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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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For the first time, GnRH peptides were isolated from testis and identified by HPLC-RIA with specific antisera and by elution position compared with synthetic standards. GnRH peptides were also present in the ovary. In addition, multiple forms of GnRH, including a form not normally detected in the brain of trout, were shown to be present in the gonads. During development, GnRH peptides were expressed only at specific stages in the gonads, which may explain the inability to detect and isolate the GnRH peptides from gonads in earlier studies.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GnRH is expressed not only in the brain but also in the gonads. Local expression of the mRNA encoding GnRH was demonstrated in various reproductive tissues including the ovary and testis in humans [11], monkeys [12], and rats [13, 14]. A GnRH peptide was reported for porcine ovary, but the results were not conclusive as blank HPLC injections and assays did not precede tissue injections [15]. In fish, a transcript encoding sGnRH has been isolated and sequenced from midshipman ovary and testis [16]. In the goldfish ovary, both sGnRH and cGnRH-II mRNAs have been identified [17], but only sGnRH peptide has been shown to be present [18]. Goldfish testis has not been studied. There are strong indications from several in vitro physiological studies that GnRH acts as a meiosis-stimulating factor in the oocytes of rats [19] and in various fresh water and marine fishes (see [20]). However, definitive evidence is lacking for any vertebrate to show that one or more forms of the mRNA are translated into peptides in the testis or that there is a specific developmental pattern for the expression of individual GnRH peptides (see [21, 22]).
In this paper, the presence of GnRH peptides in the ovary and testis of rainbow trout is demonstrated by HPLC and RIA. The ovary and testis were examined at various stages of development in rainbow trout at 17–21 mo of age. Immunological and chromatographical methods were used to determine that multiple forms of GnRH are present and are developmentally regulated in the salmonid ovary and testis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male and female rainbow trout (Oncorhynchus mykiss) used in this study were raised in an open lake fed by a natural stream in Sooke, British Columbia (Mountain Trout Sales). Most trout ovulate and spawn for the first time at 3 yr and then continue to spawn annually. However, 10–20% of trout mature precociously, beginning at about 1.5 yr of age, and may spawn at 2 yr, one year ahead of their cohorts. This true precocious maturation is a normal reproductive state in which offspring can be produced. Fish were judged to be precociously mature on the basis of the weight of the ovaries and testes (see Table 1) and of other defining characteristics. Histology was not done on the gonads. However, the precocious gonads at 17 mo were 2- to 4-fold greater in weight than those of their nonprecocious counterparts; also the testis was white and smooth, and the ovary had orange eggs, compared to small, thin translucent organs in the normal fish. At 18–20 mo, the precocious ovaries had not changed in weight from 17 mo, but the testes weight was 8-fold increased. At 21 mo, the precocious ovaries were 44 times the weight of those in normal fish and contained large eggs.
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The fish were exsanguinated for several minutes. The ovaries, testes, and brains were removed, immediately frozen on dry ice, and subsequently stored at -80°C. Ovaries, testes, and brains were taken from a total of 109 fish that were either 17, 19, or 21 mo of age (see Table 1 for details).
Tissue Extraction
The age, number of organs, and total weight of the tissue examined are listed in Table 1.
The frozen tissue was crushed to a powder using a cold mortar and pestle. For larger extractions, a Waring blender (New Hartford, CT) was used to powder the material. Peptides were extracted with an acetone-HCl mixture, and soluble lipids were removed with petroleum ether as previously described [23]. The final acetone-water soluble mixture was reduced in a vacuum centrifuge to approximately 2 ml and filtered through a 45-µm filter.
HPLC
The filtrate was loaded onto a Supelcosil LC-18 column (25.0 cm x 4.6 mm; 5-mm particle size; Supelco Canada, Oakville, ON, Canada) with a guard column of the same material attached. Each filtered extract (2 ml) was loaded in repeated injections of 600 µl each or less at 2-min intervals onto a 1-ml loop. Initially, 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 there for an additional 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).
Injections for each set of extracts were preceded by an overnight wash and a blank run in which a 600-µl volume of Milli-Q water (Millipore, Bedford, MA) was injected onto the column. A blank was injected, and the fractions were collected and assayed between each application of extract to ensure that the column was free of any contaminating residual GnRH from previous HPLC analyses. Mammalian GnRH (mGnRH) standard had never been previously injected onto this column, also ensuring that the presence of mGnRH was not due to residual synthetic mGnRH peptide. Synthetic standards were applied to the column after each set of extracts was assayed. Four GnRH forms—mGnRH, cGnRH-II, dogfish (df)GnRH, and sGnRH—were combined at 200 ng each and applied to the HPLC system as described for the above extracts. The elution positions of the standards on the chromatograph were confirmed by absorbance peaks (A = 280 nm) and a GnRH-specific RIA.
RIA
Aliquots of 100 µl from each fraction collected for each HPLC run were dried and assayed for irGnRH by methods previously described [24]. The reconstituted extracts were assayed using various antisera and 125I-labeled synthetic GnRH tracers in a competitive RIA. The three assay systems are as follows: 1) mGnRH standard, GF-6 antibody at a final dilution of 1:25 000, and mGnRH 125I tracer; 2) mGnRH standard, B-7 antibody at 1:10 000, and mGnRH 125I tracer; and 3) cGnRH-II standard, 7CR-10 antibody at 1:37 500, and cGnRH-II 125I tracer. Serial dilutions were done on brain fractions if values of irGnRH were less than B/B0 = 20%; the value closest to 50% B/B0 was used to estimate the quantity of irGnRH present.
Antisera GF-6, B-7, and 7CR-10 were raised in rabbits in our laboratory against sGnRH, mGnRH, and dfGnRH, respectively. GF-6 cross-reacts with a number of forms of GnRH. It detects mGnRH (100% cross-reactivity with mGnRH tracer), seabream (sb)GnRH (94.1%), sGnRH (23.7%), and cGnRH-II (10.5%) [5]. B-7 detects mGnRH (100% cross-reactivity with mGnRH tracer) [4]. Antibody 7CR-10 detects cGnRH-II (100% cross-reactivity with a cGnRH-II tracer), sGnRH (84.8%), dfGnRH (25.0%), and lamprey GnRH-III (12.6%) [4]. The limits of detection (B/B0 = 80%) were 2–12 pg for GF-6, 1–16 pg for 7CR-10, and 3–6 pg for B-7.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The GF-6 antibody, which detects many forms of GnRH, did not detect any peaks indicative of irGnRH-like material in the extracts of immature normal ovaries or testes (Fig. 1).
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Ovaries and Testes from Precocious Trout at 17 Months
GF-6 detected a small amount of immunoreactive material (0.075 ng) in fractions 24–25 of the extract from the precocious ovaries (not shown) but not in precocious testes.
With antiserum 7CR-10, which has a higher cross-reactivity than GF-6 for cGnRH-II, 0.282 ng (total in two fractions) of irGnRH was found in fractions 24–25 in the extract from the precocious ovaries (Fig. 1), but GnRH was not detected in the testes. Synthetic cGnRH-II standard eluted in position 24 under the same HPLC conditions. The mGnRH-specific antibody, B-7, did not detect mGnRH immunoreactive material in the extract from the precocious ovaries at this stage (not shown).
Ovaries and Testes from Precocious Trout at 18 to 20 Months
In precociously mature ovaries, the GF-6 antibody detected four peaks indicative of GnRH-like immunoreactivity: mGnRH (fractions 18–20), cGnRH-II (fractions 24–26), an unknown GnRH (fractions 30–32), and sGnRH (fractions 39–41) (Fig. 2, upper graph). In our HPLC program, mGnRH standard consistently eluted at position 20, the cGnRH-II standard at position 25, the dfGnRH standard at positions 31/32, and the sGnRH standard at positions 39/40. However, it is not clear whether the GnRH-like immunoreactivity that eluted in fractions 30–32 corresponds to dfGnRH or if it is a novel form. There was a total of 3.7 ng of immunoreactive mGnRH compared to only 308 pg immunoreactive sGnRH. The amount of immunoreactive cGnRH-II and of the unknown GnRH form were roughly the same (1.7 ng, total in three fractions). Synthetic mGnRH, cGnRH-II, and sGnRH eluted in positions corresponding to the native material under the same HPLC conditions.
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Although testes from only one precocious animal was used, analysis of the precocious testis extract showed three immunoreactive peaks. Two peaks corresponded to elution positions of synthetic mGnRH and cGnRH-II (Fig. 2, upper graph). The third peak in fractions 30 and 31 (201 pg) could represent a novel GnRH form. Immunoreactive mGnRH-like (333 pg) and cGnRH-II-like (348 pg) material was detected with GF-6 in fractions 19–21 and 24–25, respectively (Fig. 2). sGnRH-like immunoreactivity was not detected in the precocious testis extract.
All the HPLC values have been converted to picogram per organ for comparison purposes (Table 1). The total amount of immunoreactive mGnRH, cGnRH-II, unknown GnRH, and sGnRH detected in each precocious ovary was 232, 106, 106, and 19 pg, respectively. In each precocious testis the total amount of immunoreactive mGnRH, cGnRH-II, and unknown GnRH detected was 167, 174, and 100 pg, respectively.
The presence of mGnRH-like immunoreactivity in the precocious ovaries (3.5 ng, total in 16 ovaries) and testes (195 pg in 2 testes) was confirmed by the immunoreactive peaks, primarily in fractions 18 and 19, using the mGnRH-specific antiserum, B-7 (Fig. 2, lower graphs).
Ovaries and Testes from Immature or Precocious Trout at 21 Months
GF-6 antiserum did not detect any peaks of irGnRH-like material in the extracts of the normal immature ovaries or in precocious ovaries or testes from 21-mo-old fish (Fig. 3). Also, each fraction of the extract from the precocious ovaries was reassayed with 7CR-10, but irGnRH material was not detected.
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Brains from 17- or 21-Month-Old Rainbow Trout
Both cGnRH-II-like (fractions 24–28) and sGnRH-like (fractions 38–40) immunoreactivity were detected in brain extracts from 17- and 21-mo-old fish (Figs. 4 and 5). Peaks corresponding to immunoreactive mGnRH were not detected with GF-6 in brain extracts (see fractions 18–20; Figs. 4 and 5). However, a small amount of irGnRH that could correspond to the unknown form observed in the ovaries and testes is present in position 30–31 (Figs. 4 and 5). The irGnRH detected in position 33–35 has previously been analyzed and determined to be sGnRH (J.F.F. Powell, personal communication).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The demonstration of the local presence of GnRH decapeptide in these tissues has been reported for the goldfish ovary [18], but not for testis. The first report of a GnRH-like factor extracted from rat follicular fluid capable of releasing FSH and LH from rat pituitary cells in vitro was made in 1981 [26], but the authors reported later that they could not repeat the result [27]. Other HPLC studies detected GnRH-like activity in ovarian extracts from various species, but the protein products were shown not to be GnRH [28, 29]. Similar reports of GnRH-like immunoactivity in testicular tissue (see [30]) or ovarian tissue [15] have been inconclusive. Pati and Habibi clearly showed that sGnRH was present in the goldfish ovary, but they did not identify any other GnRH forms [18].
This is the first report showing that mGnRH is present in the ovary and testis, but is not present in the brain, at least not at 17 or 21 mo of age (Figs. 4 and 5). In addition, small amounts of irGnRH-like material in the brain and gonads are detected in positions 30–32, which could represent the expression of an unknown (possibly novel) form of GnRH. Clearly, cGnRH-II and sGnRH are the dominant forms of GnRH in the salmonid brain, in which they are expressed in large quantities compared to the amounts in the gonads. Nevertheless, our results show that different forms of GnRH (m, c-II, s, and unknown GnRH in females and all but sGnRH in males) are synthesized at specific stages during ovarian and testicular cell maturation.
Previously we showed the expression of two different sGnRH-encoding mRNAs and genes in both the ovary and testis of rainbow trout [25]. Isolation and characterization of neither mGnRH- nor cGnRH-II-encoding transcripts have been reported to date. In contrast to the previous study, which demonstrated expression of sGnRH mRNA in precociously mature testes from 18 to 20 mo of age, the present HPLC/RIA study found that the sGnRH peptide was not detectable, at least not at the time the tissue was collected (20 July). Whether this was due to a small sample number or to fluctuations in the levels and/or forms of GnRH remains to be clarified. Furthermore, detection of cDNA for sGnRH confirms that the gene is transcribed. The difference between these two results may be one of difference in sensitivity of the two techniques: PCR amplification is sensitive compared to RIA, for which the threshold of detection for the peptide is only 3–7 pg.
Rainbow trout were chosen because, unlike other salmonids in the same genus (Oncorhynchus), rainbow trout mature early at 3 yr; they do not die after spawning (iteroparous); and they are readily available because they remain in fresh water throughout their lives. In the second year of life, up to 20% of the population may have well-developed reproductive tissue. The precocious testes examined were very large compared to those of their immature counterparts (see Table 1). The ovaries examined were considered to be precocious because they had well-defined features indicating they were maturing (visible eggs, orange coloration, larger size).
Our HPLC/RIA data from extracts of immature normal and precociously mature gonads show that the GnRH peptides are present in a stage-specific period, i.e. GnRH peptides were evident in precocious gonads at 18–20 mo, but were not present even in large precociously ripe gonads at 21 mo. The first indication of GnRH protein was the presence of cGnRH-II in the precocious ovary at 17 mo (35 pg/organ). The greatest expression of GnRH peptide in this study was for mGnRH at 232 pg/organ, followed by 106 pg/organ for both cGnRH-II and the unknown GnRH in pooled extracts from 18- to 20-mo-old precocious ovaries. These contents were considerably less than in the brain but were easily detectable (Table 1). Only in precocious ovaries from 18- to 20-mo-old fish was sGnRH detected (19 pg/organ). The extract from the 19-mo-old precocious testis showed the presence of similar amounts of mGnRH, cGnRH-II, and the novel GnRH as found for the 18- to 20-mo-old pooled ovaries (see Table 1). If there is a requirement for GnRH at stages just before ovulation in the salmonid follicles, these tissues need to be analyzed later in the year (October or November), because GnRH was not present in any tissues examined in September.
Synthesis of the mRNAs that encode both GnRH and its receptor in ovarian and testicular cells points to a direct physiological role for this factor. It is unlikely that secreted hypothalamic GnRH acts peripherally on the gonads because of the low concentration and short half-life in the blood (see [14]). The present results suggest that GnRH is not present as a protein in detectable amounts except in specific stages, which may offer a clue to the function of gonadal GnRH.
The present experiments do not indicate the function of GnRH in the gonads of rainbow trout. However, others have used rats to show that there may be a function during early maturation. In immature granulosa cells, for example, exogenous GnRH inhibits FSH-stimulated steroidogenesis and FSH/LH receptor production [22, 31]. This FSH-suppressing action has been demonstrated to profoundly disrupt granulosa cell maturation in vitro [32]. GnRH also inhibits gonadotropin-mediated progesterone biosynthesis in the ovary and androgen biosynthesis in the testis. This GnRH-suppressing action has been shown to affect the synthesis of LH receptors and steroidogenic enzymes [31], as well as enzymes involved in cAMP production and signal transduction [22, 32, 33]. The present experiments in trout indicate that caution should be used in interpreting results from rat studies. In rats a number of effects have been reported for the gonads after the addition of GnRH in vitro. However, these effects may not be physiological if the rat gonad does not normally express GnRH peptide at that stage of development.
We show that GnRH peptide is present only at a time that coincides with oocyte and sperm maturation. In mature granulosa cells, GnRH is associated with stimulatory effects on meiotic maturation, oocyte cleavage, ovulation, and luteinization [19, 22, 32]. The precociously mature gonads in the present study are most likely at the stage at which the oocytes and sperm are undergoing maturation and will be released in 4–5 months at spawning. Stimulation of steroidogenesis is also evident in cultured adult rat Leydig cells after short-term exposure (4 h) to GnRH [34]. In the ovary, GnRH may modulate follicular development by selection of particular follicles for ovulation while promoting atresia in follicles more sensitive to early GnRH inhibition [21, 32].
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FOOTNOTES |
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2 Correspondence. FAX: 250 721 7120; nsherwoo{at}uvic.ca
Accepted: January 20, 1999.
Received: October 26, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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