA was treated to remove genomic contamination and the transcript that retains all three introns was not identified. Immature sockeye salmon in their first 2 yr used an upstream promoter for sGnRH gene-2 to produce the intron-1-retaining transcript exclusively in gonadal tissue [8]. These data suggest the alternative start sites regulate tissue-specific expression of GnRH. Our identification of the long sGnRH-2 transcript in the brain in the present study challenges this view. Instead, expression of the long transcript may be related to a specific stage of sexual maturation in the third year.
The physiological significance of the long transcript is presumably related to changes in the level of sGnRH peptide in the brain. The long sGnRH-2 transcript is first detected in May and is present from May to September but disappears before spawning, which begins in October. The sGnRH peptide also reaches its highest content in the brain in May–July. The sGnRH content, however, reflects both the peptide stored in axon terminals that are distributed to various brain regions and to peptide that is stored in the terminals in the pituitary. One possible explanation is that more sGnRH peptide will be synthesized if the long sGnRH-2 transcript is present and available for translation. However, another possibility is that the long sGnRH-2 transcript blocks translation because intron 1 is retained within the 5' UTR. The 5' UTR is essential for initiation of translation at the ribosome. This explanation would be more consistent with an earlier study by Davies et al. [31] in which a number of reproductive hormones were decreased for a few months in the summer before spawning in rainbow trout that were in a similar stage to ours (third year approaching first spawning). These authors suggest that the decrease in hormones may occur because trout are repeat spawners and need to retain some immature eggs for future seasons. To determine if the long sGnRH-2 transcript activates or blocks translation, the long and/or short sGnRH-2 transcripts could be transfected into COS cells and the output of sGnRH peptide measured.
Separate Patterns of Expression for Three sGnRH Transcripts in Gonads During Sexual Maturation
The expression pattern of sGnRH mRNA-1 and -2 is distinct in 3-yr-old mature adult rainbow trout from either the parr/smolt or immature stages [18]. As in 1- and 2-yr-old rainbow trout, the expression pattern of the sGnRH-1 gene and the splice variants of the sGnRH-2 gene in the gonads of sexually maturing 3-yr-old rainbow trout are intermittent (Fig. 3). The sGnRH transcripts are not expressed during December of the second or third year. In the testis of 3 yr olds, expression of sGnRH transcripts is shorter lived than in the ovary, which expresses some form of sGnRH in all months except December (spawning or postspawning). The sGnRH gene-1 and -2 have the potential to produce identical peptides, yet their expression is regulated independently of one another, suggesting the mRNAs transcribed from the sGnRH genes play independent roles in the rainbow trout gonads.
Salmon GnRH Protein Present in the Brain Throughout Sexual Maturation
The sGnRH peptide was present in the brain of sexually maturing rainbow trout during all 13 mo throughout the third year. The sGnRH in the preoptic area is known to control reproductive processes through the hypothalamic-pituitary-gonadal axis. GnRH causes the release of the gonadotropins from the anterior pituitary, which regulates gametogenesis and steroidogenesis.
Earlier reports also show that the GnRH peptide is expressed in the brain before and during reproduction. In seabream, females spawn daily and levels of all three GnRH forms (sbGnRH, sGnRH, and cGnRH-II) in the brain fluctuate, with highest levels at 8 h before spawning, the time at which meiotic division is resumed [7]. In rockfish, a species that is ovoviviparous, only sbGnRH fluctuates with the reproductive state; sGnRH and cGnRH-II remain at constant levels in the brain and pituitary [32]. Masu salmon spawn once yearly, as do rainbow trout. Increases in sGnRH levels in the pituitary (from GnRH nerve terminals) of masu salmon correspond to gonad maturation. The sGnRH levels in the telencephalon and preoptic area of the brain were similar or higher in prepubertal fish compared with mature fish [33, 34]. In mammals, such as rats, GnRH release into portal blood peaks at 8–10 h before ovulation [35]. Transcription of the sGnRH genes prior to gonadal maturation has been shown in rainbow trout [18], sockeye salmon [8], and masu salmon, where sGnRH mRNA-l and -2 levels are highest in prepubescent fish, prior to gonad maturation [9].
The present study shows the highest levels of sGnRH peptide in the brain of rainbow trout prior to spawning, in late spring and early summer (May, June, and July), a time of rapid growth for both ovaries and testes. As suggested for the masu salmon [33, 34], the high levels of sGnRH produced early in the third year may be required for gonad maturation or are stored for later use in reinitiation of meiosis. The sGnRH peptide increases again in October, at the beginning of spawning, which occurred from October to December (Table 1).
Salmon GnRH Protein Undetectable in the Gonads During Sexual Maturation
The lack of sGnRH peptide in the gonads of sexually maturing rainbow trout suggests that sGnRH is produced in the gonads at levels too low to be detected by RIA, that sGnRH is produced for brief periods that are not detected by monthly sampling, or that sGnRH peptide does not play a role in first spawning or maturation of rainbow trout gonads. Because sGnRH was detected in the brain at all sampling times and the detection limit for the RIAs was less than 5 pg (which is less than 1 fM per fish), it is clear that the antibody reliably detects sGnRH in the RIA. In addition, the GF antibody series has been tested for detection of different GnRH fragments [36]. In immature rainbow trout, sGnRH mRNAs were detected in gonads intermittently throughout the first 2 yr of life, but peptide was absent [18]. The sGnRH peptide was only detected in the ovary of precociously developing female rainbow trout (2 yr old). In precociously developing testis, sGnRH mRNAs were expressed, but no peptide was detected [18, 20]. These earlier results by von Schalburg and Sherwood [18] support the data of the present study, showing that, although the sGnRH genes are transcribed in 1-, 2-, and 3-yr-old rainbow trout gonads, the peptide is not usually present at detectable levels. We hypothesize that the lack of sGnRH in rainbow trout gonads shows that the sGnRH peptide, produced at a very low level, immediately binds a local receptor acting in an autocrine/paracine manner within the gonads or that peptide is not produced from the sGnRH mRNAs, precluding a functional role for sGnRH in the gonad. If the latter is true, this study provides an example of how mRNA expression levels are not an indication of protein production.
Our data and that from 1- and 2-yr-old rainbow trout [18] show sGnRH mRNA and protein expression in the brain of rainbow trout during development and spawning. In the gonad, sGnRH mRNAs are expressed but either are not translated or are translated at very low levels during the final stages of gonadal development and during first spawning.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 250 721 7120; nsherwoo{at}uvic.ca
Received: 19 February 2002.
First decision: 8 March 2002.
Accepted: 25 May 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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