Pituitary Of Fish Research Articles

Anatomical distribution of galanin-like immunoreactivity in the brain and pituitary of teleost fishes

Immunohistochemical study of brains of five teleost fishes (molly, sea bass, killifish, flounder, tilapia) revealed similar extensive systems of galanin immunoreactive (GAL-ir) neurons. Cell bodies were located in the anterior preoptic recess (where coexistence with corticotrophin-releasing factor-like-ir was found), posterior tuberal hypothalamus and vagal lobe of the medulla oblongata. Fibres in the fingers of neurohypophysial tissue penetrating the pituitary pars distalis suggested an anatomical relationship between GAL-ir terminals and the hormone secreting cells. Electron microscopic studies on sea bass pituitary revealed contacts of GAL-ir fibres with growth hormone cells and gonadotrophs. Thus a GAL-like peptide may be released from nerve terminals in the teleost pituitary, where it may act locally to modulate the secretion of one or more pituitary hormones.

Detection of Met-enkephalin and Leu-enkephalin in the posterior pituitary of the holostean fish, Amia calva

Immunohistochemical analysis of the pituitary of the holostean fish, Amia calva, indicated that enkephalin-related immunoreactivity was restricted to the pars nervosa, and was not detected in other regions of the pituitary. Fractionation of acid extracts of posterior pituitaries by reverse phase HPLC followed by RIA analysis indicated the presence of immunoreactive Met-enkephalin and Leu-enkephalin. No immunoreactive forms were detected with RIAs specific for either Met-enkephalin-RF or Met-enkephalin-RGL. The molar ratio of Met- to Leu-enkephalin in this terminal field was 3:1 ( n=4 ). HPLC fractions were also digested with trypsin and carboxypeptidase B to test for C-terminally extended forms of Met-enkephalin. A novel modified form of Met-enkephalin was detected. Extracts of the posterior pituitary, forebrain, midbrain, hypothalamus and hindbrain were screened with RIAs specific for the Pro-dynorphin end products, alpha-neo-endorphin, dynorphin A(1–17), dynorphin A(1–8) and dynorphin B(1–13). The results of these analyses were negative. Collectively, these data suggest that a Pro-enkephalin-like molecule is present in holostean fish. The holostean enkephalin precursor contains at least Met-enkephalin and Leu-enkephalin. However, Pro-dynorphin-related end products with antigenic determinants similar to mammalian dynorphin A(1–17), dynorphin A(1–8), dynorphin B(1–13) and alpha-neo-endorphin could not be detected in the brain or pituitary of this species.

Requirement of extracellular calcium in fish pituitary gonadotropin release by gonadotropin hormone-releasing hormone.

Influence of extracellular calcium on gonadotropin hormone-releasing hormone (GnRH)-stimulated gonadotropin hormone (GtH) release from a teleostean fish (Channa punctatus) pituitary was examined in vitro by preparing enzymatically dispersed pituitary cell incubation. Effect of Ca2+ on GnRH-augmented GtH release was evaluated with partially purified C. punctatus GnRH (cGnRH) and synthetic mammalian GnRH (mGnRH). Cells were dispersed by 0.3% collagenase plus 0.05% trypsin in culture medium and a high yield of viable cells were obtained. Addition of cGnRH (10 micrograms/ml) to pituitary cells in Ca2+-free medium resulted in a significant increase in GtH release, but the addition of Ca2+ (2 mM) enhanced it to about four- and threefold over cGnRH and mGnRH, respectively. Increasing concentrations of Ca2+ (0.1-2.0 mM/well) with fixed concentrations of GnRH (10 micrograms/ml) or increasing doses of GnRH (2.5 to 20 micrograms/ml) with fixed amount of Ca2+ (2 mM/well) resulted in a dose dependent increase in GtH release. EDTA or EGTA (2 mM/well) completely suppressed the Ca2+-augmenting effect of GnRH-stimulated GtH release. Addition of lanthanum (La3+, 4 mM/well), a competitive inhibitor of Ca2+, reduced 60% of the Ca2+ (2 mM/well) stimulation. Verapamil, a specific Ca2+ channel blocker, when added in increasing concentrations (1-100 microM/well) to pituitary cell incubations containing GnRH-stimulated GtH release in Ca2+-free medium could be waived by EGTA (2 mM/well), indicating availability of extracellular calcium from tissue sources. The uptake of radioactive Ca2+ by pituitary cells was greatly enhanced by GnRH while the addition of verapamil (10 microM/well) not only inhibited the GnRH-stimulated uptake, but also reduced it below the control level.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic localization of binding sites for vasotocin in the brain and pituitary of the sea bass ( Dicentrarchus labrax)

Specific binding sites for vasotocin (VT) were detected in brain and pituitary of a teleost fish, the sea bass, after in vitro incubation of tissue sections with [ 3H]arginine-vasopressin (AVP) and light microscopic autoradiography. Conditions for the binding assay were optimized and as a result the binding was saturable and specific. In the brain [ 3H]AVP binding was found to occur in the pars lateralis and the pars ventralis of the ventral telencephalon, in the pars centralis of the dorsal telencephalon, in the hypothalamic region (especially in the nucleus preopticus, in the tuberal hypothalamus and around the posterior recess), in the tectum opticum and in the noncellular layer of the corpus cerebelli. In the pituitary a high density of [ 3H]AVP binding was observed in the areas of the pars distalis (PD) occupied by ACTH-, TSH- and GH-cells and also in the pars intermedia (PI). The present study presents the first anatomical evidence for the presence of VT specific binding sites in teleost brain and pituitary.

Folliculo-Stellate Cells of the Pituitary Gland

n the early 1950s, scientists studying the ultrastructure of the rat pituitary gland described starshaped cells whose arms, or processes, extend among their neighbors (Rinehart and Farquhar 1953, 1955). Soon after, Farquhar (1957) described cells that border an irregularly shaped space, or follicle, containing gelatinous material. There followed many electron microscopy studies describing either stellate or follicular cells in various areas-the pars intermedia (PI), pars distalis (PD), and even the enigmatic pars tuberalis (PT)-of the pituitary of amphibians, fish, reptiles, and mammals (Figure 1). However, scientists gradually came to realize that they were describing two views of the same cell type, now known as folliculo-stellate (FS) cells. Even though the roles of various other cells in the pituitary have been delineated, the FS cell remains, as described by Nunez and Gershom (1981), cell in search of a function. This article will consider the ultrastructure of this cell and relate its morphology to possible suggested functions. Certainly the FS cell is not the only cell whose function is unclear. Scientists still debate the function of the

Effects of Aroclor 1254 and cadmium on reproductive endocrine function and ovarian growth in atlantic croaker

The effects of a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) and cadmium on the reproductive endocrine function in female Atlantic croaker ( Micropogonias undulatus) were investigated. Fish were fed Aroclor 1254 in the diet (0·5 mg/100 g body wt/day) for 17 days or exposed to 1 mg cadmium/liter seawater (30% S) for 40 days during the period of ovarian recrudescence. Both Cd and the PCB disrupted reproduction, but they exerted opposite effects on reproductive endocrine function and ovarian growth. PCB treatment impaired ovarian growth which was accompanied by a significant decline in vitellogenesis as assessed by a decrease in plasma estradiol levels. In addition there was a decrease in spontaneous secretion of gonadotropin (GTH) in vitro by pituitaries from PCB-treated fish. In contrast, exposure to Cd accelerated ovarian growth and elevated plasma estradiol concentrations, suggesting a stimulation of vitellogenesis. In addition there was a marked increase in the spontaneous secretion of GTH from pituitaries of Cd-exposed fish in vitro. The results show that chemicals can disrupt reproductive processes in teleosts both by inhibiting and stimulating the activity of the hypothalamus-pituitary-gonadal-liver axis.

Studies of the biological activity of LHRH analogs in the rainbow trout, landlocked salmon, and the winter flounder

Studies of gonadotropic hormone (GtH) release bioactivity by mammalian and submammalian varities of LHRH and LHRH analog were primarily conducted in vivo in testosterone-primed yearling (TPY) rainbow trout, a convenient test animal for LHRH bioassays in fish. Validation of these results, using sexually mature fish, was accomplished by examining LHRH agonist activities on release of GtH in vivo in spermiating landlocked salmon and by studying LHRH peptide hormone binding affinities using a flounder pituitary LHRH radioreceptor assay. Our surveys of LHRH analog bioactivity in vivo in TPY trout and salmon demonstrated that all types offish, bird, and mammalian LHRH agonists possess superactive properties on the fish pituitary. The most active group of LHRH analogs, based upon both LHRH receptor binding affinity and in vivo release of gonadotropin, was judged to include [ d-Nal(2) 6,Pro 9-NHEt]LHRH, [ d-Nal(2) 6-AzaGly] 10]LHRH, [ d-Ala 6,Pro 9-NHEt]-LHRH, and the fish LHRH analogs, [ d-Arg 6,Trp 7 Leug,Pro 8-NHEt]LHRH, [ d-hArg(Et 2 6,Trp 7 LeU 8, Pro 9-NHEt]LHRH.

Development of salmon GTH I and GTH II radioimmunoassays

Radioimmunoassays (RIAs) for the measurement of two gonadotropins, GTH I and GTH II, in the plasma and pituitary of salmonid fish were developed using a rabbit antiserum to β-subunits of chum salmon GTH I and GTH II. Intact GTH I and GTH II were used as standards and radioactive competitors. The displacement curves for plasma of salmonids including chum salmon, amago salmon, and rainbow trout were parallel to chum salmon GTH I and GTH II standards. Parallel displacement curves were obtained for pituitary extracts of chum salmon and amago salmon. The cross-reactivities of growth hormone, prolactin, and proopiomelanocortin (POMC)-related hormones were less than 1% in both RIAs. However, cross-reactivities of GTH I in the GTH II RIA and GTH II in the GTH I RIA were 10 and 12%, respectively. Plasma concentrations of both GTHs from salmonids at various stages of reproductive development were compared. In immature rainbow trout of both sexes (males: average (AV) gonadosomatic index (GSI) = 0.05; females: AV GSI = 0.24), plasma levels of GTH I and GTH II were low (less than 2 ng/ml). During prematurational stages of spermatogenesis and vitellogenesis in rainbow trout (males: AV GSI = 0.43; females: AV GSI = 2.8), the predominant GTH in the pituitary and plasma was GTH I. In contrast, plasma concentrations of GTH II were significantly higher than those of GTH I in postovulatory amago and chum salmon. Similarly, pituitary concentrations of GTH II were significantly higher than those of GTH I in postovulatory and spermiating amago salmon and postovulatory chum salmon. These data suggest that the synthesis and secretion of GTH I and GTH II vary during salmonid reproductive development.

Forms of immunoreactive β-endorphin in the intermediate pituitary of the holostean fish, Amia calva

Acid extracts of the intermediate pituitary of the holostean fish, Amia calva, were fractionated by gel filtration chromatography and analyzed with radioimmunoassays specific for N-acetylated β-endorphin and C-terminally amidated α-MSH. In these extracts β-endorphin-related immunoreactive material and α-MSH-related immunoreactive material were present in roughly equimolar amounts. The immunoreactive β-endorphin-sized material was tested for opiate receptor binding activity using a β-endorphin radioreceptor assay. The results of these studies were negative. The immunoreactive β-endorphin-sized material was further analyzed by cation exchange chromatography at pH 2.5. Two major and three minor peaks of immunoreactive material were isolated. Peak 5 exhibited a net charge of +7 at pH 2.5 and represented 53% of the total immunoreactivity recovered. Peak 2 with a net charge of +3 at this pH represented 38% of the total immunoreactivity recovered. The minor forms, Peaks 1, 3 and 4, exhibited net charges of +2, +4 and +6, respectively. The apparent molecular weights of Peaks 2 and 5 were determined on a Sephadex G-50 column. Peak 2 had an apparent molecular weight of 2.7 Kd and Peak 5 had an apparent molecular weight of 3.5 Kd. Reverse phase HPLC analysis of Peak 5 indicates that this form of Amia β-endorphin had chromatographic properties similar to salmon β-endorphin II. These results would suggest that N-terminal acetylation and C-terminal proteolytic cleavage are important post-translational modifications of the forms of Amia β-endorphin.

Localization of CRF-like immunoreactivity in the brain and pituitary of teleost fish

Immunocytochemical techniques were applied to brain and pituitary sections of eleven teleost species. A corticotropin-releasing factor (CRF)-antiserum allowed the identification of a CRF-like system in these species. Perikarya were labeled in the preoptic nucleus. Labeled fibers were traced laterally, then ventrally close to the optic chiasma, forming two symmetrical tracts running through the basal hypothalamus. These ended in the rostral neurohypophysis (NH) close to ACTH cells as shown by double immunostaining. Other fibers, often more variquous, ended in the caudal NH close to melanocorticotropic cells. In Salmo fario, small perikarya also stained in the nucleus lateralis tuberis. The CRF-like system appears distinct from that of somatostatin. In Anguilla, adjacent sections stained with CRF- and vasotocin (AVT)-antisera respectively showed that these two peptides coexist in some perikarya. As few fibers containing only AVT end in the rostral NH, they probably do not control ACTH cells directly. AVT fibers terminate mostly in the caudal NH close to melanocorticotropic cells. Some extra-hypothalamic fibers suggest that CRF may also act as a neurotransmitter. The plurality of hormones showing a CRF-like activity in teleosts is considered.

Identification and distribution of the cell types in the pituitary gland of Austromenidia laticlavia (Teleostei, Atherinidae)

By using antisera against human pituitary hormones in immunocytochemistry in combination with classical cytochemical techniques, we have been able to identify the different cell types in the adenohypophysis of the Austromenidia laticlavia and to determine their location. Antisera against prolactin and growth hormones did not stain cells in the pituitary of Austromenidia, whereas antisera against β-endorphin, LH, and β-TSH selectively crossreacted with cells which have a specific location within the adenohypophysis. The β-endorphin antiserum stained the periodic acid-Schiff (PAS)-negative cells in the pars intermedia and also, though faintly, the PAS-negative cells in the internal border of the rostral pars distalis (RPD). Human β-TSH antiserum showed a discrete population of small PAS-positive cells in the proximal pars distalis (PPD). Antiserum against human LH stained PAS-positive cells located in the most ventral zone of the PPD and around the pars intermedia (PI). The distribution of the different cell types is similar to that of other teleosts. The phylogenetic implications of the degree of cross-reactivity of the antisera against human pituitary hormones with specific cells of the teleost fish pituitary is discussed.

Brain distribution of radioimmunoassayable gonadotropin-releasing hormone in female goldfish: Seasonal variation and periovulatory changes

A radioimmunoassay (RIA) for [Trp 7, Leu 8]gonadotropin-releasing hormone (sGnRH) was developed to determine the gonadotropin-releasing hormone (GnRH) content in discrete brain areas of female goldfish at different stages of ovarian development. Temporal changes in serum gonadotropin (GtH) and GnRH concentrations in discrete brain areas were measured during spontaneous ovulation. There were no clear parallel changes in brain GnRH with seasonal ovarian development in goldfish. However, under a 10° temperature acclimation regimen, the GnRH content in the hypothalamus and pituitary decreased as the ovary progressed from the regressed to the mature condition; on the other hand, GnRH content in the spinal cord increased in sexually mature fish compared with that in regressed fish. Significant decreases in GnRH concentration were observed in certain brain areas (olfactory bulbs, telencephalon, hypothalamus, and pituitary) of fish undergoing spontaneous ovulation compared with those of nonovulatory fish. The simultaneous changes of GnRH concentration in these brain areas suggested that the GnRH neuronal system may function as an integrated unit for the activation of GtH secretion during ovulation in goldfish.

Ovarian and body responses of the African catfish ( Clarias gariepinus) to human chorionic gonadotropin (Chorulon R) and carp pituitary suspension, used in a bioassay for estimating the gonadotropic activity of a crude carp powder preparation

Teleostean in vivo bioassays used for estimating the gonadotropic activity of fish pituitaries and gonadotropins are not standardized. Ovarian and body responses of the African catfish ( Clarias gariepinus) to human chorionic gonadotropin (hCG) and a carp pituitary suspension (cPS) were used in a bioassay for estimating the gonadotropic activity of a crude carp powder preparation. The relationship between log-dose hCG and ovarian and body response was examined. Amongst the responses studied the stripped egg weight and stripping of ovary (wet weight of stripped eggs/wet weight of stripped eggs+wet weight of ovary after stripping × 100) are the most useful variables for a bioassay. The dose interval of 1103–1558 IU hCG and 0.724-1.025 mg cPS (per fish of 510 g, range ±50g) can be used for these responses in a bioassay. Whether or not the three-point parallel line assay provides a suitable model in the African catfish to perform a routine bioassay is not clear yet. Three fish per dose appears, in general, to be too small a number to get significant regression. In the case of significant regression, both for standard (hCG) and unknown preparation (cPS), tests for curvature and parallelism were carried out. For stripped egg weight and the stripping of ovary responses, the tests of curvature and parallelism were not significant, so the potency ratio was calculated for both variables and found to be 1 mg cPS equivalent to 1445 (1272–1613) IU hCG and 1 mg cPS equivalent to 1402 (1301–1496) IU hCG, respectively.

The Role of Calcium in Prolactin Release from the Pituitary of a Teleost Fishin Vitro*

The release of PRL from the pituitary of a teleost fish, the tilapia (Oreochromis mossambicus), has been previously shown to be dependent on calcium. However, the source(s) and specific action(s) of calcium in the secretory process have not been identified. Also undefined are the mechanisms by which regulators of PRL cell function may alter calcium distribution. In the present investigation, the elevation of medium K+ concentration during static incubations to a depolarizing concentration (56 mM) produced no change in cumulative PRL release over control levels during the 18-20 h of incubation. During perifusion incubation, exposure to high K+ concentrations briefly stimulated (less than or equal to 10 min) and then depressed PRL release. In contrast, reduced medium osmotic pressure elicited a rapid elevation in PRL release that was sustained for 2 h or more. D600, a calcium entry blocker, at 10(-5) M diminished the K+-induced pulses of PRL release. The same concentration, however, did not alter the release of PRL evoked by reduced osmotic pressure. In contrast, CoCl2, which blocks a range of calcium-mediated processes in addition to calcium influx, suppressed PRL release during perifusion and static incubations in hyposmotic medium. These findings suggest that while PRL secretion from the tilapia pituitary is calcium dependent, calcium entry through voltage-regulated plasmalemma channels may not be a prerequisite to the actions of reduced osmotic pressure.

Ultrastructural characterization of neurosecretory fibres immunoreactive for vasotocin, isotocin, somatostatin, LHRH and CRF in the pituitary of a teleost fish, Poecilia latipinna.

Using the electron-microscopic immunogold method, vasotocin, isotocin, somatostatin (SRIF), gonadotrophin-releasing hormone (LHRH) and corticotrophin-releasing factor (CRF)-like immunoreactivities were localized in separate neurosecretory fibres in the pituitary of a teleost fish Poecilia latipinna. Antigenicities were preserved in sections of conventionally fixed tissue, except in the case of LHRH and CRF-like substances which were sensitive to osmium postfixation. Under the same fixation conditions, ultrastructural differences were observed between the 5 fibre types, and morphometric analysis of their granule sizes revealed significant differences in mean diameter except between vasotocin and isotocin fibres. Terminal-like regions of each type were identified on blood vessels, glial cells or other fibres in the neurohypophysis, on the basement lamina of the adenohypophysis, or directly on adenohypophysial endocrine cells. The fibres containing the two neurohypophysial hormones, originating from separate preoptic perikarya, were intermingled with, and may form endings near all the adenohypophysial cell types except those secreting prolactin. Although both types had similar mean granule diameters, the granules in the vasotocin fibres (mean 135 nm) were markedly less electron dense than those in the isotocin fibres (mean 140 nm). SRIF-immunoreactive fibres (mean 101 nm) appeared to form synapse-like endings on the somatotrophs, and a few thyrotrophs in the proximal pars distalis, and near the pars intermedia cells. An LHRH-positive type (mean 103 nm) contacted only the gonadotrophs of the proximal pars distalis. The rarer CRF-like fibres (mean 116 nm) appeared to project mainly towards the pars intermedia, but a few appeared to terminate rostrally near the adrenocorticotrophic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical changes in serotonin in the forebrain and pituitary of aging fish

The immunocytochemical distribution of immunoreactive serotonin (ir-5HT) was studied in the forebrain of male platyfish ranging in age from 5 to 30 months (mean life span, 30 months). In fish at all ages, ir-5HT is found in the forebrain in the wall of the third ventricle and its lateral and posterior recesses and in the pituitary in PAS-positive cells of the pars intermedia (PI). With increasing age, ir-5HT first appears in perikarya of the nucleus preopticus (18 months) and in gonadotropes of the caudal pars distalis. Between 8 and 12 months of age, some fish display pale 5HT immunoreactivity in pituitary gonadotropes while in all fish of 18 months and older, the gonadotropes as well as the PI cells show intense 5HT immunostaining. It is suggested that these modifications in serotonin localization may be related to reproductive senescence in platyfish and that they may be associated with other age-related changes in neurohormone and neurotransmitter immunoreactivity. These results are compared with similar age-related changes in the metabolism of neurotransmitters which have been found in mammals.

Isolation and partial characterization of a pair of prolactins released in vitro by the pituitary of a cichlid fish, Oreochromis mossambicus.

The pituitary of the cichlid fish tilapia secretes two prolactins (PRLs) of molecular masses 20 kDa and 24 kDa. The 20-kDa PRL has an isoelectric point in the range of those of mammalian PRLs (pI 6.7), but the 24-kDa PRL is unusually basic (pI 8.7). Partial sequence information indicates that the PRLs are homologous but distinct proteins, differing by five amino acids within the first 29 NH2-terminal residues. Homology in the known region is higher with chum salmon PRL than with known mammalian PRLs. Reversed-phase HPLC permits isolation of these two PRLs and a single tilapia growth hormone from culture medium or from the pituitary in a single step. HPLC and radio-HPLC analysis of [3H]leucine pulse-chase experiments reveal that each PRL is secreted in vitro at remarkably high rates (21 pmol per gland per hr) and that the two PRLs are released in approximately equimolar amounts, suggesting the coordinate regulation of the secretion. Both PRLs exert characteristic PRL activity in that they prevent the loss of Na+ from the plasma of hypophysectomized tilapia in fresh water.

Development and validation of a salmon prolactin radioimmunoassay

A highly specific radioimmunoassay (RIA) for the measurement of prolactin (PRL) in the plasma and pituitary of salmonid fishes was developed using a rabbit antiserum to chinook salmon ( Oncorhynchus tschawytscha) PRL. The PRLs purified from chinook salmon and chum salmon ( O. keta) pituitaries showed exactly the same competitive inhibition curves in the RIA, regardless of iodination of either hormone. The displacement curves for pituitary extracts and plasma from several salmonids, including chum, coho, and amago salmon, rainbow trout, and Japanese charr, were parallel to the salmon PRL standard, whereas those from the eel, goldfish, carp, and tilapia showed negligible cross-reactivity. Negligible cross-reactivity was also seen with plasma from hypophysectomized rainbow trout or coho salmon. None of the mammalian PRL or growth hormone (GH) preparations, bullfrog PRL, or presumptive chum salmon “gonadotropin” and eel “PRL” cross-reacted in the PRL RIA. Presumptive chum salmon GH showed less than 0.05% cross-reactivity. The RIA sensitivity was less than 0.1 ng of the salmon PRL standard per milliliter. The immunoreactive reactive plasma PRL levels in mature chum salmon were below 1 ng/ml in seawater. The plasma PRL in females increased to about 8 ng/ml 1 day after transfer to fresh water, and high levels (2–4 ng/ml) were maintained during 3–7 days after the transfer. In contrast, when males were transferred to fresh water, an increase in plasma PRL was seen only 1 day after the transfer. A significant decrease in plasma osmolality was observed in both males and females after transfer to fresh water. No change was observed either in plasma PRL or osmolality, when fish were transferred from seawater to seawater.

Ultrastructural autoradiographic localization of serotonin in the pituitary of a teleost fish, Poecilia latipinna

Light- and electron-microscopic autoradiographic studies of pituitaries of the molly Poecilia latipinna, after their incubation with tritiated serotonin, revealed the presence of labelled cells in the proximal pars distalis, together with cell processes or nerve fibres throughout all regions of the gland except the prolactin cell zone. The serotonincon-centrating cells and most of the fibres contained small dense-cored vesicles, but some labelled fibres contained larger granules similar in ultrastructure to those of vasotocinergic fibres.

Application of radioreceptor assay to the purification of silver carp gonadotropin.

Radioreceptor assay was successfully used to detect glycoprotein gonadotropin of silver carp Hypophthalmichthys molitrix with high sensitivity and precision. Purification was carried out by ethanol extraction, ion exchange chromatography, chromatofocusing and gel filtration. The gonadotropic potency was measured by bitterling ovipositor bioassay and radioreceptor assays which were used for the discrimination of bullfrog FSH and LH. Gonadotropic activity was detected only in acidic fractions. Throughout the purification processes, all the three activities were always detected in the same fractions at about the same ratio and there was no separation of the gonadotropin activities among fractions. Thus, even though we employed the radioreceptor assay methods specific to each of frog FSH and LH, we failed to show two types of gonadotropins such as FSH and LH in the silver carp pituitary gland. These results favour an idea that fish pituitary contains a single type of gonadotropin.The molecular weight of the silver carp gonadotropin was estimated to be 38, 000 by gel filtra-tion. Isoelectrofocusing of the final preparation showed several components with different pIs (4.7, 4.8 and 5.1).