Evidence that gonadotropin-releasing hormone (GnRH) functions as a prolactin-releasing factor in a teleost fish (Oreochromis mossambicus) and primary structures for three native GnRH molecules.

Three forms of gonadotropin-releasing hormone (GnRH) are isolated and identified here by chemical sequence analysis for one species of tilapia, Oreochromis niloticus, and by HPLC elution position for a second species of tilapia, O. mossambicus. Of the three GnRH forms in O. mossambicus, chicken GnRH-II (cGnRH-II) and sea bream GnRH (sbGnRH) are present in greater abundance in the brain and pituitary than salmon GnRH (sGnRH). These three native forms of GnRH are shown to stimulate the release of prolactin (PRL) from the rostral pars distalis (RPD) of the pituitary of O. mossambicus in vitro with the following order of potency: cGnRH-II > sGnRH > sbGnRH. In addition, a mammalian GnRH analog stimulated the release of PRL from the pituitary RPD incubated in either iso-osmotic (320 mosmol/l) or hyperosmotic (355 mosmol/l) medium, the latter normally inhibiting PRL release. The response of the pituitary RPD to GnRH was augmented by co-incubation with testosterone or 17 beta-estradiol. The effects of GnRH on PRL release appear to be direct effects on PRL cells because the RPD of tilapia contains a nearly homogeneous mass of PRL cells without intermixing of gonadotrophs. Our data suggest that GnRH plays a broad role in fish, depending on the species, by affecting not only gonadotropins and growth hormone, but also PRL.

Sequence and expression of cDNA for pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH)-like peptide in catfish.

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase activating polypeptide (PACAP) are two neuropeptides that are associated with the release of pituitary growth hormone. Here a cDNA of 2501 base pairs encoding both a PACAP and a GHRH-like peptide was isolated from a brain cDNA library made from Thai catfish (Clarias macrocephalus). The organization is unlike that of the mammalian gene where PACAP and PACAP-related peptide (PRP) are encoded in one gene, and the GHRH peptide is on a separate gene. Northern analysis of catfish brain mRNA indicated that PACAP/GHRH-like mRNA has three sizes; bands of 6000, 2500, and 1000 bases suggest alternative splicing of the gene. Reverse transcriptase/PCR assay detected PACAP/GHRH-like mRNA in tissues from the brain, testis, ovary, and stomach, but not from the pancreas, pituitary, muscle, and liver. Our hypothesis that the two mammalian genes encoding GHRH or PACAP originated from a gene duplication between fish and tetrapods is supported by the present findings of similar mRNA organization and pattern of expression for the one fish gene and two mammalian genes.

Structure-function studies of five natural, including catfish and dogfish, gonadotropin-releasing hormones and eight analogs on reproduction in Thai catfish (Clarias macrocephalus).

Five distinct forms of gonadotropin-releasing hormone (GnRH) and their analogs, six of which are newly designed, were used to study reproduction in Thai catfish, Clarias macrocephalus. Determination was made for the percentage of fish that ovulated within 16-18 h; the percentage of eggs fertilized; and the percentage of larva that hatched and survived for 7 days. The results show, firstly, that natural chicken GnRH-II, which is identical with catfish GnRH-II, was significantly more effective at a dose of 300 micrograms/kg than the control injection for the induction of ovulation. Dogfish GnRH at the same dose was also significantly more effective than the control, but was not significantly different from chicken (catfish) GnRH-II for ovulation induction. The novel catfish GnRH-I, mammalian GnRH and salmon GnRH were not effective at 100, 150 or 300 micrograms/kg in Thai catfish. Secondly, 5 of 8 analogs of GnRH at a dose of 20 micrograms/kg resulted in a significantly higher percentage of ovulating fish compared with the control fish. Among these five analogs, the most effective were the two analog forms of chicken GnRH-II (D-Arg6,Pro9 NEt and D-Nal6,Pro9 NEt), followed by the salmon GnRH analog (D-Arg6,Pro9 NEt), a dogfish GnRH analog (D-Arg6,Pro9 NEt) and the mammalian GnRH analog (D-Ala6,Pro9 NEt). Not significantly different from the controls were the two catfish GnRH-I analogs and one of the dogfish (D-Nal6,Pro9 NEt) analogs. The six new analogs had not been previously tested in any animal. Thirdly, the number of fish ovulating was the same whether GnRH was administered in one or two injections.

Gonadotropin-releasing hormone from thai catfish: chromatographic and physiological studies.

Two forms of immunoreactive gonadotropin-releasing hormone (GnRH) were extracted from brain-pituitary tissues of Thai catfish, Clarias macrocephalus and C. batrachus. The peptides were detected using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). In both the HPLC systems, catfish GnRH-I eluted earlier than catfish GnRH-II and also eluted before the synthetic standards of mammalian, lamprey, chicken I, chicken II, and salmon GnRH. Hence, catfish GnRH-I appears to be the most hydrophilic GnRH family member because of this early elution from the HPLC. Catfish GnRH-II eluted in a position similar to that of chicken GnRH-II. This study suggests that catfish GnRH-I is a novel form of GnRH, whereas catfish GnRH-II is the same as chicken GnRH-II. Indirect evidence suggests that the catfish molecule is 10 amino acids in length and has an amide at the C-terminus. Moreover, the novel catfish GnRH appears to be different within the domain of amino acids 5 to 10 compared with mammalian GnRH because it is not recognized by antiserum B-6. An injection of native chicken GnRH-II was more effective than salmon or mammalian GnRH for induced ovulation in C. macrocephalus.

Distinct sequence of gonadotropin-releasing hormone (GnRH) in dogfish brain provides insight into GnRH evolution.

In vertebrates, gonadotropin-releasing hormone (GnRH) belongs to a family of decapeptides characterized by the conservation of residues 1, 2, 4, 9, and 10. In the jawed vertebrates only positions 5, 7, and 8 in the GnRH molecules vary. We have now purified two forms of GnRH from the brains of spiny dogfish (Squalus acanthias) by using reverse-phase high-performance liquid chromatography. The primary structures were established by automated Edman degradation and mass spectral analysis. The distinct structure of the first form (dogfish GnRH) is pGlu-His-Trp-Ser-His-Gly-Trp-Leu-Pro-Gly-NH2 (pGlu represents pyroglutamyl). The second peptide is identical to a form of GnRH originally isolated from chicken brains (chicken GnRH-II; pGlu-His-Trp-Ser-His-Gly-Trp-Tyr- Pro-Gly-NH2) and is widespread throughout the vertebrates. We are aware of no other species of cartilaginous fish in which the primary structures of two forms of GnRH have been determined. The presence of chicken GnRH-II in dogfish supports the idea that chicken GnRH-II is the oldest GnRH to evolve in jawed vertebrates. With the addition of the dogfish GnRH structure to the family, two main structural branches of GnRH can be delineated. The physiological effects of dogfish GnRH included the release of not only gonadotropin but also growth hormone from goldfish pituitary fragments.

Primary structures of two forms of gonadotropin-releasing hormone, one distinct and one conserved, from catfish brain.

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the Thai catfish, Clarias macrocephalus, using reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The amino acid sequences of both forms of catfish GnRH (catfish GnRH-I and -II) were determined using Edman degradation. The presence of the N-terminal pGlu residue in both peptides was established by digestion with pyroglutamyl aminopeptidase. In addition, catfish GnRH-I was studied by mass spectrometry. The primary structure of catfish GnRHI is pGluHisTrpSerHisGlyLeuAsnProGlyNH2 and catfish GnRH-II is identical to chicken GnRH-II, pGluHisTrpSerHisGlyTrpTyrProGlyNH2. Functional studies showed that synthetic catfish GnRH-I released not only gonadotropin but also growth hormone from an in vitro preparation of goldfish pituitaries. Catfish GnRH-II is identical to the widely distributed and highly conserved chicken GnRH-II. Indirect evidence has suggested its presence in bony fish, but this is the first report of its primary sequence. The distinct structure of catfish GnRH-I increases the number of GnRH family members to six.