Catfish Gonadotropin-releasing Hormone Research Articles

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus.

Simple SummaryGonadotropin-releasing hormone (GnRH) plays a pivotal role in fish reproduction. In the present study, transcription activator-like effector nuclease (TALEN) plasmids targeting the catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). After low fertility was observed, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants (individuals with human-induced sequence changes at the cfGnRH locus). Gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0–50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3–74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Cloning, localization and differential expression of Neuropeptide-Y during early brain development and gonadal recrudescence in the catfish, Clarias gariepinus

Neuropeptide-Y (NPY) has diverse physiological functions which are extensively studied in vertebrates. However, regulatory role of NPY in relation to brain ontogeny and recrudescence with reference to reproduction is less understood in fish. Present report for the first time evaluated the significance of NPY by transient esiRNA silencing and also analyzed its expression during brain development and gonadal recrudescence in the catfish, Clarias gariepinus. As a first step, full-length cDNA of NPY was cloned from adult catfish brain, which shared high homology with its counterparts from other teleosts upon phylogenetic analysis. Tissue distribution revealed dominant expression of NPY in brain and testis. NPY expression increased during brain development wherein the levels were higher in 100 and 150days post hatch females than the respective age-matched males. Seasonal cycle analysis showed high expression of NPY in brain during pre-spawning phase in comparison with other reproductive phases. Localization studies exhibited the presence of NPY, abundantly, in the regions of preoptic area, hypothalamus and pituitary. Transient silencing of NPY-esiRNA directly into the brain significantly decreased NPY expression in both the male and female brain of catfish which further resulted in significant decrease of transcripts of tryptophan hydroxylase 2, catfish gonadotropin-releasing hormone (cfGnRH), tyrosine hydroxylase and 3β-hydroxysteroid dehydrogenase in brain and luteinizing hormone-β/gonadotropin-II (lh-β/GTH-II) in pituitary exhibiting its influence on gonadal axis. In addition, significant decrease of several ovary-related transcripts was observed in NPY-esiRNA silenced female catfish, indicating the plausible role of NPY in ovary through cfGnRH-GTH axis.

Endosulfan and flutamide impair testicular development in the juvenile Asian catfish, Clarias batrachus

Endosulfan and flutamide, a widely used pesticide and a prostate cancer/infertility drug, respectively, have an increased risk of causing endocrine disruption if they reach water bodies. Though many studies are available on neurotoxicity/bioaccumulation of endosulfan and receptor antagonism of flutamide, only little is known about their impact on testicular steroidogenesis at molecular level. Sex steroids play an important role in sex differentiation of lower vertebrates including fishes. Hence, a small change in their levels caused by endocrine disruptors affects the gonadal development of aquatic vertebrates significantly. The aim of this study was to evaluate the effects of endosulfan and flutamide on testis-related transcription factor and steroidogenic enzyme genes with a comparison on the levels of androgens during critical period of catfish testicular development. We also analyzed the correlation between the above-mentioned genes and catfish gonadotropin-releasing hormone (cfGnRH)-tryptophan hydroxylase2 (tph2). The Asian catfish, Clarias batrachus males at 50 days post hatch (dph) were exposed to very low dose of endosulfan (2.5μg/L) and flutamide (33μg/L), alone and in combination for 50 days. The doses used in this study were far less than those used in the previous studies of flutamide and reported levels of endosulfan in surface water and sediments. Sampling was done at end of the treatments (100dph) to perform testicular germ cell count (histology), measurements of testosterone (T) and 11-ketotestosterone (11-KT) by enzyme immunoassay and transcript quantification by quantitative real-time PCR. In general, treatments decreased the expression of several genes including testis-related transcription factors (dmrt1, sox9a and wt1), steroidogenic enzymes (11β-hsd2, 17β-hsd12 and P450c17), steroidogenic acute regulatory protein and orphan nuclear receptors (nr2c1 and Ad4BP/SF-1). In contrast, the transcripts of cfGnRH and tph2 were elevated in the brain of all treated groups with maximum elevation in the endosulfan group. However, combination of endosulfan and flutamide (E+F) treatment showed minor antagonism in a few results of transcript quantification. Levels of T and 11-KT were elevated after flutamide and E+F treatments while no change was seen in the endosulfan group signifying the effect of flutamide as an androgen receptor antagonist. All the treatments modulated testis growth by decreasing the progression of differentiation of spermatogonia to spermatocytes. Based on these results, we suggest that the exposure to endosulfan and flutamide, even at low doses, impairs testicular development either directly or indirectly at the level of brain.

Endosulfan and flutamide, alone and in combination, target ovarian growth in juvenile catfish, Clarias batrachus

Juvenile Catfish(es), Clarias batrachus of 50days post hatch (dph) were exposed to endosulfan (2.5 parts per billion [ppb]) and flutamide (33ppb), alone and in combination for 50days to access their impact on ovarian development. The doses used in this study were nominal considering pervious reports. Sampling was done at 100 dph to perform histology and measurement of various transcripts, estradiol-17β and aromatase activity. In general, treatments enhanced expression of ovary-specific transcription factors, steroidogenic enzymes steroidogenic acute regulatory protein and aromatases while transcripts of tryptophan hydroxylase2 (tph2) and catfish gonadotropin-releasing hormone declined in the brain of all treated groups with maximum reduction in the endosulfan group. Significant reduction of tph2 immunoreactivity in the forebrain/telencephalon–preoptic area endorsed our results. Increased number of pre-vitellogenic and less immature oocytes in the treated groups indicated hastened ovarian growth. Elevated ovarian aromatase activity and plasma estradiol-17β levels were noticed in the treated groups with maximum being in the endosulfan group. These data together demonstrate that the exposure of endosulfan causes synchronous precocious ovarian development better than flutamide, alone or in combination. Our results suggest that both endosulfan and flutamide alter ovarian growth by triggering precocious development in catfish.

Thiourea-induced thyroid hormone depletion impairs testicular recrudescence in the air-breathing catfish, Clarias gariepinus

We used thiourea-induced thyroid hormone depletion as a strategy to understand the influence of thyroid hormones on testicular recrudescence of the air-breathing catfish, Clarias gariepinus. Treatment with 0.03% thiourea via immersion for 21 days induced hypothyroidism (thyroid hormone depletion) as evidenced by significantly reduced serum T 3 levels. Thiourea-treated males had narrowed seminiferous lobules with fewer spermatozoa in testis, very little or no secretory fluid, reduced protein and sialic acid levels in seminal vesicles when compared to controls. The histological changes were accompanied by reduction in serum and tissue levels of testosterone (T) and 11-ketotestosterone (11-KT), a potent male specific androgen in fish. Qualitative changes in the localization of catfish gonadotropin-releasing hormone (cfGnRH) and luteinizing hormone (LH, heterologous system) revealed a reduction in the distribution of immunoreactive neuronal cells and fibers in thyroid depleted fish. Interestingly, thiourea-withdrawal group showed physiological and histological signs of recovery after 21 days such as reappearance of spermatozoa and partial restoration of 11-KT and T levels. These data demonstrate that thyroid hormones play a significant role in testicular function of catfish. The mechanism of action includes modulating sex steroids either directly or through the hypothalamo (GnRH)-hypophyseal (LH) axis.

Cupular organs in two species of Corella (Tunicata: Ascidiacea)

Abstract. Simple cupular organs similar to those described in Ciona intestinalis were observed in Corella eumyota. They consist of a macula containing the cell bodies of 20–30 primary sensory neurons whose cilia project into a dome‐ or finger‐shaped structure, the cupula. Rather than being found in the mantle lining as in C. intestinalis, the organs were located on the atrial surface of the branchial sac. The sensory innervation was examined in whole‐mount preparations using anti‐tubulin immunohistochemistry. Sensory neurons in C. eumyota showed no immunoreactivity with antisera raised against gonadotropin‐releasing hormone (GnRH).A novel, elongated sense organ termed the cupular strand was found in Corella inflata. It has the same basic components as the simple type of cupular organ but consists of a single, long structure containing ∼1500 sensory cells. Located on the atrial surface of the branchial sac, it extends along the midline of the dorsal fold, from the gonoduct openings almost as far as the brain. Preparations were examined using optical and electron microscopy. Nerves and cilia were visualized by anti‐tubulin immunofluorescence microscopy. It was possible to follow the sensory axons from the macula of the cupular strand to points where they joined branches of the visceral nerve, which enters a nerve root at the back of the brain.In C. inflata the sensory cell bodies and their axons were immunoreactive not only with anti‐tubulin but also with an antiserum raised against Tunicate I GnRH. There was no immunoreactivity, however, with Chicken II and catfish GnRH antisera. All three GnRH antisera labeled the dorsal strand plexus, a structure associated with production of GnRH in its role as a reproductive hormone. We concluded that the GnRH‐like molecule labeled in sensory neurons differs from the form of GnRH found in the dorsal strand plexus, and may have a different function, perhaps in the neural control of ciliary activity.The function of the cupular organs in species of Corella has not yet been investigated physiologically, but by analogy with such structures in other metazoans, cupular organs are probably hydrodynamic sensors registering local disturbances or changes in water flow through the atrial cavity.

Chimaeric gonadotropin-releasing hormone (GnRH) peptides with improved affinity for the catfish (Clarias gariepinus) GnRH receptor

The gonadotropin-releasing hormone (GnRH) receptor in catfish differs from its mammalian counterparts in showing a very low affinity for the hypothalamic GnRH form [i.e. catfish GnRH (cfGnRH)] and a very high affinity for the highly conserved mesencephalic GnRH, chicken GnRH-II (cGnRH-II). In the present study we investigated the molecular interactions between ligand and receptor involved in determining the ligand selectivity of the catfish GnRH receptor. Studies on the binding characteristics of the catfish GnRH receptor for cfGnRH and cGnRH-II as well as for mammalian GnRH (mGnRH) and synthetic chimaeric GnRHs, differing at positions 5, 7 and 8, revealed that the low affinity of the catfish receptor for cfGnRH can be improved by replacing Leu7 by a tryptophan residue and/or Asn8 by either a tyrosine or an arginine residue. Testing cfGnRH and cGnRH-II as well as mGnRH and the chimaeric GnRHs on Asp304 → Ala, Asp304 → Glu and Asp304 → Asn mutant catfish GnRH receptors revealed that Asp304 of the catfish receptor mediates the recognition of Arg8 in mGnRH, as well as in the chimaeric peptides [Arg8]cfGnRH and [Arg8]cGnRH-II, but seems to be less important for the recognition of Tyr8 in cGnRH-II. On the basis of these results, a three-dimensional model for the binding of [Arg8]cGnRH-II to the catfish GnRH receptor is proposed.

Chimaeric gonadotropin-releasing hormone (GnRH) peptides with improved affinity for the catfish (Clarias gariepinus) GnRH receptor.

The gonadotropin-releasing hormone (GnRH) receptor in catfish differs from its mammalian counterparts in showing a very low affinity for the hypothalamic GnRH form [i.e. catfish GnRH (cfGnRH)] and a very high affinity for the highly conserved mesencephalic GnRH, chicken GnRH-II (cGnRH-II). In the present study we investigated the molecular interactions between ligand and receptor involved in determining the ligand selectivity of the catfish GnRH receptor. Studies on the binding characteristics of the catfish GnRH receptor for cfGnRH and cGnRH-II as well as for mammalian GnRH (mGnRH) and synthetic chimaeric GnRHs, differing at positions 5, 7 and 8, revealed that the low affinity of the catfish receptor for cfGnRH can be improved by replacing Leu(7) by a tryptophan residue and/or Asn(8) by either a tyrosine or an arginine residue. Testing cfGnRH and cGnRH-II as well as mGnRH and the chimaeric GnRHs on Asp(304)-->Ala, Asp(304)-->Glu and Asp(304)-->Asn mutant catfish GnRH receptors revealed that Asp(304) of the catfish receptor mediates the recognition of Arg(8) in mGnRH, as well as in the chimaeric peptides [Arg(8)]cfGnRH and [Arg(8)]cGnRH-II, but seems to be less important for the recognition of Tyr(8) in cGnRH-II. On the basis of these results, a three-dimensional model for the binding of [Arg(8)]cGnRH-II to the catfish GnRH receptor is proposed.

Differences in structure—function relations between nonmammalian and mammalian GnRH receptors: what we have learnt from the African catfish GnRH receptor

Mammalian gonadotropin-releasing hormone (GnRH) receptors differ from other G-protein coupled receptors (GPCRs) in lacking the intracellular C-terminal tail and in showing an exchange of two otherwise highly conserved aspartate protease (Asp) and asparaginyl (Asn) residues in TM 2 and 7, respectively. However, the first GnRH receptor characterized from a nonmammalian vertebrate, the African catfish, contains an intracellular C-terminal tail and has Asp residues. Next to structural variations, differences between mammalian and nonmammalian GnRH receptors are found in their pharmacology and their regulation. In addition, the results of studies on catfish and mammalian GnRH receptor expression, regulation and activation, and ligand binding are summarized. To this end, mammalian-nonmammalian chimeric GnRH receptors, site-directed mutagenesis of GnRH receptors, various GnRH analogs, and a three-dimensional model of the receptor-ligand complex were used. It is demonstrated that the catfish GnRH receptor is susceptible to agonist-induced phosphorylation and that Ser in the C terminal tail is the major phospho-acceptor site in this process. Mammalian GnRH receptors, on the contrary, are resistant to agonist-dependent phosphorylation due to the lack of a C-terminal tail.

Development of three distinct GnRH neuron populations expressing two different GnRH forms in the brain of the African catfish (Clarias gariepinus)

The early development of both the catfish gonadotropin-releasing hormone (cfGnRH)- and the chicken GnRH-II (cGnRH-II) system was investigated in African catfish by immunocytochemistry by using antibodies against the GnRH-associated peptide (GAP) of the respective preprohormones. Weakly cfGnRH-immunoreactive (ir) neurons and fibers were present at 2 weeks after hatching (ph) but only in the ventral telencephalon and pituitary. Two weeks later, cfGnRH fibers and neurons were also observed in more rostral and in more caudal brain areas, mainly in the preoptic area and hypothalamus. Based on differences in temporal, spatial, and morphologic appearance, two distinct cfGnRH populations were identified in the ventral forebrain: a population innervating the pituitary (ventral forebrain system) and a so-called terminal nerve (TN) population. DiI tracing studies revealed that the TN population has no neuronal connections with the pituitary. The cGnRH-II system is present from 2 weeks ph onward in the midbrain tegmentum and only their size and staining intensity increased during development. Based on the comparison of GnRH systems amongst vertebrates, we hypothesize that during fish evolution, three different GnRH systems evolved, each expressing their own molecular form: the cGnRH-II system in the midbrain, a hypophysiotropic GnRH system in the hypothalamus with a species-specific GnRH form, and a salmon GnRH-expressing TN population. This hypothesis is supported by phylogenetic analysis of known GnRH precursor amino acid sequences. We hypothesize, because the African catfish is a less advanced teleost species, that it contains the cfGnRH form both in the ventral forebrain system and in the TN population.

Proper receptor signalling in a mutant catfish gonadotropin-releasing hormone receptor lacking the highly conserved Asp 90 residue

The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein-coupled receptors, except in mammalian gonadotropin-releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp 90 of the catfish GnRH receptor can be substituted by a neutral Asn 90 without abolishing receptor signalling if another negatively charged Glu 93 is introduced in a proximal region of the receptor interior, thereby mimicking the Glu 90–Lys 121 salt bridge of mammalian GnRH receptors.

Gonadal steroids and the maturation of the species-specific gonadotropin-releasing hormone system in brain and pituitary of the male African catfish (Clarias gariepinus)

The effect of testosterone (T), 11-ketotestosterone (KT) and estradiol (E2) on the development of the catfish gonadotropin-releasing hormone system (cfGnRH) of male African catfish (Clarias gariepinus), at the onset of puberty [between 10 and 12 weeks post hatching (ph)] was investigated. The cfGnRH neurons, located in the ventral forebrain, were visualized by immunofluorescence and their numbers were determined and the amounts of cfGnRH-associated peptide (cfGAP) in the pituitary were measured by RIA. Steroid treatments did not significantly alter the numbers of immunoreactive GnRH neurons. However, T and E2 caused an increase in the amount of GnRH, demonstrated by the intensity of the immunostaining of GnRH neurons and fibers in the brain and the amount of cfGAP in the pituitary. Treatment with KT, the main circulating androgen in adult male catfish, neither changed the number of cfGnRH neurons, nor elevated the cfGnRH content in the pituitary. In previous experiments with younger, prepubertal fish (2–6 weeks ph), T caused an elevation of the number of cfGnRH neurons to the same level as present in pubertal fish of 12–14 weeks. We conclude that the onset of puberty in the male African catfish coincides with the completion of the steroid-dependent structural maturation of the cfGnRH system in the brain. T and/or E2, however, are still able to exert a positive influence on the amounts of cfGnRH during the later stages of pubertal development, thus still playing a role in the control of the cfGnRH system.

Gonadotropin-Releasing Hormone Fibers Innervate the Pituitary of the Male African Catfish (Clarias gariepinus) during Puberty

The development of the catfish gonadotropin-releasing hormone (cfGnRH) fiber network in the pituitary of male African catfish (Clarias gariepinus) was investigated in relation to puberty. Double immunolabeling studied by confocal laser scanning microscopy revealed a concomitant development of gonadotropes and of pituitary cfGnRH innervation during the first wave of spermatogenesis. Catfish GnRH-immunoreactive fibers in the proximal pars distalis (PPD) of the pituitary were initially observed at the age of 10 weeks (onset of spermatogonial proliferation) and gradually reached the adult pattern at the age of 20 weeks (spermatozoa present in the testis). The content of cfGnRH-associated peptide (cfGAP, part of the prohormone) in the pituitary similarly increased during puberty. At the electron microscopical level, fibers containing cfGAP-ir granules came into close proximity of the gonadotropes at 18 weeks of age. In vitro studies indicated a progressively increasing basal and cfGnRH-stimulated luteinizing hormone (LH) secretion during pubertal development. The LH secretion patterns were similar in response to exogenous cfGnRH (0.1 µM) or to endogenous cfGnRH, the release of which was induced by forskolin (1 µM). Castration experiments demonstrated that the innervation of the pituitary with cfGnRH fibers continued after surgery, accompanied by an increase in the cfGAP levels. However, gonadotrope development was retarded, suggesting a differential regulation of the two maturational processes. Since testosterone stimulates both processes, other testicular factors may also be involved. Puberty-associated changes in LH release patterns appear to reflect changes in the GnRH sensitivity and in the pool of releasable LH, while availability of cfGnRH does not appear to be a limiting factor.

Molecular Cloning and Tissue-Specific Expression of a Gonadotropin-Releasing Hormone Receptor in the Japanese Eel

Gonadotropin-releasing hormone (GnRH) is a key regulatory neuropeptide involved in the control of reproduction in vertebrates. In the Japanese eel, one of the most primitive teleost species, two molecular forms of GnRH, mammalian-type GnRH and chicken-II-type GnRH (cGnRH-II), have been identified. This study has isolated a full-length cDNA for a GnRH receptor from the pituitary of the eel. The 3233-bp cDNA encodes a 380-amino acid protein which contains seven hydrophobic transmembrane domains and N- and C-terminal regions. The exon/intron organization of the open reading frame of the eel GnRH receptor gene was also determined. The open reading frame consists of three exons and two introns. The exon–intron splice site is similar to that of the GnRH receptor genes of mammals reported so far. Expression of the eel GnRH receptor was detected in various parts of the brain, pituitary, eye, olfactory epithelium, and testis. This result suggests that GnRH has local functions in these tissues in addition to its actions on gonadotropin synthesis and release in the pituitary. This tissue-specific expression pattern is similar to that of the eel cGnRH-II. Furthermore, the present eel receptor shows very high amino acid identity with the catfish and goldfish GnRH receptors, which are highly selective for the cGnRH-II. These results suggest that the cGnRH-II acts through binding to the present receptor in the eel.

GnRH stimulates LH release directly via inositol phosphate and indirectly via cAMP in African catfish.

In African catfish, two gonadotropin-releasing hormone (GnRH) peptides have been identified: chicken GnRH (cGnRH)-II and catfish GnRH (cfGnRH). The GnRH receptors on pituitary cells producing gonadotropic hormone signal through inositol phosphate (IP) elevation followed by increases in intracellular calcium concentration (¿Ca(2+)(i)). In primary pituitary cell cultures of male African catfish, both cGnRH-II and cfGnRH dose dependently elevated IP accumulation, ¿Ca(2+)(i), and the release of the luteinizing hormone (LH)-like gonadotropin. In all cases, cGnRH-II was more potent than cfGnRH. The GnRH-stimulated LH release was not associated with elevated cAMP levels, and forskolin-induced cAMP elevation had no effect on LH release. With the use of pituitary tissue fragments, however, cAMP was elevated by GnRH, and forskolin was able to stimulate LH secretion. Incubating these fragments with antibodies against cfGnRH abolished the forskolin-induced LH release but did not compromise the forskolin-induced cAMP elevation. This suggests that cfGnRH-containing nerve terminals are present in pituitary tissue fragments and release cfGnRH via cAMP signaling on GnRH stimulation, whereas the GnRH receptors on gonadotrophs use IP/¿Ca(2+)(i) to stimulate the release of LH.

Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation.

Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment receptors due to the absence of an intracellular C-terminal tail frequently important for internalization and/or desensitization of other G protein-coupled receptors. The recent cloning of nonmammalian (i.e. catfish, goldfish, frog, and chicken) GnRHRs shows that these contain an intracellular C terminus. Addition of the 51-amino acid intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR (rGnRHR) did not affect rGnRHR binding affinity but elevated receptor expression by about 5-fold. Truncation of the added C terminus impaired the elevated receptor-binding sites by 3- to 8-fold, depending on the truncation site. In addition, introducing the C terminus to rGnRHR altered the pattern of receptor regulation from biphasic down-regulation and recovery to monophasic down-regulation. The extent of down-regulation was also enhanced. The alteration in receptor regulation due to the addition of a C terminus was reversed by truncation of the added C terminus. Furthermore, addition of the cfGnRHR C terminus to rGnRHR significantly augmented the inositol phospholipid (IP) response of transfected cells to Buserelin, but this did not result from the elevation of receptor-binding sites. Addition of the C terminus did not affect Buserelin-stimulated cAMP and PRL release. GH3 cells transfected with wild-type cfGnRHR did not show measurable Buserelin binding or significant stimulation of IP, cAMP, or PRL in response to Buserelin (10[-13]-10[-9] M). GH3 cells transfected with C terminus-truncated cfGnRHR showed no IP response to Buserelin (10[-13]-10[-7] M). These results suggest that addition of the cfGnRHR intracellular C terminus to rGnRHR has a significant impact on rGnRHR expression and regulation and efficiency of differential receptor coupling to G proteins.

Addition of Catfish Gonadotropin-Releasing Hormone (GnRH) Receptor Intracellular Carboxyl-Terminal Tail to Rat GnRH Receptor Alters Receptor Expression and Regulation

Addition of Catfish Gonadotropin-Releasing Hormone (GnRH) Receptor Intracellular Carboxyl-Terminal Tail to Rat GnRH Receptor Alters Receptor Expression and Regulation

Internal and external factors controlling reproduction in the African catfish,Clarias gariepinus

The African catfish, Clarias gariepinus, is a highly appreciated species for aquaculture, because of its favourable food conversion, its resistance to discases, its relatively low requirements for water quality, the possibility for high stocking density and the excellent meat quality. For those reasons even in the Netherlands, there is a modest, but Europe's largest and still expanding, African catfish farming activity. Although this species grows and matures in captivity, there is no spontaneous reproduction. We could demonstrate that the failure to reproduce resides in the brain-pituiary-gonad axis. Hormones required for oogenesis and spermatogenesis are being produced in sufficient quantities. However, final oocyte maturation, ovulation, spermiation and spawning behaviour do not occur, due to the lack of a gonadotropin surge. In nature, the prespawning gonadotropin surge is induced by environmental factors such as the water level in the spawning area. Under farming conditions the environmental cues are hard to identify and/or to mimic. In combination with unavoidable stress this causes a blockade of the release of gonadotropin releasing hormone (GnRH). Consequently, gonadotropin surge release fails to occur, which is enforced by an effective hypothalamic dopaminergic inhibition. The gonadrotropin surge induces the conversion of 17 αOH-progesterone into 17 αhydroxy-20 β-dihydroprogesterone, the final maturation inducing substance. Based on these data, several protocols for artificial propagation could be developed. They include either a treatment with a GnRH analogue in combination with a dopamine receptor antagonist, a treatment with homologous gonadotropin or HCG, or a treatment with 17 αOH-progesterone. Since a number of years we have used the African catfish as a model for fundamental research on fish reproductive endocrinology. Till now one gonadotropic hormone (GTH) could be demonstrated. Its amino acid composition and sequence was analysed and appeared to be homologous with known forms of the maturational GTH (GTH-II). Specific radioimmuno assays for the complete hormone and its α- and β-subunit respectively, have been developed. cDNAS encoding the subunits have been cloned. They are applied now for Northern blotting and in situ hybridization. GnRHs were fully characterised (a specific catfish-GnRH and chicken-GnRH-II). Specific antibodies against these peptides were raised and the cDNAS encoding the hormone precursor molecules were cloned and used for respectively immunocytochemical localisation and radioimmunoassays, and in situ hybridisation. The importance of the two GnRH forms for gonadotropin release was studied. Chicken-GnRH-II appears to be 10 to 100 times more potent than catfish GnRH, probably due to its higher receptor affinity. Catfish GnRH, however, is present in the brain and pituitary about 100 times more than chicken GnRH-II. Steroid hormone synthesis by ovaries, testis and seminal vescicles was analysed. The sex sieroids that play a role in the negative feedback control of gonadotopin release were identified (11-keto-testosterone and testosterone) and their interaction with hypothalamic dopamine metabolism was demonstrated as one of the possible mechanisms of action. Several steroid conjugates from the seminal vesicles were shown to have pheromonal activities, involved in reproductive behaviour. They induce under certain physiological conditions attraction between conspecifics and synchronization of ovulation.

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His 5,Trp 7,Leu 8]-GnRH, dogfish (df) GnRH; [His 5,Asn 8]-GnRH, catfish (cf) GnRH; and [His 5,Trp 7,Tyr 8]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro ( d)-isomers d-arginine ( d-Arg) or d-naphthylalanine ( d-Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of d-Nal 6 into [His 5,Asn 8]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of d-Nal 6 or d-Arg 6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [ d-Nal 6,Pro 9NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [ d-Nal 6,Pro 9NEt]-mGnRH ( k d = 0.40 ± 0.04 and 0.55 ± 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.

Isolation, characterization and expression of cDNAs encoding the catfish‐type and chicken‐II‐type gonadotropin‐releasing‐hormone precursors in the African catfish

The cDNAs encoding the catfish prepro-gonadotropin-releasing hormone and the chicken prepro-gonadotropin-releasing hormone II of the African catfish (Clarias gariepinus) have been isolated and sequenced. The catfish gonadotropin-releasing-hormone precursor and the chicken gonadotropin-releasing-hormone-II precursor have the same overall architecture as other gonadotropin-releasing-hormone precursors identified so far; each is composed of a signal peptide, gonadotropin-releasing hormone and a gonadotropin-releasing-hormone-associated peptide which is connected to gonadotropin-releasing hormone and chicken gonadotropin-releasing hormone II, in combination with the Gly-Lys-Arg sequence, are highly conserved during evolution when compared with the corresponding regions of mammalian, avian (chicken gonadotropin-releasing hormone I) and other fish gonadotropin-releasing-hormone precursors. However, the gonadotropin-releasing-hormone-associated peptide regions are markedly divergent. Northern-blot analysis revealed the presence of a single catfish gonadotropin-releasing-hormone mRNA species of about 470 bases, and the presence of a single chicken gonadotropin-releasing-hormone-II mRNA species of about 650 bases in the African catfish brain. In situ hybridization revealed catfish gonadotropin-releasing-hormone cell bodies rostro-caudally scattered in the olfactory nerve, along both sides of the midline of the telencephalon, in the preoptic area of the ventral hypothalamus, and in the infundibular stalk close to the pituitary. Chicken gonadotropin-releasing-hormone-II cell bodies, however, were exclusively found in the midbrain tegmentum.