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Biology of Reproduction 62, 1329-1334 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Article

Differential Splicing of Three Gonadotropin-Releasing Hormone Transcripts in the Ovary of Seabream (Sparus aurata)1

Massimo Nabissia, Laura Soverchiaa, Alberta M. Polzonetti-Magni2,a, and Hamid R. Habibib

a Dipartimento di Scienze Morfologiche e Biochimiche Comparate, University of Camerino, 62032 Camerino (MC), Italy b Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies demonstrated the presence of high-affinity GnRH binding sites and compounds with GnRH-like activity in the ovary of seabream, Sparus aurata, providing evidence for the role of GnRH as a paracrine/autocrine regulator of ovarian function in this species. In the present study, the expression of three forms of GnRH (salmon, chicken-II, and seabream) genes in this marine teleost species was demonstrated for the first time. Moreover, there is evidence for differential splicing and intronic expression of cGnRH-II and sbGnRH. Treatment of seabream follicle-enclosed oocytes with salmon GnRH stimulated reinitiation of oocyte meiosis, whereas chicken GnRH-II treatment was without effect. Novel information was also provided about organization of cGnRH-II and seabream GnRH transcripts, confirming that GnRH gene organization is maintained through evolution, despite changes in the size and sequence of exons and introns.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GnRH plays a pivotal role in reproduction by regulating pituitary gonadotropin synthesis and release, which in turn modulate gonadal activity [1, 2], including that of seabream [3]. 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 [1–4], including placental mammals [5, 6]. There has also been parallel evolution of the GnRH receptors, resulting in diverse regulatory functions of GnRH peptides in the brain, pituitary, and other peripheral tissues. In particular, GnRH molecules have been shown to function as neurotransmitters, as well as a local signal in the gonads [7]. There is also evidence for the presence of GnRH in the ovary of a number of mammalian and nonmammalian species [8, 9] including seabream [10]. The cDNA sequences encoding the precursor of mammalian GnRH (mGnRH), salmon GnRH (sGnRH), chicken GnRH-I, chicken GnRH-II (cGnRH-II), catfish GnRH, and seabream GnRH (sbGnRH) have been isolated and characterized from different species [4, 11–13], and GnRH gene expression has been demonstrated in the ovary of a number of mammalian species [14–18] and other vertebrates including fish [19, 20].

Primary structure of GnRH is largely conserved, and GnRH precursors in all species so far studied show the same general organization, containing regions for the signal peptide, GnRH, amide-donating glycine, processing site, and GnRH-associated peptide (GAP). Whereas the segment coding for the mature GnRH has a high degree of similarity among different species, there is less than 50% similarity in the signal peptide and GAP regions. Previous studies in seabream demonstrated the presence of high-affinity GnRH-binding sites and compounds with GnRH-like activity in the ovary, providing evidence for the role of GnRH as a paracrine regulator of ovarian function in this species [10]. The present study extends this information and demonstrates the expression of GnRH genes and action of GnRH peptides on reinitiation of oocyte meiosis in the seabream ovary.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Adult female seabream (Sparus aurata, 0.7–1.5 kg BW) were purchased from a commercial fish farm (La Rosa, Orbetello, west coast of Italy; lat 42°28'N, long 11°12'E). Fish were maintained in aquaria at 22–23°C under natural photoperiod (10L:14D) and 35 parts per thousand salinity, and fed with commercial fish food. All fish were netted, anesthetized in MS-222 (100 mg/L; Sigma, Milan, Italy), and decapitated before removal of ovaries. Ovaries were immediately frozen in liquid nitrogen and stored at -70°C until use in molecular biology studies.

Hormones

sGnRH-A (D-Arg6,Trp7,Leu8,Pro9]-N[Et]-GnRH), SGnRH ([Trp7,Leu8]-GnRH), and cGnRH-II ([His5,Trp7,Tyr8]-GnRH) were purchased from Peninsula Laboratories Ltd., Belmont, NY. Peptides were solubilized in 0.1 M acetic acid (10 µg/20 µl), stored at -20°C, and used within 4 wk. Appropriate concentrations of the peptide were prepared by diluting the stock solutions immediately before use in an experiment.

Follicle Incubation

Ovaries obtained from five different animals in the recrudescence period were dissected out and washed in minimum essential medium (MEM; Gibco/BRL, Gaithersburg, MD) in a sterile Petri dish. Pooled follicles were manually separated under a stereomicroscope, and only 0.6- to 0.7-mm follicles were used for in vitro incubation. Follicles were incubated in MEM (with 0.05% streptomycin) in sterile multiwell plates; each well contained 20 follicle-enclosed oocytes in 1 ml of MEM plus the appropriate compounds. Incubations were performed for 24, 36, 48, 60, 72, 84, and 96 h at 18°C in the dark. After each period, the incubation was stopped by addition of acetic acid (50% final concentration) to each well, and the incubate was allowed to stand for 10 min at 18°C to clear the oocyte cytoplasm. The cleared oocytes were observed under the stereomicroscope, and absence of the germinal vesicle (oocyte nucleus) indicated breakdown of the nuclear envelope (germinal vesicle breakdown [GVBD]) and reinitiation of meiosis, as described previously for goldfish [21].

Total RNA Extraction

Total RNA was extracted, for each reproductive period, from 5 g of an ovarian pool from five fish, using Trizol RNA isolation reagent (Gibco/BRL) based on the acid guanidinium thiocyanate-phenol-chloroform extraction method [22]. Final RNA concentration was determined by optical density (OD) reading at 260 {lambda}, and integrity was verified by ethidium bromide staining of 28S and 18S RNA bands on a denaturing agarose gel.

Oligonucleotides

GnRH primers were designed on the basis of the sequence of the salmon, chicken-II, and seabream GnRH cDNAs of seabream (U30311; U30325; U30320) [12], and were purchased from Gibco/BRL. The 19-mer sGnRH sense primer (5' ATGGAGGCGAGCAGCAGAG 3') corresponded to nucleotide regions 233–251, and the 20-mer sGnRH antisense primer was complementary to nucleotide region 477–496 of sGnRH cDNA. The 19-mer cGnRH-II sense primer (5' ATGTGTGTATCTCGGCTGG 3') corresponded to nucleotide region 118–137, and the 19-mer cGnRH-II antisense primer (5' CTTCCTCTTCTGGAGCTCT 3') was complementary to nucleotide region 355–373 of cGnRH-II cDNA. The 19-mer sbGnRH sense primer (5' CTCAAACCTCTGGATCCTG 3') corresponded to nucleotide region 52–70, and the 19-mer sbGnRH antisense primer (5' TTCTGTGTCCGATGTCCCG 3') was complementary to nucleotide region 303–311 of sbGnRH cDNA.

Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)

Ten micrograms of total RNA from seabream ovary were reverse-transcribed into cDNA in a total volume of 25 µl containing 1 µg of oligo(dT) 12–18 primers (Gibco/BRL) and 100 IU of Moloney murine leukemia virus (MMLV) reverse transcriptase (Gibco/BRL). The reaction was carried out for 15 min at 25°C, and then for 90 min at 37°C. After deoxyribonuclease (DNase) treatment of the RT mixture, an aliquot (1 µl) of the resulting cDNA products was subsequently amplified with 2.5 IU Taq DNA polymerase (Gibco/BRL) in 50 µl of master mix containing single-strength PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 0.5 mM dNTP, and GnRH primers (50 pmol each). The PCR amplification, for all sets of primers, was carried out for 35 cycles in an automated thermocycler (MJ Research, Watertown, MA) with thermocycle profile (denaturation at 94°C for 40 sec, primer annealing at 58°C for 40 sec, primer extension at 72°C for 1 min) followed by a post-PCR incubation at 72°C for 7 min. PCR products were then extracted with phenol-chloroform-isoamyl alcohol, and an aliquot of each sample was electrophoresed on 2% agarose gel in 0.5-strength TAE buffer (Tris-HCl 20 mM pH 7.5, acetic acid 20 mM, EDTA 0.5 mM, pH 8.0). Different negative controls were prepared. First, the same amount of total RNA as that used for the RT-PCR assay was added to the RT reaction mixture without reverse transcriptase and was subsequently amplified, to confirm the absence of genomic contamination. Second, all the components of the RT reaction were prepared without RNA and subsequently amplified to confirm the absence of contamination in the reagents used. The data were considered useful only if no bands were observed in the negative controls.

Cloning and Sequencing

Twenty nanograms of PCR products from sGnRH and 28 ng of PCR products from cGnRH-II and sbGnRH primer amplifications were cloned using pGEM-T vector system (Promega, Madison, WI). Ten positive clones for each sample were screened to confirm the presence of an insert and subsequently sequenced. DNA sequence analysis was performed by dye-labeled terminators using a DNA sequencing kit (Perkin Elmer, Irvine, CA) and M13 universal primers that flank the inserted DNA. The nucleotide sequences were read in both directions.

Statistical Analysis

Follicle incubation results were analyzed by one-way ANOVA with Stat View 512+ (Brain Power, Calabasas, CA). P < 0.05 was taken to indicate a statistically significant difference between means. Results are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Experiments on Oocyte Maturation

Previous studies provided characterization of three forms of GnRH (sGnRH, cGnRH-II, and sbGnRH) in the seabream brain [23], as well as evidence for the presence of high-affinity GnRH binding sites and compounds with GnRH-like activity in the seabream ovary [10]. We used isolated seabream follicle-enclosed oocytes, in vitro, to investigate direct action of sGnRH and cGnRH-II on the reinitiation of oocyte meiosis as indicated by GVBD. Administration of sGnRH at 10-6 M concentration significantly increased the GVBD response, while GVBD responses at lower (10-7 M, 10-8 M) concentrations of sGnRH were not significantly different from that of MEM (Fig. 1). Treatment with cGnRH-II did not affect GVBD response in the seabream ovary. A significantly high GVBD response was observed after treatment with 10-7 M of sGnRH-A (used as positive control) compared to sGnRH (Fig. 1).



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  		FIG. 1. Effect of sGnRH and cGnRH-II on reinitiation of meiosis determined by means of GVBD in cultured follicle-enclosed seabream oocytes. GVBD values were determined after 96 h of incubation, and nonstatistically significant incubation periods are not shown. Each value represents a percentage of GVBD determined using 60 oocytes (20 oocytes per well). Values are means ± SE. *Significantly different from control (P < 0.05). sGnRH-A was used as positive control, MEM (culture medium)

GnRH Gene Expression and Characterization

Three sets of primers were designed on the basis of sGnRH, cGnRH-II, and sbGnRH cDNA sequences characterized previously in the seabream brain [12, 13]. The primers amplified regions between the signal peptide and GAP for all three GnRH forms. For each set of primers, the predicted PCR products were 264 base pairs (bp) for sGnRH, 255 bp for cGnRH-II, and 259 bp for sbGnRH. Using sGnRH primers, amplification of cDNA from seabream ovarian total RNA at the recrudescence phase resulted in a PCR product of 264 bp, while the use of cGnRH-II and sbGnRH primer sets resulted in larger than expected products of 549 bp and of 551 bp, respectively (Fig. 2). Two independently amplified 264-bp fragments were cloned, and nucleotide sequences were found to have 100% identity with the appropriate region of sGnRH cDNA characterized from seabream brain. Cloning of the 549-bp fragments (from cGnRH-II primer) and nucleotide sequence analysis of two different clones revealed in each case a number of matching regions with 100% similarity to cGnRH-II cDNA characterized in seabream brain. The regions of similarity in the 549-bp fragment were 1–135, 272–355, and 514–549, corresponding to cGnRH-II cDNA regions of 119–253, 254–337, and 338–373, respectively (Fig. 3A). The genetic information contained in the nonmatching regions of the 549-bp PCR product corresponded to an intervening sequence (introns) in-frame with exons, due to the presence of invariant GT and AG dinucleotides found at the 5' and 3' intron borders, which are part of the largest consensus sequence characteristic of nuclear pre-mRNA introns. These regions had higher A+T contents than their neighboring exons, and the sequence upstream of 3' ends was preceded by a stretch of pyrimidine and T-run, T, and A homopolymers. The branch point sequences were also determined, and the presence of inverted repeats that could interfere with splicing was revealed (Fig. 4). Cloning and sequencing of the 551-bp fragments (from sbGnRH primer) also revealed a number of matching regions with 98% similarity to sbGnRH cDNA characterized in the seabream brain. The regions of similarity in the 551-bp fragment were 1–133, 314–412, and 525–551, corresponding to sbGnRH cDNA regions 52–184, 185–283, and 284–311, respectively (Fig. 3B). Similarly, the nonmatching regions of the 551-bp PCR product contained genetic information corresponding to the intervening sequence (introns) because of the presence of invariant GT and AG dinucleotides found at the 5' and 3' intron borders (Fig. 4).



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  		FIG. 2. PCR results from analyses in different reproductive periods. NR, Nonreproductive; R, reproductive; PS, postspawning; T, PCR from total RNA; N, PCR without template. s, c-II, sb: RT-PCR products using sGnRH, cGnRH-II, sbGnRH sets of primers, respectively. M, Amplisize agarose markers (Bio-Rad, Milan, Italy). The data were considered useful only if no bands were observed in the negative controls



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  		FIG. 3. A) The cDNA nucleotide sequence encoding the partial cGnRH-II precursor and the deduced amino acid sequence. The 5' and 3' intron borders are in boldface; the 100% identity regions with cGnRH-II cDNA from seabream pituitary are underlined. *Stop codon. The length of each region is numbered in the schematic representation. B) The cDNA nucleotide sequence encoding the partial sbGnRH precursor and the deduced amino acid sequence. The 5' and 3' intron borders are in boldface; the 98% identity regions with sbGnRH cDNA from seabream pituitary are underlined. *Stop codon. The length of each region is numbered in the schematic representation



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  		FIG. 4. Analysis of intron regions of cGnRH-II and sbGnRH partial transcript sequence. Top) Sequence and nucleotide position of intron junctions. Invariant GT dinucleotides (at 5' intron border) and terminal AG dinucleotide (at 3' end of the pre-mRNA introns) are in boldface. The characteristic sequence of introns is represented as nucleotide positions in the cGnRH-II and sbGnRH partial transcript sequences. Bottom) Pyrimidine stretch (pyrimidine stretch that preceded 3' end of introns); T/A homop. (polymers composed only of A or T nucleotides); Inv.repeat (inverted repeat sequences between branch points and 3' splice sites); % ex/int TA (percentage of T and A nucleotides in exons and introns)

The predicted amino acid sequence derived from the ovarian cGnRH-II and sbGnRH partial transcript nucleotide sequence was compared to the amino acid sequence of brain cGnRH-II and sbGnRH [12, 13]. In the case of cGnRH-II, the presence of a stop codon (TAA) in nucleotide position 286–288 may give rise to the translation of a shorter GAP peptide than the pre-cGnRH-II peptide produced in the seabream brain. Similarly, a stop codon (TAA) in nucleotide position 212–214 of ovarian sbGnRH may result in a shorter GAP peptide compared to the pre-sbGnRH peptide produced in the seabream brain. From the sequence data, we can also indicate, in terms of exon/intron, the organization of the cGnRH-II and sbGnRH genes in seabream (Fig. 3). Analysis of size distribution of exons and introns suggests that the cGnRH-II and sbGnRH genes in seabream ovary maintain the same exon/intron distribution as in the other GnRH genes characterized (in human, rat, and salmon). In particular, from the sequences of cGnRH-II and sbGnRH partial transcripts, we may predict the gene organization in those regions. As shown in Figure 3, the coding region for signal peptide, GnRH, and processing sites is separated from the coding regions for GAP peptide by two introns, both in cGnRH-II and in sbGnRH partial transcripts.

To investigate possible seasonal variation in splicing of these three GnRH genes in seabream, we performed RT-PCR from total RNA obtained during the nonreproductive and postspawning periods. The results indicated no seasonal variations in the processing of these pre-mRNA in the seabream ovary (Fig. 2). In these studies, we were not able to demonstrate ovarian expression of GnRH by Northern hybridization, presumably because of low levels of expression at the time of the experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The expression of the GnRH gene was previously reported in human and rat ovary [11, 14, 15]. The study in the rat suggests that a proportion of the ovarian GnRH transcripts contain intronic sequences and utilize a different transcription start, compared to hypothalamic GnRH [14]. In the light of previous studies demonstrating expression of GnRH genes in the ovary of various vertebrates [14–20], and the existence of sGnRH, cGnRH-II, and sbGnRH in the seabream brain [23], we investigated the expression of these three different GnRH forms in the seabream ovary by RT-PCR, demonstrating that cGnRH-II and sbGnRH transcripts undergo differential splicing compared to the same GnRH mRNA forms expressed in the seabream brain. Most nuclear messenger RNA precursors (pre-mRNA or heteronuclear (hn) RNA) in higher eukaryotes contain multiple introns, which must be precisely excised by RNA splicing [24]. Alternative RNA splicing is the process that allows the selection of different combinations of splice sites within an hnRNA, and is part of the expression program of a large number of genes implicated in cell growth and differentiation [24, 25]. Patterns of alternative splicing can be very complex and can involve alternative introns and exons as well as variations in the position of individual splice junctions [24]. Regulated alternative splicing can lead to the production of different proteins from a single hnRNA or can function as an on/off switch during development [24, 25]. In this context, the presence of sGnRH, cGnRH-II, and sbGnRH transcripts has been demonstrated previously in the seabream brain [10], and our findings demonstrate that cGnRH-II and sbGnRH are alternatively spliced in different tissues, presumably because of different splicing mechanisms. It has also been demonstrated that the insertion of inverted repeats between the branch point and 3' splice sites of yeast and mammals interferes with splicing [26], which might also be responsible for differential splicing of cGnRH-II and sbGnRH in seabream ovary. The differential splicing observed in seabream ovary was not influenced by reproductive stages, suggesting a major role of the intron/exon element in controlling the differential splicing rather than a variation in the abundance of a specific factor regulating alternative splicing [27]. It should be noted, however, that the present results are based on studies carried out during a rather narrow period of seabream life history, and we cannot rule out the possibility of seasonal variations encompassing different stages of sexual development in this species. The involvement of macro (pituitary gonadotropins) and micro (ovarian peptides) regulatory systems is particularly important in species such as seabream, which contain asynchronous ovary continuously undergoing change and development. While the relationship between GAP and GnRH may not be clear, there is evidence that GAP and GnRH may have related functions [28] and that other cryptic peptides associated with neurohormones may be involved in the correct processing and packaging of the hormone [29]. In this respect, the presence of a stop codon in the introns of the cGnRH-II and sbGnRH partial transcripts may be related to the biological activity of GnRH molecular forms. Study of paracrine ovarian function in seabream is very interesting, since this species starts development as a hermaphrodite and undergoes significant changes, from being functional males in puberty to becoming functional females at later stages.

The present results also provide information about organization of the cGnRH-II and sbGnRH genes. In this context, the exon/intron organization of cGnRH-II and sbGnRH partial transcripts was found to be similar to that of the other GnRH genes characterized previously, indicating that GnRH gene organization, in terms of exon/intron location, is maintained through evolution, in spite of changes in the size and sequence of exons and introns [30].

In conclusion, the present findings strongly support the hypothesis that sGnRH, and possibly other forms of GnRH variants, are involved in paracrine/autocrine regulation of seabream ovarian function.


   FOOTNOTES
 
First decision: 6 April 1999.

1 This study was supported by a grant to Prof. Alberta M. Polzonetti-Magni from the MURST (PRIN) Italy. Back

2 Correspondence: Alberta Maria Polzonetti-Magni, Dipartimento di Scienze Morfologiche e Biochimiche Comparate, University of Camerino, Via Camerini no 3, 62032 Camerino (MC), Italy. FAX: 39 0737 403217; alberta{at}camserv.unicam.it Back

Accepted: December 20, 1999.

Received: March 10, 1999.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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