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Characterization of Complementary DNA Encoding the Precursor for Gonadotropin-Releasing Hormone and Its Associated Peptide from a Teleost Fish Chris T. Bond, Richard C. Francis, Russell D. Fernald, and John P. Adelman Vollum Institute (C.T.B., J.P.A.) and the Department of Cell Biology and Anatomy (J.P.A.) Oregon Health Sciences University Portland, Oregon 97201 Department of Human Biology Stanford University (R.C.F., R.D.F.) Stanford, California 94305 Reproductive maturity among male African cichlids Haplochromis burtoni is cued by a series of environmental and social interactions and is mediated physiologically by GnRH. A cDNA clone encoding the precursor for GnRH was isolated from this teleost. The molecular architecture of the predicted prohormone is analogous to that of the previously characterized mammalian forms; however, the predicted sequence of the associated peptide is strikingly different. Attempts to isolate a putative second precursor using low stringency hybridization were not successful despite evidence that a second related decapeptide exists in at least some teleost species. (Molecular Endocrinology 5: 931-937, 1991) most stable region, residues 1-3, is responsible for releasing gonadotropins, while the region with the most changes, residues 5-8, is thought to mediate receptor binding (2). In some vertebrates, including several teleosts, there is considerable chromatographic and immunological evidence that more than one form of GnRH exists, and in the chicken two distinct forms have been isolated, and the amino acid sequences determined (25). These decapeptides differ by three amino acids; one form is similar to lamprey (chicken II), while the other is more similar to mammalian GnRH (chicken I), suggesting that chicken II may be the older form. However, DNA coding sequences have been determined for the prohormone only in human (6, 7), rat (7), and mouse (8) species. In the teleost Haplochromis burtoni, as in many vertebrates, sexual development is regulated by social interactions (9-11). A normal population of H. burtoni contains two distinct male types: territorial (machos) and nonterritorial (wimps). Macho males are brightly colored, sexually mature, and establish and defend territories for feeding and breeding. In contrast, wimps are cryptically colored, sexually immature, and do not reproduce. In social encounters, machos are dominant over wimps, and this social dominance is correlated with the size of forebrain magnocellular neurons in the preoptic area which are immunoreactive for GnRH (1214). In males that gain territories and become macho, these GnRH neurons enlarge significantly, while in males that lose territories and become wimps, GnRH cells shrink. Thus, an extraordinary neuronal plasticity, dependent upon social cues and interactions, remains throughout the adult life of H. burtoni males (Francis, R. C , and R. D. Fernald, in preparation). As a first step to understanding the cellular and molecular basis of this plasticity, we report the isolation INTRODUCTION In vertebrates, GnRH plays a central role in development and maintainance of reproductive function. This decapeptide regulates the release of pituitary gonadotropins, which stimulate the release of steroid hormones from the gonads. In turn, these steroids feedback to both the hypothalamus and the pituitary, completing the hypothalamic-pituitary-gonadal axis, and act to attenuate the release of GnRH and its effects on gonadotroph cells (1). During 500 million yr of vertebrate evolution, the primary structure of GnRH has been remarkably conserved. The length of GnRH has remained at 10 amino acids, the amino (pGlu) and carboxy (Gly-amide) residues are unchanged, and five residues are identical from lamprey to mammals. The 0888-8809/91/0931 -0937$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society 931 MOL ENDO-1991 932 Vol 5 No. 7 shown in Fig. 2. The most 5' AUG initiates an open reading frame of 90 amino acids. The N-terminal 23 residues comprise a hydrophobic signal sequence that bears significant homology to cloned mammalian prepro-GnRH and is directly followed by the GnRH decapeptide sequence, identical to that determined from peptide sequencing of the salmon decapeptide. Analogous to the mammalian forms, a glycine residue in the 11 position, thought to donate the amide group found at the C-terminus of mature GnRH, is followed by dibasic residues characteristic of trypsin-like proteolytic substrates found in many polyprotein hormone precursors (18). The remainder of the open reading frame predicts a novel associated peptide, cichlid GnRH-associated peptide (cGAP). This peptide is 54 amino acids in length and bears no homology to the predicted mammalian GnRH-associated peptides (mGAPs). The preprohormone is predicted to have a mol wt of 10.1 kilodaltons; there are no consensus sites for N-linked glycosylation. The structures of the three known mammalian precursors and the teleost precursor are compared in Fig. 3. and characterization of a cDNA encoding GnRH in this African cichlid. RESULTS Isolation of H. burtoni Pro-GnRH cDNA The enlarged hypothalamic neurons characteristic of macho males may reflect an increased level of proGnRH mRNA, and therefore, a cDNA library was constructed from mRNA extracted from the brains of macho males. Approximately 5 x 105 primary clones were screened with two pools of oligonucleotides, 17 bases in length, comprised of all possible sequences encoding the first eight amino acids of salmon GnRH (Fig. 1) (15). Hybridization specificity was obtained by washing the filters in 3 M tetramethylammonium chloride (16, 17), and a single positively hybridizing clone was isolated. The 435 bases of nucleotide sequence and the predicted translation product of the cDNA insert are 1a 10 pGlu Lamprey pGlu Chicken II pGlu Salmon pGlu Chicken I pGlu Mammal pGlu - His His X - - Tyr - Ser - x - x - x - x - Ser - Leu - Glu - Trp - Lys - Pro - GlyNH 2 Pro - GlyNH 2 His - Trp - Ser His - Gly Trp - Tyr - Pro - GlyNH 2 His - Trp - Ser Tyr - Gly Trp - Leu - Pro - GlyNH 2 - His - Trp - Ser Tyr - Gly Leu - Gin - Pro - GlyNH 2 - His - Trp - Ser Tyr - Gly Leu - Arg - Pro - GlyNH 2 - 1b pGlu - His - Trp - Ser - Tyr - Gly - Trp - Leu - Pro - GlyNH, CA" CA; TGG AG GG C TCX TGG AG, TA, GGX TGG ;TT TCX Fig. 1. a, Evolution of the GnRH Decapeptide Family Primary structure of the five known forms of GnRH. Positions 1, 2, 4, 9, and 10 are invariant, and positions 3 and 7 show only conservative changes, while positions 5 and 8 tolerate dramatic differences, b, Oligonucleotides used as probes to isolate cichlid prepro-GnRH cDNA clones. Pools consisted of overlapping 17-mers comprised of all possible coding combinations for eight amino acids of salmon GnRH. 933 cDNA for a Teleost GnRH Precursor _23 SIGNAL SEQUENCE _10 _j CAGAGTTCTA ATG GAA GCA GGC AGC AGA GTT ATA ATG CAG GTG TTG TTG CTG GCG TTG GTG GTT CAG GTC ACC CTG TCC MET Glu Ala Gly Ser Arg Val H e Met Gin Val Leu Leu Leu Ala Leu Val Val Gin Val Thr Leu Ser Processing +1 GnRH Site cGAP 10 20 CAG CAC TGG TCC TAT GGA TGG CTA CCA GGT GGA AAG AGA ACT GTG GGA GAG CTT GAG GCA ACC ATT AGG ATG ATG GGC ACA | Gin His Trp Ser Tyr Gly Trp Leu Pro Gly] Gly Lys Arg Ser Val Gly Glu Leu Glu Ala Thr H e Arg Met Met Gly Thr 30 40 SO GGA GGG GTG GTG TCT CTT CCC GAT GAG GCA AAT GCC CAA ATC CAA GAG AGA CTT AGA CCA TAC AAT ATA ATT AAT GAT GAT Gly Gly Val Val Ser Leu Pro Asp Glu Ala Asn Ala Gin H e TCC AGT CAC TTT GAC CGA AAA AAA AGG TTC CCT AAT AAT TGA Gin Glu Arg Leu Arg Pro Tyr Asn lie H e Asn Asp Asp AGAGCTACATCATTATCAGCAGCACATGGGAAAGATCGCCCGGCAACAAGA Ser Ser His Phe Asp Arg Lys Lys Arg Phe Pro Asn Asn Fig. 2. Nucleotide and Amino Acid Sequence of Cloned H. burtoni cDNA Encoding Prepro-GnRH Amino acid numbers appear above the sequences, with negative numbers corresponding to the presumed signal sequence. The functional domains of the precursor are indicated, and the polyadenylation signal is underlined. It is interesting to note that introduction of a single base frameshift within the cGAP domain generates a predicted peptide sequence that contains a significant block of homology to mGAP, within a region which itself is highly conserved among the mammalian species. To assure that the isolated cichlid cDNA did not represent a cloning artifact in this region, three independent oligo(dT)-primed reverse transcription reactions were performed using the same mRNA from which the original cDNA library was constructed. Two independent aliquots from each of these reactions were subjected to enzymatic amplification [polymerase chain reaction (PCR)] (19, 20) in the presence of oligonucleotide primers specific for untranslated sequences in the proGnRH mRNA, a total of six separate PCR reactions. The PCR products were cloned and subjected to nucleotide and predicted protein sequence analysis. A total of four independent clones from each PCR reaction (24 subclones) were analyzed, and although occasional disagreements between the sequences were observed, usually resulting from transitions, incorporation of C in positions where T was present in the original cDNA clone and presumed to represent PCR mistakes, in no case was the reading frame altered. Northern Blot Analysis of Cichlid Pro-GnRH mRNA To examine the full-length pro-GnRH mRNA, total and poly(A)+ RNAs were extracted from whole brains of macho males and prepared as a Northern blot. Hybridization to a radiolabeled DNA probe derived from the pro-GnRH cDNA detected a single RNA species approximately 550 nucleotides in length (Fig. 4). Reprobing of this blot at lower stringencies failed to detect additional RNA species. Gene Copy Analysis Chromatographic and immunological data have provided considerable evidence implicating the existence of a second form of GnRH in several teleost species (2) (Sherwood, N., personal communication). However, a second decapeptide form was not found encoded on the precursor described above. To investigate whether this second form is encoded by a partially homologous mRNA transcribed from a distinct gene, 2 x 106 primary clones from the H. burtoni macho cDNA library were rescreened at reduced stringency, using a radiolabeled DNA probe derived from the cichlid pro-GnRH cDNA. Even employing extremely permissive hybridization conditions, no related sequences were detected, although several independent pro-GnRH cDNA clones were isolated. Further, H. burtoni genomic DNA was digested with a variety of restriction endonucleases, prepared as a Southern blot, and probed at high and low stringencies. The results, shown in Fig. 5, indicate that the gene encoding GnRH in H. burtoni is a single copy gene; no additional hybridizing bands were detected at any stringency. DISCUSSION The cDNA encoding pro-GnRH has been isolated and characterized for the first time from a nonmammalian species, the cichlid teleost H. burtoni. The molecular architecture of the predicted prohormone is identical to that of the previously described mammalian forms: a 23-amino acid signal sequence in direct linkage with the decapeptide, which, in turn, is followed by residues Vol 5 No. 7 MOL ENDO-1991 934 3a SIGNAL SEQUENCE n r—i QHWSYG il PGGKR SVGE : !ATIR> <G rGGWSLPDEANAQIQERIR ?YNIINDDSSHFDRKKRFPNN 1 1 QHWSYG LR PGGKR NTEH 1 reSFQE <G (EVDQMAEPQHFECTVHWER 3PLRDLRGALESLIEEEARQKKM (LMAHVViLITVCLEGCS 3HWSYG LF PGGKR NTEH 1 rtlSFQE <G (EEDQMAEPQNFECTVHWERSPLRDLRGALERLIEEEAGQKKM QHWSYG Lf PGGKR DAEN;CDSFQEIVKEVGQLAETQRFECTTHQE R SPLRDLKGALESLIEEETGQKKI :J*/IMdVLLl MIL(LMAGILLITVCLEGCS < LMAGI L: IQ < LLAGLI L] 3b CICHLID ...ERLRPYNIINDD... . . .HW >RSPI *DIRG. . . . . .HW >RSPI . . .HC >RSPI 1DIKG. . . CICHLID-alt Fig. 3. a, Comparison of the Mammalian and Cichlid Precursor Proteins for Prepro-GnRH The single letter code is used to designate amino acids. The three domains of signal sequence, GnRH, and GAP are set apart. Amino acids that represent the site for enzymatic precursor cleavage and carboxy-terminal amidation (18) are joined to the GnRH decapeptide. Boxes do not represent homologies between the various mammalian sequences, b, Comparison of GAP domain in which a frameshift in the cichlid sequence (Cichlid-alt) produces a region of significant homology to the mammalian forms. This domain is highly conserved among the mammalian species. involved in proteolytic processing and maturation of GnRH, and a 54-residue associated peptide. Eight of 23 residues in the signal sequence are shared between H. burtoni and at least one of the mammalian forms, not including an analogously placed arginine in the fish and corresponding lysine in all mamalian sequences. The known decapeptide sequences vary at two positions, 7 and 8, while the three residues involved in posttranslational maturation, Gly11, Lys12, and Arg13, are completely conserved. Although the signal sequence and decapeptide show remarkable homology to the mammalian forms, the associated peptide, cGAP, shows little if any relation to mGAP; five residues may be appropriately aligned, with no more than two contiguously. However, a single base insertion within cGAP generates a potential peptide sequence with a significant block of homology to a conserved region of the mammalian forms; a total of nine residues may be aligned, and allowing substitution of a lysine for an arginine, this yields a stretch of six of seven matches. However, analysis of an independent population of prohormone sequences by PCR demonstrated that the bonifide cGAP does not possess such homology. It is, therefore, possible that the mammalian GAP arose by a frameshift event at some point between the appearance of fish and mammals. Mammalian GAP has been implicated in the control of PRL release from pituitary lactotrophs (21). Teleost fish have two forms of PRL (22,23) which are controlled by hypothalamic factors (24, 25), and both forms are thought to function in osmoregulation (26,27). Although the physiological properties regulated by mammalian PRL are not germain to teleost physiology (lactation, parturition), it is possible that cGAP functions to regulate the release of cichlid PRL. Planas et al. (28) recently reported that human GAP effects PRL release from the pituitary of the Tilapia, Oreochromis mossambicus. Since the structure of cGAP, as determined from the experiments described here, does not resemble that of mGAP, these results are enigmatic. Alternatively, it is possible that cGAP does not effect PRL secretion and that mGAP arose in the tetrapod lineage as PRL came to serve new functions. Experiments are in progress to examine the physiological role of this novel peptide, cGAP. Chromatographic and immunological methods have identified two forms of GnRH in several teleost species, including salmon, trout, goldfish, herring, mullet, siganids, milkfish, and sea bass (2). However, the exact amino acid sequence of only one form, salmon I, is known (15). This peptide sequence was used to design the oligonucleotides that succeeded in detecting the cDNA encoding pro-GnRH. Reduced stringency cDNA screening and Northern and Southern analyses failed to provide evidence for a related gene that may encode the putative second form of cichlid GnRH. If a second form is expressed in H. burtoni, it is encoded by DNA that does not bear strong enough homology to the proGnRH cDNA to be detected by hybridization. Although two peptides coelute from HPLC columns and demonstrate cross-reactivity with antiserum raised against the sequenced form of GnRH, data presented here suggest that the mRNAs encoding these peptides may be very different in sequence. Without the exact amino acid 935 cDNA for a Teleost GnRH Precursor H. burtoni Genomic DNA Blot a a E2 • < , PQ H 5 O o PQ u g u w 1400- 780530- 1— 400280- kb Fig. 5. Southern Blot Analysis of H. burtoni Genomic DNA Each lane contains 20 ^g liver DNA cleaved to completion with the indicated restriction endonucleases. Random primed cDNA fragment was used as a probe under high or low stringency conditions, as described in Fig. 4. At lowered stringency, no additional bands were detected. Fig. 4. Northern Blot Analysis of Cichlid Prepro-GnRH mRNA Lane 1 contains 5 ^g poly(A)+, and lane 2 contains 20 /ug total RNA isolated from the brains of macho males. H. burtoni pro-GnRH cDNA was random primed and used as probe under high (50% formamide; 37 C) or low (30% formamide; 25 C) stringency conditions. At lowered stringency, no additional bands were detected. sequence of the second decapeptide form it is difficult to speculate about possible physiological functions. However, in the chicken, in which the amino acid sequences of two distinct GnRH-like peptides have been determined, both forms function to release gonadotropins, although chicken II is 10 times more potent than chicken I (5). Such a situation is similar to the relationship between the enkephalins and the endorphins; two peptides with similar biological activities are encoded by genes that are not able to recognize each other by cross-hybridization (29, 30). Questions concerning the comparative biological activities of the GnRH peptide encoded on the precursor described here and that of the putative second form, and the evolutionary relatedness of the genes encoding these peptides can only be addressed, however, when the cDNA encoding the second form has been isolated and characterized, allowing direct comparison of the peptide moieties, the nucleic acid milieu harboring each decapeptide-encoding sequence, and experiments to determine the potency of the second form as a GnRH. MATERIALS AND METHODS Tissue and RNA Preparation Social dominance in H. burtoni, a prerequisite for sexual reproduction in the males, is advertised through territorial defense and bright body colorations (12, 13). Behavioral observations and these distinctive color patterns were used to select males from breeding colonies for collection of brain tissue. Sixty animals were chosen and killed by rapid heart puncture; brains were quickly removed and stored in liquid nitrogen. MOL ENDO-1991 936 Total RNA was extracted using guanidine thiocyanate and purified by centrifugation through a cesium chloride pad, as previously described (31). The poly(A)+ fraction was purified by oligo(dT)-cellulose chromatography, as previously described (32). Complementary DNA Library Construction and Screening Complementary DNA was synthesized as previously described (7). After ligation to EcoRI adaptors, synthesized on an Applied Biosystems PCR Mate DNA synthesizer (Foster City, CA), the cDNA was size-selected for molecules over 300 basepairs in length by agarose gel electrophoresis. Eluted material was ligated to Xgt10 arms (Stratagene, La Jolla, CA) and incorporated into phage particles by in vitro packaging according to manufacturer's instructions (Stratagene, La Jolla, CA). Recombinant clones were plaqued on E. Coli C600 hfl at a density of 40,000/plate, from which filter lifts were prepared (Genescreen, New England Nuclear-DuPont, Boston, MA.). Oligonucleotide pools including all possible coding sequences for the first eight amino acids of the known salmon GnRH sequence (15) were synthesized on the PCR mate. Chains were radiolabeled with [7-32P]ATP and polynucleotide kinase (New England Nuclear-DuPont); filters were prehybridized and probed at room temperature in 1 M NaCI, 1 % sodium dodecyl sulfate, 100 ng/m\ yeast RNA (Sigma, St. Louis, MO), and 30% formamide (Bethesda Research Laboratories, Gaithesburg, MD). After a brief rinse at room temperature in 2 x standard salt citrate solution (SSC), filters were washed with 3 M tetramethylammonium chloride (Aldrich Chemicals, Milwaukie, Wl) at 51 C and exposed to x-ray film for 5 days. Northern and Southern Blot Analyses For Northern analysis, 5 ^g poly(A)+ or 20 ^9 total H. burtoni macho brain RNA were electrophoresed through 1 % agaroseformaldehyde gels and transferred by capillary to Magna NT nylon membranes (Micro Separations, Inc., Westboro, MA). For Southern analysis, H. burtoni genomic DNA was extracted from liver tissue, which was quickly frozen and pulverized in liquid nitrogen, followed by treatment with proteinaseK (Boehringer Mannheim, Indianapolis, IN). Nucleic acids were ethanol precipitated after adjustment to 0.3 M KOAC. The pellet was resuspended in distilled sterile water, phenol extracted three times, chloroform extracted twice, and ethanol precipitated in the presence of 0.3 M NaoAc. The pellet was resuspended in 10 mM Tris (pH 7.5), 1 mM EDTA, 25 ng/rr\\ pancreatic RNase (Sigma), and incubated at 37 C. After 1 h, the reactions were phenol and chloroform extracted, and the DNA precipitated. After centrifugation, the DNA was dialyzed overnight against excess 10 mM Tris (pH 7.5), 1 mM EDTA, and 10 mM NaCI, reprecipitated, and stored in distilled water. Twenty micrograms of this material were digested with the indicated restriction endonucleases (Bethesda Research Laboratories), electrophoresed through a 1 % agarose gel (FMC, Rockland, ME), and prepared as a Southern blot by capillary transfer. Probes for Northern and Southern blots were prepared from a DNA fragment generated by enzymatic amplification, using oligonucleotides specific for sequences encoding cichlid GnRH and Replinase (Bethesda Research Laboratories). After fragment purification over an agarose gel, the eluted DNA was radiolabeled by random priming with E. coli DNA polymeraseI, Klenow fragment (Bethesda Research Laboratories) to a specific activity of 5 x 108 cpm/^g. Routinely, 108 cpm were applied as probe. cGAP PCR and DNA Sequence Analysis Reverse transcription and PCRs reactions were performed as previously described (20), using Replinase. Oligonucleotides specific for GnRH sequences were initially present at 0.5 ^ M ; Vol 5 No. 7 these chains contained restriction sites flanking GnRH sequences. After amplification, the reactions were phenol and chloroform extracted and ethanol precipitated. PCR products or cDNA clones were cut with appropriate restriction enzymes, gel purified, and cloned into M13 vectors. DNA sequencing was performed by the dideoxy chain terminating method (33), using Sequenase according to manufacturer's instructions (U.S. Biochemical Corp., Cleveland, OH). Acknowledgments We thank Ms. Shiela Vollmer for nurturing the macho male fish, Drs. Nancy Sherwood and Imogen Coe for fruitful discussions, and the Vollum Illustrations staff for artwork. Received March 21,1991. Revision received April 22,1991. Accepted April 22, 1991. Address requests for reprints to: Dr. John P. Adelman, Vollum Institute for Advanced Biomedical Research/Oregon Health Sciences University, L-474, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. This work was supported by NIH Grants HD-23799 (to R.D.F.) and HD-24562-02 (to J.P.A.). REFERENCES 1. Grumbach MM 1980 The neuroendocrinology of puberty. In: Krieger DT, Hughes JC (eds) Neuroendocrinology. Sinauer, Sunderland, pp 249-258 2. Sherwood N 1987 The GnRH family of peptides. Trends Neurosci 10:129-132 3. King JA, Millar RP1982 Structure of chicken hypothalamic luteinizing hormone releasing hormone. II. Isolation and characterization. J Biol Chem 257:10729-10732 4. Miyamoto K, Hasegawa Y, Minegishi T, Nomura M, Takahashi Y, Igarashi M, Kangawa K, Matsuo H 1982 Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone. Biochem Biophys Res Commun 107:820-827 5. 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