The dopaminergic regulation of gonadotropin-releasing hormone receptor binding in the pituitary of the African catfish, Clarias gariepinus

In several teleost species, including the African catfish, dopamine acts as an endogenous inhibitor of gonadotropin-releasing hormone (GnRH)-stimulated gonadotropin (GTH) release. The present in vivo study was carried out to investigate whether this inhibitory action of dopamine can be explained by an effect on the pituitary GnRH receptors. To that end, sexually mature female catfish were treated with dopamine and the dopamine antagonist pimozide (PIM), respectively. At different time intervals after injection, the pituitaries were collected, and in a GnRH receptor assay the GnRH-binding parameters were determined. The dopamine treatment affected neither GnRH-binding capacity nor affinity. The PIM treatment resulted in a two-fold increase in pituitary GnRH-binding capacity without affecting binding affinity. The time course of this effect coincided with the potentiating effect of PIM of the GTH-releasing activity of a GnRH analog. It is concluded that the stimulatory effect of PIM on the action of GnRH might, in part, be due to an increased pituitary GnRH-binding capacity. Reversely, these results suggest that the endogenous dopaminergic inhibition of GnRH-stimulated GTH release may be mediated, at least in part, through down-regulation of the pituitary GnRH receptors.     

Glucagon-like immunoreactivity in the forebrain and pituitary of the teleost, Clarias batrachus (Linn.)

The organization of glucagon-like immunoreactivity (GLI) in the olfactory system, forebrain, and pituitary was investigated in the teleost Clarias batrachus. Weak to moderate GLI was seen in some olfactory receptor neurons and basal cells of the olfactory epithelium. Intense GLI was seen in the olfactory nerve fascicles that ran caudally to the bulb, spread over in the olfactory nerve layer, and profusely branched in the glomerular layer to form tufts organized as spherical neuropils; some of the immunoreactive fibers seem to closely enfold the mitral cells. In the inner cell layer of the bulb, some granule cells were intensely immunoreactive. Although there were thick fascicles of immunoreactive fibers in the medial olfactory tracts (MOT), the lateral olfactory tracts were generally devoid of immunoreactivity. Immunoreactive fibers in the medial olfactory tract penetrated into the telencephalon from its rostral pole and entered into the area ventralis telencephali/pars ventralis where the compact fiber bundles loosen somewhat and course dorsocaudally into the area ventralis telencephali/pars supracommissuralis just above the anterior commissure. While some immunoreactive fibers decussated in the anterior commissure, fine fibers were seen in the commissure of Goldstein. Isolated immunoreactive fibers of the medial olfactory tract were traced laterally into the area dorsalis telencephali/pars lateralis ventralis and mediodorsally into the area dorsalis telencephali/pars medialis. However, a major component of the MOT continued dorsocaudally into the thalamus and terminated in the habenula. Two immunoreactive neuronal groups and some isolated cells were seen in the periventricular region of the thalamus. Although nucleus preopticus showed no immunoreactivity, some neurons of the nucleus lateralis tuberis displayed moderate GLI. Several immunoreactive cells were seen in the pars intermedia of the pituitary gland; few were encountered in the rostral pars distalis and proximal pars distalis. Immunoreactive fibers were seen throughout the pituitary gland.     

Effects of neuropeptide Y on the in vitro release of gonadotropin-releasing hormone, luteinizing hormone, and beta-endorphin and pituitary responsiveness to gonadotropin-releasing hormone in female macaques

The objectives of these studies were to examine the release of gonadotropin-releasing hormone (GnRH) and beta-endorphin-like activity (beta-EP) from macaque hypothalami, and the release of luteinizing hormone (LH) and GnRH-induced LH from macaque anterior pituitaries in response to neuropeptide Y (NPY) treatment. Anterior hypothalamic (AH) and mediobasal hypothalamic (MBH) blocks of tissues and the adenohypophysis were bisected along the midline into two equal-sized fragments. Fragments were superfused with medium for 3 h, followed by 3 h of either NPY (80 nM) or medium alone. In a separate experiment, adenohypophyseal (AP) fragments were superfused in accordance with the same protocol (3 h medium - 3 h NPY or medium) except that exogenous GnRH (352 nM) was added for 30 min at the beginning of hour 3 and again at the beginning of hour 6. Immunoactive GnRH, beta-EP, and LH levels were measured in superfusate samples (400 microliters) collected at 10-min intervals. GnRH levels rose within 20-30 min of initiation of NPY treatment, and elevated GnRH release was sustained for the duration of NPY exposure of both AH and MBH fragments from ovarian intact (INT) rhesus (Macaca mulatta: n = 8; p less than 0.05) or Japanese (Macaca fascicularis; n = 4; p less than 0.01) macaques. NPY treatment had no effect on either AH or MBH fragments isolated from ovariectomized (OVX) rhesus macaques (n = 4 for AH, and n = 5 for MBH). In AP fragments isolated from INT rhesus macaques (n = 8), NPY stimulated LH release within 1 h of treatment (p less than 0.05), whereas NPY had no effect on pituitaries from OVX animals (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-endorphin-like immunoreactivity in the forebrain and pituitary of the teleost Clarias batrachus (Linn.)

The organization of beta-endorphin-like immunoreactivity in the olfactory system, forebrain, and pituitary of the teleost Clarias batrachus was investigated. Immunoreactivity was prominently seen in the sensory neurons and basal cells in the olfactory epithelium and in some cells in the periphery and center (granule cells) of the olfactory bulb. Immunoreactive fibers in the olfactory nerve enter the olfactory nerve layer of the olfactory bulb and branch profusely to form tufts organized as spherical neuropils in the glomerular layer. While fascicles of immunoreactive fibers were seen in the medial olfactory tracts, the lateral olfactory tracts showed individual immunoreactive fibers. Immunoreactive fibers in the medial olfactory tract extend into the telencephalon and form terminal fields in discrete telencephalic and preoptic areas; some immunoreactive fibers decussate in the anterior commissure, while others pass into the thalamus. While neurons of the nucleus lateralis tuberis revealed weak immunoreactivity, densely staining somata were seen at discrete sites along the wall of the third ventricle. Although a large population of immunoreactive cells was seen in the pars intermedia of the pituitary gland, few were seen in the rostral pars distalis and proximal pars distalis; immunoreactive fibers were seen throughout the pituitary gland.     

Immunocytochemical localization of LH-RH in the brain and pituitary of the catfish, Clarias batrachus (Linn.)

An elaborate organization of luteinizing hormone-releasing hormone (LH-RH) immunoreactive (ir) cells and fibers was encountered in the olfactory system of Clarias batrachus. In addition to the ir structures in the olfactory nerve, peripheral area of the olfactory bulb, and the medial olfactory tract (MOT), ir cells and fibers were prominently seen in the lamellae of the olfactory organ. Perikarya showing varying degrees of intensity of immunoreaction were observed along the base of the forebrain in the nucleus preopticus basalis lateralis, nucleus preopticus periventricularis, nucleus preopticus, nucleus lateralis tuberis pars posterior, and the pituitary. Some cells were also noticed in the midbrain tegmentum. A well-defined system of ir fibers from the MOT penetrated the telencephalon and curved dorsocaudally into the pars supracommissuralis above the anterior commissure (AC); while some fibers decussate in the AC, others extended posteriorly into the diencephalon. A fairly dense network of beaded ir fibers was seen in the basal forebrain, conspicuous around the organum vasculosum laminae terminalis and caudally traceable as far as the neurohypophysis; some immunostained fibers appear to be directly contacting with the cells of the proximal pars distalis. Fibers were also witnessed in the optic chiasma and in the inner plexiform layer of the retina. Solitary fibers were noticed in certain circumscribed telencephalic areas, caudal hypothalamus, posterior commissure, midbrain tegmentum, cerebellum, and ventral medulla oblongata. The highly organized LH-RH containing system in C. batrachus is indicative of its elaborate role in synchronization of the reproductive processes and the environmental cues.     

Seasonal changes in beta-endorphin-like immunoreactivity in the olfactory system of the female catfish, Clarias batrachus (Linn)

In the olfactory system of the catfish Clarias batrachus, beta-endorphin-like immunoreactivity was seen in several olfactory receptor neurons (ORN) and their fiber projections extending caudally over the olfactory nerve to the olfactory bulb (OB). With beta-endorphin-like immunoreactivity as a cellular marker, the olfactory system in the female fish was investigated at different stages of its annual reproductive cycle. The reproductive cycle of the fish is divisible into four distinct phases: preparatory (February-April), prespawning (May-June), spawning (July-August), and postspawning (September-January). The gonosomatic index and the immunocytochemical profile of beta-endorphin-like immunoreactivity showed distinct changes as the fish progressed from one phase to another. In the preparatory phase, limited immunoreactivity was seen in the periphery of the bulb. However, the immunoreactivity showed a robust increase as the immunolabeled fibers extended progressively deeper into the bulb toward the mitral cell layer during the prespawning and spawning phases. Significant reduction in the immunoreactivity was noticed in the olfactory nerve layer of the fish in the postspawning phase. Several granule cells showed poor to moderate immunoreactivity during the spawning phase, although no immunoreactivity was seen in the inner cell layer during the rest of the year. The beta-endorphin-like immunoreactivity in the ORN also showed season-related changes, although these were less distinct. Whereas weak immunoreactivity confined to a few ORN was noticed in the fish collected in the preparatory phase, those in the prespawning phase showed conspicuous augmentation in immunoreactivity. During the spawning phase, the sensory layer of the olfactory epithelium showed reduced, homogenous immunoreactivity. In the postspawning phase, several ORN revealed distinct granular immunoreactivity, suggesting possibilities of de novo synthesis. These annual cyclic changes in the beta-endorphin-like immunoreactivity were consistently observed over a 30-month study period that spanned three consecutive spawning phases. The results suggest that the beta-endorphin-containing ORN, their fiber projections to the OB, and several granule cells in the inner cell layer may be involved in the processing of reproduction/reproductive behavior-related signals.     

Involvement of neuropeptide Y Y1 receptors in the regulation of LH and GH cells in the pituitary of the catfish, Clarias batrachus: an immunocytochemical study

Although neuropeptide Y (NPY) has been known to influence the release of luteinizing hormone (LH) and growth hormone (GH) from the pituitary gland of teleosts, the NPY receptor subtypes involved in the regulatory processes have not been fully defined. An attempt has been made to study the involvement of NPY Y1 receptors, if any, in mediating the NPY-triggered stimulation of the LH and GH secreting cells in the pituitary of the catfish, Clarias batrachus. NPY (10 ng/g of body wt) or NPY Y1 receptor agonist (Leu(31)-Pro(34)-NPY, 3 ng/g of body wt) were administered by the intracranial route and the responses by the LH and GH cells in the pituitary were investigated with the help of immunocytochemistry. Both the agents caused a highly significant decrease (P<0.001) in the immunoreactivity of LH cells. However, the treatment with NPY Y1 receptor antagonist (BIBP 3226, 1 ng/g of body wt), prior to NPY or NPY Y1 agonist, blocked the response by the LH cells; the profile of the cells was quite similar to that of the saline-injected control fish. GH cells also showed similar pattern of responses to these treatments. While NPY and NPY Y1 receptor agonist caused significant (P<0.001) decrease in the GH immunoreactivity, pretreatment with the NPY Y1 antagonist blocked the response. These results suggest that NPY may exercise a secretogogue-like action on the LH and GH cells in the pituitary of C. batrachus via NPY Y1 receptors.     

Role of calcium ions in action of gonadotropin-releasing hormone on gonadotropin secretion in the African catfish, Clarias gariepinus

The aim of the present study was to establish the role of calcium ions in the mechanism of action of gonadotropin-releasing hormone (GnRH) in stimulating gonadotropin (GTH) release in the African catfish, Clarias gariepinus. For that purpose, GTH release from pituitary fragments was monitored in a perifusion system. GTH release, induced by the GnRH analog Buserelin, was strongly diminished in the absence of Ca2+, as well as in the presence of the Ca2+ channel antagonist nifedipine. In addition, the Ca2+ ionophore A23187 stimulated GTH secretion in the absence of GnRH. These results indicate that calcium ions play an intermediate role in the mechanism of action of GnRH in the African catfish.     

Nitric oxide: An autocrine regulator of Leydig cell steroidogenesis in the Asian catfish, Clarias batrachus

Nitric oxide has been recognized as an important inter- and intra-cellular modulator of testicular steroidogenesis in higher vertebrates with conflicting results. Moreover, its role in regulation of testicular steroidogenesis in ectothermic vertebrates is non-existent. The present study was, therefore, undertaken to examine whether Leydig cells of a freshwater catfish, Clarias batrachus produce nitric oxide (NO), if so, does it regulate its steroidogenic activity? The purified Leydig cells were stained histochemically for NADPH-diaphorase (NADPH-d) activity, and immunocytochemically for neuronal and inducible nitric oxide synthase (nNOS and iNOS) like molecules. Leydig cells were also incubated with NOS inhibitor, N-nitro-l-arginine methyl ester (l-NAME), and NO donor, sodium nitroprusside (SNP). NO and testosterone released in incubation medium were analyzed. A distinct positive NAPDH-d staining was observed in Leydig cells. These cells also exhibited immunoprecipitation of variable intensity with nNOS and iNOS antibodies. Further, l-NAME treatment caused significant suppression in NO production and elevation in testosterone secretion by Leydig cells. On the contrary, exposure of Leydig cells to SNP resulted in increased NO production with concomitant decline in testosterone level. Thus, the present study reports NO production by Leydig cells in fish for the first time, which appears to inhibit its own androgen production.     

FMRFamide-like immunoreactive nervus terminalis innervation to the pituitary in the catfish, Clarias batrachus (Linn.): demonstration by lesion and immunocytochemical techniques.

Certain thick FMRFamide-like immunoreactive fibers arising from the ganglion cells of nervus terminalis in the olfactory bulb of Clarias batrachus can be traced centripetally through the medial olfactory tract, telencephalon, lateral preoptic area, tuberal area, and hypothalamohypophysial tract to the pituitary. Following 6 days of bilateral olfactory tract transection, the immunoreactivity in the thick fibers, caudal to the lesion site, was partially eliminated, whereas after 10 and 14 days, it was totally abolished in the processes en route to the pituitary. The results indicate a direct innervation of the pituitary gland by the FMRFamide-like peptide containing fibers of the nervus terminalis.     

Interaction of alpha-melanocyte-stimulating hormone with beta-endorphin to influence anterior pituitary hormone secretion in the female rat

The interaction of alpha MSH and beta-endorphin on the secretion of PRL, GH, and LH was determined in the ovariectomized rat. The potent stimulatory effect on PRL release of injection of 20 ng (5.8 pmol) beta-endorphin into the third cerebral ventricle was completely blocked by 100 ng (60 pmol) alpha MSH. The same dose of alpha MSH partially blocked the effect of 150 ng (44 pmol) beta-endorphin on PRL secretion. Intraventricular injection of either 20 ng beta-endorphin or 100 ng alpha MSH had no effect by itself on plasma LH. However, coinjection of these doses of beta-endorphin and alpha MSH suppressed plasma LH levels significantly within 15 min. beta-Endorphin (150 ng) produced a significant suppression of plasma LH levels. Furthermore, coadministration of 100 ng alpha MSH with 150 ng beta-endorphin lowered plasma LH for a longer period of time than this dose of beta-endorphin alone. Low doses of beta-endorphin (20 ng) or alpha MSH (100 ng) alone or in combination did not alter plasma GH levels. A higher dose of 150 ng beta-endorphin produced a slight elevation of plasma GH. This effect was potentiated 5-fold when 150 ng beta-endorphin were injected in combination with 100 ng alpha MSH. These results indicate that alpha MSH acts as an antagonist to beta-endorphin in regard to the secretion of PRL, whereas it potentiates the effect of beta-endorphin in stimulation of GH and inhibition of LH secretion.     

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 microM) or to endogenous cfGnRH, the release of which was induced by forskolin (1 microM). 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.     

Photoperiodic adjustments in hypothalamic amines, gonadotropin-releasing hormone, and beta-endorphin in the white-footed mouse

This study was undertaken to examine short photoperiod (SD; 8 h of light, 16 h of darkness)-induced alterations in reproductive endocrine and neuroendocrine parameters in the male white-footed mouse, Peromyscus leucopus. Exposure to SD for 8 weeks caused dramatic reductions in testis and seminal vesicle weights, decreased circulating LH and testosterone levels, and lowered the content of LH in the pituitary gland relative to those in mice under long photoperiod (LD; 16 h of light, 8 h of darkness). These changes were associated with significant increases in content of radioimmunoassayable GnRH in the mediobasal hypothalamus (MBH) and anterior hypothalamus at two time points in the light/dark cycle: 2100 h (dark phase) and 0900 h (light phase), respectively. Exposure to SD also caused an increase in radioimmunoassayable beta-endorphin in the MBH and preoptic area of the hypothalamus (POA) at 2100 h, but not at 0900 h. Mice exposed to SD also had a significantly higher metabolism of serotonin in the MBH at 0900 and 2100 h compared to mice under LD. The concentration of noradrenaline in the hypothalamus was unaffected by exposure to SD. However, the metabolism of dopamine (DA) in the POA at 0900 h was significantly increased relative to that in mice maintained under LD at this time. This increase in DA metabolism was associated with enhanced immunocytochemical staining for tyrosine hydroxylase in nerve fibers of the POA. Conversely, staining for tyrosine hydroxylase in tuberoinfundibular DA cell bodies of the arcuate nucleus was less intense under SD exposure. From these data it is concluded that exposure to SD caused regional and time-dependent alterations in the activities of hypothalamic amines (serotonin and DA) and neuropeptides (beta-endorphin and GnRH). These changes may be part of the neuroendocrine mechanism for SD-induced seasonal adaptations.     

GnRH-LH secreting cells axis in the pituitary of the teleost Clarias batrachus responds to neuropeptide Y treatment: an immunocytochemical study

An attempt has been made to immunocytochemically visualize the effect of neuropeptide Y (NPY) on the GnRH fibers and luteinizing hormone (LH) secreting cells in the pituitary of the catfish, Clarias batrachus. Two hours following the intracranial administration of NPY at the dose of 20 ng/g body wt, a significant increase in the area occupied by the GnRH-like immunoreactive fibers, and population and size of the LH cells was observed. The treatment also resulted in considerable augmentation of immunoreactivity in the LH cells. Double immunolabeling revealed NPY fibers directly terminating on the LH cells. The results suggest that NPY may (a) stimulate some GnRH containing hypophysiotropic neuronal group in the brain, (b) promote anterograde transport of GnRH to the pituitary gland, and (c) up-regulate the LH cells.     

The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors

Gonadotropin-releasing hormone (GnRH) induces both synthesis and release of pituitary gonadotropins, but rapid or slow frequencies of stimulation result in reduced LH and FSH secretion. We determined the effects of frequency of GnRH stimulation on pituitary GnRH receptors (GnRH-R). Castrate male rats received testosterone implants (cast + T) to inhibit endogenous GnRH secretion. GnRH pulses were injected by a pump into a carotid cannula and animals received GnRH (25 ng/pulse) at various frequencies for 48 h. In control animals (saline pulses) GnRH-R was 307 +/- 21 fmol/mg protein (+/- SE) in cast + T and 598 +/- 28 in castrates. Maximum GnRH-R was produced by 30-min pulses and was similar to that seen in castrate controls. Faster or slower frequencies resulted in a smaller GnRH-R response and GnRH given every 240 min did not increase GnRH-R over saline controls. Equalization of the total GnRH dose/48 h (6.6 ng/pulse every 7.5 min or 200 ng/pulse every 240 min) did not increase receptors to the maximum concentrations seen after 30-min (25 ng) pulses. Serum LH responses after 48 h of injections were only present after 30-min pulses, and peak FSH values were also seen after this frequency. Serum LH was undetectable in most rats after other GnRH frequencies, even though GnRH-R was increased. These data show that GnRH pulse frequency is an important factor in the regulation of GnRH-R. A reduction of GnRH-R is part of the mechanism of down-regulation of LH secretion by fast or slow GnRH frequencies, but altered frequency also exerts effects on secretory mechanisms at a site distal to the GnRH receptor.     

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.     

Correlative changes of the gonadotropin-releasing hormone and gonadotropin-releasing-hormone-associated peptide immunoreactivities in the pituitary portal plasma in female rats

The precursor protein of gonadotropin-releasing hormone (GnRH) contains a 56-amino acid peptide, known as GnRH-associated peptide (GAP), and GnRH. Both of these peptides are localized in the same neurons and coprocessed under varieties of physiological conditions. In the present study, we evaluated whether these two peptides are cosecreted into the pituitary portal blood in female rats under the conditions in which the secretion of hypothalamic GnRH and pituitary luteinizing hormone (LH) are known to be altered. The immunoreactivities of GAP-like peptide (IR-GAP-LI) and GnRH (IR-GnRH) in the portal plasma were 2- to 15-fold higher than those observed in peripheral plasma of female rats. In the pubertal females, the preovulatory LH surge which occurred in the afternoon of the day before vaginal opening (puberty) was found to coincide with surges of IR-GAP-LI and IR-GnRH in the pituitary portal plasma. The surges of IR-GAP-LI and IR-GnRH in portal plasma corresponded with a fall in the preoptic and hypothalamic contents of these peptides. In the adult rats, the levels of IR-GAP-LI and IR-GnRH in portal plasma and LH in peripheral plasma were significantly higher during the afternoon of proestrus than those in the afternoon of diestrus. Ovariectomy increased the portal plasma levels of IR-GAP-LI and GnRH and peripheral plasma levels of LH as compared to the level of these hormones in diestrous females. These results indicate that both GnRH and GAP-LI are cosecreted into pituitary portal blood and that changes in the endocrine environment similarly affect both GnRH and GAP secretion.     

Purification of gonadotropic hormone from the pituitary of the African catfish, Clarias gariepinus (Burchell), and the development of a homologous radioimmunoassay

Glycoprotein gonadotropic hormone (GTH) was purified from 2000 pituitaries of male and female African catfish, Clarias gariepinus. The first step was chromatography on concanavalin A-Sepharose followed by filtration on Ultrogel Aca 54, chromatography on DEAE-cellulose, and filtration on Ultrogel Aca 54, respectively. Finally, the purified fractions were analyzed by polyacrylamide gel electrophoresis. The gonadotropic activity in the different fractions was characterized using two tests: the radioimmunoassay for carp gonadotropin-beta subunit was used to quantify the immunoreactive GTH and a cAMP accumulation test was applied to measure the GTH biological activity. The purified glycoprotein GTH was used to raise antibodies and to develop a radioimmunoassay. This resulted in an assay with a variation between assays of approximately 4%, a precision of 4-8%, and an accuracy of 4-8%. GTH levels can be measured over a range of 0.8 to 12.5 ng/ml.