Molecular analysis of the koala reproductive hormones and their receptors: gonadotrophin-releasing hormone (GnRH), follicle-stimulating hormone ß and luteinising hormone ß with localisation of GnRH.

During evolution, reproductive hormones and their receptors in the brain-pituitary-gonadal axis have been altered by genetic mechanisms. To understand how the neuroendocrine control of reproduction evolved in mammals, it is important to examine marsupials, the closest group to placental mammals. We hypothesised that at least some of the hormones and receptors found in placental mammals would be present in koala, a marsupial. We examined the expression of koala mRNA for the reproductive molecules. Koala cDNAs were cloned from brain for gonadotrophin-releasing hormones (GnRH1 and GnRH2) or from pituitary for GnRH receptors, types I and II, follicle-stimulating hormone (FSH)ß and luteinising hormone (LH)ß, and from gonads for FSH and LH receptors. Deduced proteins were compared by sequence alignment and phylogenetic analysis with those of other vertebrates. In conclusion, the koala expressed mRNA for these eight putative reproductive molecules, whereas at least one of these molecules is missing in some species in the amniote lineage, including humans. In addition, GnRH1 and 2 are shown by immunohistochemistry to be expressed as proteins in the brain.

Expression of pejerrey gonadotropin-releasing hormone in three orders of fish.

Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.

Targeted disruption of the pituitary adenylate cyclase-activating polypeptide gene results in early postnatal death associated with dysfunction of lipid and carbohydrate metabolism.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hormone belonging to the glucagon superfamily of hormones. These hormones are known to play important roles in metabolism and growth. PACAP is a neuropeptide that causes accumulation of cAMP in a number of tissues and affects the secretion of other hormones, vasodilation, neural and immune functions, as well as the cell cycle. To determine whether PACAP is essential for survival and to evaluate its function(s), we have generated mice lacking the PACAP gene via homologous recombination. We found that most PACAP null mice died in the second postnatal week in a wasted state with microvesicular fat accumulation in liver, skeletal muscle, and heart. Gas chromatography-mass spectrometry showed that fatty acid beta-oxidation in liver mitochondria of PACAP(-/-) mice was not blocked based on the distribution of 3-hydroxy-fatty acids (C6-16) in the plasma. Instead, increased metabolic flux through the beta-oxidation pathway was suggested by the presence of ketosis. Also, serum triglycerides and cholesterol were significantly higher (2- to 3-fold) in PACAP null mice than littermates. In the fed state, both serum insulin and blood glucose were normal in 5-d-old null mice compared with their littermates. In contrast, fasted PACAP null pups had a significant increase in insulin, but a decrease in blood glucose compared with littermates. Glycogen in the liver was reduced. These results suggest PACAP is a critical hormonal regulator of lipid and carbohydrate metabolism.

Presence of luteinizing hormone-releasing hormone fragments in the rhesus monkey forebrain.

Previously, we have shown that two types of luteinizing hormone-releasing hormone (LHRH) -like neurons, "early" and "late" cells, were discernible in the forebrain of rhesus monkey fetuses by using antiserum GF-6, which cross-reacts with several forms of LHRH. The "late" cells that arose from the olfactory placode of monkey fetuses at embryonic days (E) 32-E36, are bona fide LHRH neurons. The "early" cells were found in the forebrain at E32-E34 and settled in the extrahypothalamic area. The molecular form of LHRH in "early" cells differs from "late" cells, because "early" cells were not immunopositive with any specific antisera against known forms of LHRH. In this study, we investigated the molecular form of LHRH in the "early" cells in the nasal regions and brains of 13 monkey fetuses at E35 to E78. In situ hybridization studies suggested that both "early" and "late" LHRH cells expressed mammalian LHRH mRNA. Furthermore, "early" cells predominantly contain LHRH1-5-like peptide and its cleavage enzyme, metalloendopeptidase E.C.3.4.24.15 (EP24.15), which cleaves LHRH at the Tyr5-Gly6 position. This conclusion was based on immunocytochemical labeling with various antisera, including those against LHRH1-5, LHRH4-10, or EP24.15, and on preabsorption tests. Therefore, in primates, a group of neurons containing mammalian LHRH mRNA arises at an early embryonic stage before the migration of bona fide LHRH neurons, and is ultimately distributed in the extrahypothalamic region. These extrahypothalamic neurons contain LHRH fragments, rather than fully mature mammalian LHRH. The origin and function of these neurons remain to be determined.

Developmental expression, alternative splicing and gene copy number for the pituitary adenylate cyclase-activating polypeptide (PACAP) and growth hormone-releasing hormone (GRF) gene in rainbow trout.

Both growth hormone-releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) are encoded on the same gene in fish, but not in mammals. Our objective was to examine the onset and pattern of expression for the grf/pacap gene and to determine whether there is more than one gene in rainbow trout. The results show that grf/pacap mRNA is first expressed at 4 days after fertilization and continues through to hatching. Alternative splicing at all developmental stages produces a full-length transcript and one lacking exon four, which encodes GRF. Thus, independent regulation of the hormones occurs throughout development. Southern analysis shows that two grf/pacap genes exist in trout, but only one gene is responsible for the two identified transcripts. Overexpression of the grf/pacap gene in transgenic fish was attempted, but did not succeed. We conclude that the early and continued expression of grf/pacap mRNA in trout embryos and regulation of the neuropeptide ratio suggests they have a role in early brain development apart from their later role in releasing pituitary hormones.

Structure and biological activity of gonadotropin-releasing hormone isoforms isolated from rat and hamster brains.

Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp(9)]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp(9)]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp(9)]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp(9)]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.

Gonadotropin-releasing hormone content in the brain and pituitary of male and female grass rockfish (Sebastes rastrelliger) in relation to seasonal changes in reproductive status.

The involvement of individual molecular forms of GnRH in the regulation of reproductive cyclicity in a viviparous marine teleost, the grass rockfish (Sebastes rastrelliger), was evaluated by relating the brain and pituitary content of the neuropeptide to reproductive status. The presence of sea bream (sb) GnRH, chicken GnRH-II, and salmon GnRH in the brain was confirmed by their elution pattern on HPLC and RIA. In addition, HPLC elution profiles suggest that there may be a fourth form of GnRH. All forms of GnRH were found in male and female brains in all reproductive conditions. However, only sbGnRH could be detected in appreciable amounts in the pituitary. Of the four forms of GnRH found in the rockfish, only sbGnRH fluctuated during the reproductive cycle and large accumulations were detected in the brains and pituitaries of postspawn females and regressed males. The accumulation of sbGnRH at the end of the reproductive cycle is suggested to reflect a decline in GnRH secretion relative to synthesis. The dominance of sbGnRH in the pituitary and its individual fluctuation in relation to seasonal changes in reproductive status suggests that sbGnRH is an important regulator of gonadotropin-mediated reproductive activity in rockfish.

Primary structure of a novel gonadotropin-releasing hormone in the brain of a teleost, Pejerrey.

The neuropeptide GnRH is the major regulator of reproduction in vertebrates acting as a first signal from the hypothalamus to pituitary gonadotropes. Three GnRH molecular variants were detected in the brain of a fish, pejerrey (Odontesthes bonariensis), using chromatographic and immunological methods. The present study shows that one form is identical to chicken GnRH-II (sequence analysis and mass spectrometry) and the second one is immunologically and chromatographically similar to salmon GnRH. The third form was proven to be a novel form of GnRH by isolating the peptide from the brain and determining its primary structure by chemical sequencing and mass spectrometry. The sequence of the novel pejerrey GnRH is pGlu-His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly-NH(2), which is different from the known forms of the vertebrate and protochordate GnRH family. The new form of GnRH is biologically active in releasing gonadotropin and GH from pituitary cells in an in vitro assay.

Early expression of pituitary adenylate cyclase-activating polypeptide and activation of its receptor in chick neuroblasts.

To investigate the involvement of pituitary adenylate cyclase- activating polypeptide (PACAP) and GH-releasing factor (GRF) during early chick brain development, we established neuroblast- enriched primary cell cultures derived from embryonic day 3.5 chick brain. We measured increases in cAMP generated by several species-specific forms of the peptides. Dose-dependent increases up to 5-fold of control values were measured in response to physiological concentrations of human/salmon, chicken, and tunicate PACAP27. Responses to PACAP38 were more variable, ranging from 5-fold for human PACAP38 to 4-fold for chicken PACAP38, to no significant response for salmon PACAP38, compared with control values. The responses to PACAP38 may reflect a greater difference in peptide structure compared with PACAP27 among species. Increases in cAMP generated by human, chicken, and salmon/carp GRF were not statistically significant, whereas increases in response to lower-range doses of tunicate GRF27-like peptide were significant, but small. We also used immunocytochemistry and Western blot to show synthesis of the PACAP38 peptide. RT-PCR was used to demonstrate that messenger RNAs for PACAP and GRF and a PACAP-specific receptor were present in the cells. This is a first report suggesting an autocrine/paracrine system for PACAP in early chick brain development, based on the presence of the ligand, messages for the ligand and receptor, and activation of the receptor in neuroblast-enriched cultures.

Characterization of the gene encoding both growth hormone-releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the zebrafish.

The purpose of this research was to isolate and characterize the gene encoding growth hormone-releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) from the zebrafish. The gene is comprised of five exons with two distinct peptides encoded on separate exons, GRF on exon 4 and PACAP on exon 5. Our evidence indicates that the zebrafish genome contains a single copy of the GRF-PACAP gene. The tissue distribution pattern of the mRNA transcript shows expression in the brain, eye, gastrointestinal tract, ovary and testis; each transcript was sequenced and found to be identical to the gene. This is the first report of GRF-PACAP mRNA expression in the eye of a non-mammalian species. Evidence that a duplication of the PACAP gene gave rise to the vasoactive intestinal peptide (VIP) gene is supported by the high amino acid sequence identity between PACAP in zebrafish and VIP in other fish species.

Biological and immunological characterization of multiple GnRH in an opisthobranch mollusk, Aplysia californica.

Gonadotropin-releasing hormone (GnRH) is a neurohormone central to the regulation of reproductive functions in vertebrates. Recently, several studies have reported the presence of GnRH immunoreactivity (IR) in a number of mollusks, suggesting that the distribution of GnRH may not be restricted to Phylum Chordata. In the present study, we extend our investigations to an opisthobranch mollusk, Aplysia californica, to characterize the source, chemical nature, and biological activity of molluscan GnRH-related molecules. Specific radioimmunoassays (RIAs) of various tissue extracts of Aplysia revealed that only ovotestis, hemocytes, and hemolymph contained significant amounts of GnRH that crossreacts with antisera raised against tunicate-I (tI) and mammalian (m) GnRH. Further RIAs and extractions revealed that the GnRH-IR in the hemolymph is biochemically and immunologically distinct from the GnRH-IR in the hemocytes and ovotestis. Using reverse-phase high-performance liquid chromatography coupled with RIAs, the GnRH-IR in the hemolymph was resolved into two major peaks. The first peak eluted earlier than most known forms of vertebrate GnRH, and the later peak coeluted with m, lamprey I, chicken II, and tI-GnRH. However, both peaks were broad and may contain a heterogeneous mixture of GnRH-IR. Immunocytochemical study showed that tI-GnRH-IR was present in the connective sheath surrounding the central nervous system, with a strong presence in what appeared to be vascular space, again suggesting the close association between Aplysia GnRH-IR and circulation. Finally, treatment of the neuroendocrine bag cells with chicken II GnRH significantly decreased the duration of the afterdischarge (AD, a characteristic pattern of electrical firing in bag cell neurons) and the number of action potentials fired during an AD, indicating the presence of a corresponding GnRH receptor in the Aplysia central nervous system. Overall, the results demonstrated the presence of multiple forms of GnRH-IR that crossreact with tI- and mGnRH antisera in A. californica and the ability of a vertebrate GnRH to alter Aplysia neural activity. Together, these data suggest that GnRH may be a factor released by the ovotestis and hemocytes into the circulation to alter neural functions. GnRH-IR produced by the latter may serve as a novel mediator of the neural and immune functions in Aplysia.

Primary structure and function of three gonadotropin-releasing hormones, including a novel form, from an ancient teleost, herring.

The evolution of GnRH and the role of multiple forms within the brain are examined. Three forms of GnRH were purified from the brain of Pacific herring (Clupea harengus pallasi) and characterized using Edman degradation and mass spectrometry. Two forms correspond with the known structures of chicken GnRH-II and salmon GnRH that are found in many vertebrate species. The third form, designated herring GnRH (hrGnRH), has a primary structure of pGlu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-NH2. This novel peptide is a potent stimulator of gonadotropin II and GH release from dispersed fish pituitary cells. The content of hrGnRH in the pituitary was 8-fold that of salmon GnRH and 43-fold that of chicken GnRH-II, which provides supporting evidence that hrGnRH is involved in the release of gonadotropin. Herring is the most phylogenetically ancient animal in which three forms of GnRH have been isolated and sequenced. Our evidence suggests that the existence of three GnRHs in the brain of one species 1) is an ancestral condition for teleosts, 2) has the potential for separate regulation of the distinct GnRHs, and 3) may be an evolutionary advantage for refined control of reproduction in different environments.

The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily.

The pituitary adenylate cyclase-activating polypeptide (PACAP)/ glucagon superfamily includes nine hormones in humans that are related by structure, distribution (especially the brain and gut), function (often by activation of cAMP), and receptors (a subset of seven-transmembrane receptors). The nine hormones include glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, glucose-dependent insulinotropic polypeptide (GIP), GH-releasing hormone (GRF), peptide histidine-methionine (PHM), PACAP, secretin, and vasoactive intestinal polypeptide (VIP). The origin of the ancestral superfamily members is at least as old as the invertebrates; the most ancient and tightly conserved members are PACAP and glucagon. Evidence to date suggests the superfamily began with a gene or exon duplication and then continued to diverge with some gene duplications in vertebrates. The function of PACAP is considered in detail because it is newly (1989) discovered; it is tightly conserved (96% over 700 million years); and it is probably the ancestral molecule. The diverse functions of PACAP include regulation of proliferation, differentiation, and apoptosis in some cell populations. In addition, PACAP regulates metabolism and the cardiovascular, endocrine, and immune systems, although the physiological event(s) that coordinates PACAP responses remains to be identified.

Regulation and expression of gonadotropin-releasing hormone gene differs in brain and gonads in rainbow trout.

The GnRH gene is transcribed in both the brain and gonads. GnRH in the brain is critical for reproduction, but the function and importance of GnRH in the ovary and testis is not clear. In this study we examine whether regulation of the GnRH gene is distinct in the brain and gonads, whether the regulation of the GnRH gene in the gonads is altered after genome duplication, and whether the regulatory region of the GnRH gene is tightly conserved in vertebrates. From ovary and testis, we isolated and sequenced for the first time two different genes and their complementary DNAs that encode the identical peptide known as salmon GnRH. Rainbow trout were selected because they are tetraploid due to genome duplication. A downstream promoter is used in the brain and gonads by salmon GnRH messenger RNA1 (mRNA1) and mRNA2, but mRNA2 also uses an upstream promoter only in the gonads. Two types of long mRNA2 transcripts in ovary and testis both use an alternative start site at position 323; one of these types also retains intron 1. This long 5'-untranslated region is a likely site for distinct regulation of mRNA in the gonad. Additional evidence for separate regulation is that a different expression pattern exists in brain and gonads for GnRH mRNAs during development and maturation. Gene duplication did not alter the encoded peptide, but changed the expression pattern and resulted in complete divergence of the promoter sequence from position -215. A comparison of the mammalian and trout GnRH genes reveals that the promoters are without sequence identity except for a few consensus sites in both regulatory regions. The duplicated trout genes provide a model to study a critical gene whose product controls reproduction in all vertebrates.

Evidence for gonadotropin-releasing hormone peptides in the ovary and testis of rainbow trout.

GnRH is usually classified as a neuropeptide that is synthesized in the brain. Recent evidence indicates that GnRH mRNA is present also in the ovary and testis. However, isolation of the peptide from testis has not been reported. We used HPLC and specific RIAs to determine whether the GnRH peptide can be detected in gonads, the developmental stage at which the peptide is expressed, and the number of molecular forms of GnRH that are present in the ovary and testis. Extracts of immature and mature ovarian and testicular tissue were examined from 17- to 21-mo-old rainbow trout (Oncorhynchus mykiss). 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.

Gonadotropin-releasing hormone in mulberry cells of Saccoglossus and Ptychodera (Hemichordata: Enteropneusta).

Mulberry cells are epidermal gland cells bearing a long basal process resembling a neurite and are tentatively regarded as neurosecretory cells. They occur scattered through the ectoderm of the proboscis, collar, and anterior trunk regions of the acorn worms Saccoglossus, usually in association with concentrations of nervous tissue. They contain secretion granules that appear from electron micrographs to be released to the exterior. The granules are immunoreactive with antisera raised against mammalian and salmon gonadotropin-releasing hormone (GnRH). Similar results were obtained with another enteropneust, Ptychodera bahamensis, using antisera raised against tunicate-1 and mammalian GnRH. Mulberry cells were not found in either Cephalodiscus or Rhabdopleura (Hemichordata: Pterobranchia). Extracts of tissues from 4200 Saccoglossus contain an area of immunoreactive GnRH that is detected by an antiserum raised against lamprey GnRH when characterized by high-performance liquid chromatography and radioimmunoassay. This is the first report of the occurrence of GnRH in hemichordates, probably the most primitive group clearly belonging to the chordate lineage. The physiological function of GnRH in enteropneusts is unknown, but an exocrine function appears more likely than an endocrine or neurotransmitter role.

Regulation and expression of gonadotropin-releasing hormone in salmon embryo and gonad.

This study examines whether gonadotropin-releasing hormone (GnRH) expression in gonads differs from that in brain as to use of alternative promoters and time of expression in early development and reproductive maturation. Within ovary, testis, and embryos, the transcripts from sGnRH gene 1 or gene 2 were expressed using either a conventional or upstream start site and conventional and alternative splicing. In immature sockeye salmon gonads, sGnRH mRNA was expressed briefly during the first 2 years, but in precociously mature fish was expressed for 2 months in the second year. Also, a cDNA encoding another form of GnRH (chicken GnRH-II) was isolated from the gonads. We conclude that differential regulation of GnRH occurs in embryos, ovary and testis compared to brain in salmonids and that more than one form of GnRH is expressed in the salmonid gonad.

Gonadotropin-releasing hormone (GnRH) in ancient teleosts, the bonytongue fishes: putative origin of salmon GnRH.

The molecular forms of gonadotropin-releasing hormone (GnRH) were examined in the bonytongue fishes (Osteoglossomorpha), one of the most ancient living teleost groups. These fish represent a phylogenetic link between the early ray-finned fishes and the modern teleosts. Five representative species from four of six bonytongue families were examined for GnRH using high-performance liquid chromatography and radioimmunoassay techniques with antisera raised against salmon (s), chicken-II (c-II), and mammalian (m) forms of GnRH. Salmon GnRH and cGnRH-II were identified in four of the species (arawana, elephantnose, false featherfin, Asiatic featherfin) whereas in the butterfly fish, mGnRH and cGnRH-II were identified. Our data suggest that teleosts such as eels and butterfly fish, which have mGnRH like that of even earlier ray-finned fishes, may have evolved before fish with sGnRH. We also suggest that sGnRH first appeared in the Osteoglossomorpha. The phylogenetic relationship of the eels (Anguillidae), butterfly fish (Pantodontidae), and bonytongue fish among other teleosts needs to be reexamined using additional characteristics.

A second form of gonadotropin-releasing hormone (GnRH) with characteristics of chicken GnRH-II is present in the primate brain.

The primate brain was thought to contain only the GnRH known as mammalian GnRH (mGnRH). This study investigates whether a second form of GnRH exists within the primate brain. We found that brain extracts from adult stumptail and rhesus monkeys contained two forms of GnRH that were similar to mGnRH and chicken GnRH-II (cGnRH-II) based on the elution position of the peptides from HPLC and on cross-reactivity with antisera that are specific to mammalian or chicken GnRH-II in RIAs. The fetal brain of rhesus monkeys also contained mGnRH and a cGnRH-II-like peptide by the same criteria. Immunocytochemistry with a cGnRH-II-specific antiserum in adult and fetal rhesus monkeys showed immunopositive neurons generally scattered in the periaqueductal region of the midbrain, with a few positive cells in the posterior basal hypothalamus. Neurons immunopositive for cGnRH-II were fewer in number and smaller in size, with less defined nuclei and thinner neurites compared with those for mGnRH. Administration of synthetic cGnRH-II to adult rhesus monkeys resulted in a significant increase in the plasma LH concentration during the luteal phase of the menstrual cycle, but not during the midfollicular phase. We conclude that the primate brain contains mGnRH and a cGnRH-II-like molecule, although the function of the latter is unknown.

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.