Steroidogenic activities of follicle-stimulating hormone in the ovary of Japanese eel, Anguilla japonica

To clarify the physiological functions of follicle-stimulating hormone (FSH) during oogenesis in Japanese eel, Anguilla japonica, the steroidogenic activities of recombinant Japanese eel FSH (rjeFSH) were assessed in the eel ovary. Female eel were injected with salmon pituitary homogenate to enhance the ovarian development, and the ovaries at different developmental stages were subjected to steroidogenic bioassay. These ovaries could be classified into three types according to oocyte growth and development of ovarian follicular cells. The type-A ovary possessed poorly developed follicular cells around pre- or early vitellogenic oocytes, and rjeFSH did not induce sex steroid secretion. Testosterone (T) secretion was stimulated by rjeFSH in the type-B ovary with developed theca cells and undeveloped granulosa cells around early to mid-vitellogenic oocytes, whereas estradiol-17beta (E2) secretion was not enhanced. The rjeFSH stimulated both T and E2 secretion in a dose-dependent manner from the type-C ovary with fully developed theca and granulosa cells around mid-vitellogenic oocytes. Salmon GTH fraction (sGTH) and a membrane permeable cAMP analogue, 8-bromo-cAMP (8-Br-cAMP) also enhanced T and E2 secretion from the type-C ovary. Human chorionic gonadotropin (hCG) similarly enhanced T secretion, but failed to stimulate E2 secretion from the type-C ovary, suggesting different effects on steroidogenic activities between eel FSH and hCG in eel ovary. There was a positive correlation between the oocyte diameter and E2 secretion from eel ovaries stimulated by rjeFSH. These results suggest that aromatase activity is accelerated by eel FSH in the granulosa cells, which develop following theca cell development in this species.     

Investigation into the duality of gonadotropic cells of Mediterranean yellowtail (Seriola dumerilii,Risso 1810): immunocytochemical and ultrastructural studies

Two antisera against the follicle-stimulating hormone-like gonadotropin (FSH) of Mediterranean (M.) yellowtail, anti-My FSHa and anti-My FSHb, were obtained. Anti-My FSHa serum specifically recognized FSH cells and did not react with any other pituitary cell type, while anti-My FSHb serum recognized the alpha-subunit of the pituitary glycoprotein hormones and immunostained FSH, luteinizing hormone-like gonadotropin (LH), and thyrotropin (TSH) cells. Anti-My FSHa serum, together with a previously obtained anti-My LHbeta serum, were used to further investigate the duality of gonadotropic cells in M. yellowtail by light and electron microscopic immunocytochemistry; three immunologically different gonadotropic cell populations expressing FSH, LH, or both hormones, were revealed. The three cell populations had the same regional distribution in the pituitary gland: the proximal pars distalis, including the thin ring surrounding the pars intermedia. However, while FSH cells were found isolated or forming small clusters, LH cells formed strands or compact groups, and were more numerous than FSH cells. FSH/LH cells were scarce. At the ultrastructural level, vesicular, granular, and intermediate FSH, LH, and FSH/LH cells were found; secretory granules and globules, on the one hand, or conspicuous dilated cisternae of rough endoplasmic reticulum (or both) predominated, respectively, in these cell types. The production of either FSH or LH, or both hormones, was not reflected in the ultrastructural features of gonadotropic cells. Thus, a single morphological cell type of varying ultrastructure depending on the functional stage seemed to encompass all gonadotropic cells in M. yellowtail. All forms of FSH, LH, and FSH/LH cells were found in involution.     

Separation of two distinct gonadotropins from the pituitary gland of the snapping turtle (Chelydra serpentina)

Two chemical fractionation schemes were used in conjunction with various non-mammalian bioassays to isolate gonadotropin from the pituitary gland of the snapping turtle (Chelydra serpentina). Ammonium sulfate fractionation, effective in separating mammalian FSH from LH, and gel filtration on Sephadex G-100 concentrated all the turtle gonadotropin into a single fraction. A second fractionation method employing ion exchange chromatography on Amberlite IRC-50 and DEAE-cellulose effected considerable purification of gonadotropin and also resulted in the distinct separation of two different kinds of gonadotropic activities from the turtle glands. The two kinds of gonadotropic activities were distinguished by tests that are relatively specific for mammalian FSH (an Anolis lizard assay) and LH (an anuran in vitro ovulation assay). Both of the turtle fractions were active in frog spermiation and the uptake of ³²P by cockerel tests which is consistent with the lack of specificity for mammalian FSH and LH shown by these assays. The turtle hormones had low potencies in mammalian assays. Growth hormone and thyrotropin were also purified during fractionation, but the latter activity was not effectively separated from the gonadotropins. These results support the existence of two separate gonadotropins having FSH- and LH-like biological and chemical properties in the turtle pituitary.     

Studies on the feedback regulation of gonadotropin concentrations in male rats. (III). The effects of testosterone and its metabolites on gonadotropin secretion from rat pituitary cells in culture

To determine whether dihydrotestosterone (DHT) or estradiol (E2) exerts negative or positive feedback effects on rat pituitary gland, Testosterone (T) metabolite (T, DHT, 5 alpha-androstane-3 alpha, 17 beta-diol:3 alpha-diol or E2) was added to the cultured pituitary cells. Anterior pituitary glands were obtained from 6-week-old male rats. Pituitary cells were prepared by trypsin digestion and incubated with various concentrations of steroid hormones for 72 h to determine the effects of steroid hormones on basal secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) after 48 h preculture without steroids. Then 10 nM luteinizing hormone-releasing hormone (LH-RH) with appropriate concentrations of these steroid hormones was added to the pituitary cells in culture and incubated for another 6h to determine the effects of steroid hormones on LH-RH induced gonadotropin release. After the incubation, pituitary cells were lysed with 0.1% Triton X100 to measure the intracellular gonadotropin content. The concentration of LH and FSH was determined by radioimmunoassay. T, DHT and 3 alpha-diol stimulated basal FSH but not basal LH secretion, and inhibited both the release of FSH and LH from cultured pituitary cells during incubations with LH-RH in a dose-dependent fashion. Intracellular content of both FSH and LH were increased, and total FSH and not LH was also increased by the addition of DHT in a dose-dependent manner. E2 did not exert any of such effects on pituitary cells in culture. These studies suggest that 5 alpha-reduced metabolites but not aromatized metabolite of T play an important role on feedback regulation of gonadotropin secretion at pituitary level. DHT directly acts on pituitary gland not only to stimulate the production of FSH but also to suppress FSH and LH secretion induced by LH-RH.     

Zebrafish as a model for vertebrate reproduction: characterization of the first functional zebrafish (Danio rerio) gonadotropin receptor

Vertebrate reproduction is tightly regulated by conserved glycoprotein hormones produced by the pituitary gland. Follicle-stimulating hormone (FSH) in tetrapods and gonadotropic hormone I (GTH-I) in fishes are orthologous glycoprotein hormones that control the timing of egg production and the number of eggs produced. Zebrafish, a well-established genetic model for developmental biology, also offers potential advantages for studies of reproductive toxicology, especially for modeling the impact of pollutants on fish reproductive processes. To facilitate these studies we have identified, expressed, and characterized the zebrafish GTH-I receptor. This receptor (zfGTHR-I)exhibits strong sequence similarity to the tetrapod FSH receptors and to GTHR-I from salmon and catfish. Human 293 cells transfected with zfGTHR-I exhibit increased cAMP levels after treatment with carp pituitary extracts or human FSH, but not when treated with a ligand to a related receptor (human chorionic gonadotropin). Northern blotting and RT-PCR analyses indicate that zfGTHR is expressed in ovaries from sexually mature fish, but not in immature fish. Several alternative splice variants of the receptor affecting putative exons 2-4 that encode dramatically shortened receptor fragments lacking the transmembrane domain as well as regions previously implicated in ligand binding were identified by RT-PCR. The zfGTHR-I sequence opens the way to study effects of genetic mutations or chemicals on ovarian zfGTHR-I expression and function in zebrafish.     

Structural and functional characterization of neuropeptide Y in a primitive teleost, the Japanese eel (Anguilla japonica)

In the present study, the first full-length cDNA encoding Neuropeptide Y (NPY) was cloned from the brain of Japanese eel (Anguilla japonica). The open reading frame of Japanese eel NPY gene is 294bp in length, encoding a precursor protein of 97 amino acids, which contains a 36-amino-acid mature peptide. Sequence analysis showed that the Japanese eel NPY peptide is similar to that of other species. Real-time PCR revealed that NPY in Japanese eel is mainly expressed in the brain, especially in the hypothalamus and the optic tectum thalamus. The effect of a negative energy balance on NPY gene expression was examined subsequently. The mRNA level of NPY in the hypothalamus and the optic tectum thalamus showed a pronounced increase after 4days of food deprivation. The biological activities of Japanese eel NPY were further investigated in vivo and in vitro. Intraperitoneal injection of the NPY peptide into Japanese eel could potently elevate the expression of the mammalian gonadotropin-releasing hormone (mGnRH) in hypothalamus and the follicle-stimulating hormone beta (FSHβ), the luteinizing hormone beta (LHβ) and growth hormone (GH) in pituitary. In static incubation studies, the stimulatory effects of NPY on mGnRH expression in hypothalamic fragments and on FSHβ, LHβ and GH expression in pituitary cells were also observed. However, in vivo and in vitro studies showed that NPY exhibits an inhibitory action on the expression of thyroid-stimulating hormone beta (TSHβ) in pituitary. The results indicate that NPY is involved in the regulation of multiple physiological processes in Japanese eel.     

First evidence for a direct inhibitory effect of kisspeptins on LH expression in the eel, Anguilla anguilla

The kisspeptin system has emerged as one of the main puberty gatekeepers among vertebrates. The European eel (Anguilla anguilla) is a remarkable model due to its phylogenetical position at the basis of teleosts, and its unique life cycle with a blockade of puberty before reproductive migration. We cloned the full-length coding sequence of a kisspeptin receptor (Kissr) in the eel. Comparison of Kissr sequences assigned the eel Kissr to a basal position in a clade including most of the known teleost Kissr, in agreement with the eel phylogenetical position. Eel Kissr tissue distribution was analyzed by quantitative real-time PCR. Eel Kissr was highly expressed in the brain, especially in the telencephalon and di-/mes-encephalon, while a very low or undetectable expression was observed in various peripheral organs. A high expression of Kissr was also found in the pituitary indicating a possible direct pituitary role of kisspeptin. Primary cultures of eel pituitary cells were performed to investigate the direct effects of kisspeptin on pituitary hormone expression. Human/lamprey kisspeptin exerted a time- and dose-dependent inhibitory effect on LHβ expression. All other tested kisspeptins had a similar inhibitory effect on LHβ expression. The inhibitory effect of kisspeptins was exerted specifically on LHβ as no change was induced on the expression of other glycoprotein hormone subunits (GPα, FSHβ and TSHβ) nor of growth hormone. These data provide the first evidence for the existence, in the European eel, of a kisspeptin system, which may play a direct inhibitory role on pituitary LHβ expression.     

Regulation of gonadal steroidogenesis in Fundulus heteroclitus by recombinant salmon growth hormone and purified salmon prolactin

The effects of recombinant salmon growth hormone (sGH) on plasma sex steroid levels and gonadal function were investigated in hypophysectomized Fundulus heteroclitus. Effects of sGH were compared to those of purified chum salmon prolactin (sPRL), Atlantic salmon gonadotropin (sGTH), and salmon pituitary extract (sPE). Treatment with sGH significantly increased plasma concentrations of testosterone in males and estradiol-17 beta in females; sPRL had similar effects on testosterone levels in males. Further, treatment with these hormones prevented the decline in gonadal weight observed after hypophysectomy in both males and females. In vivo treatment of male fish with sGH also augmented testosterone and 11-ketotestosterone production by testis tissue subsequently incubated in vitro. Direct action(s) on gonadal steroidogenesis was examined by incubating gonadal tissues from hypophysectomized fish in vitro with various hormones. sGH significantly stimulated the in vitro production of testosterone and 11-ketotestosterone by testis, and estradiol-17 beta by ovary. sPE and sGTH also stimulated gonadal steroidogenesis, whereas sPRL and bovine GH had no significant effect. By comparison, rainbow trout gonads also produced increased amounts of steroids when treated with sGH in vitro. The use of a cloned GH rules out contamination by other pituitary hormones. These results, therefore, demonstrate that recombinant salmon growth hormone possesses steroidogenic and gonadotropic activity. Purified sPRL also has steroidogenic and gonadotropic actions. However, the significance of these effects of teleost GH and PRL is not known.     

Estradiol has inverse effects on pituitary glycoprotein hormone alpha-subunit messenger ribonucleic acid in the immature European eel and the gonadectomized rat

In teleosts, the pituitary contains a single glycoprotein gonadotropic hormone (GTH) composed of two dissimilar alpha- and beta-subunits. The European eel, Anguilla anguilla L, is sexually immature at the silver stage due to a deficiency in GTH synthesis and secretion. In previous studies we (S.D., YA.F.) have demonstrated a strong stimulatory action of estradiol (E2) on eel pituitary GTH content. In contrast, we (R.C., M.J.) have shown that in the rat E2 negatively regulates gonadotropin subunit synthesis via changes in specific mRNA levels. The purpose of our present work was to check for such effects of E2 on the synthesis of GTH alpha- and beta-subunits in the European eel. Eel pituitary mRNA was translated in a cell-free system in the presence of [35S]Met + Cys. We demonstrate that one of the translated polypeptides, characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, cross-reacts with an antiserum to denatured bovine alpha-subunit. Its apparent mol wt (18.5K), which is slightly higher than that of the corresponding rat alpha-precursor, suggests that it represents the precursor of the alpha-subunit of eel glycoprotein hormones. The specificity of immunoprecipitation was confirmed by competition with ovine alpha (but not with ovine LH beta or bovine TSH beta). Quantitative evaluation of the putative eel alpha-subunit precursor showed that it represents 0.2% of the total protein translated by RNA from the normal silver eel. Chronic treatment of eels for 3 weeks with 17 beta-E2 increased by 8.0- to 8.5-fold the proportion of the putative alpha-subunit precursor among translation products. Due to the lack of cross-reactivity with the presumed GTH beta precursor, no radioactive material could be specifically detected in translation medium of eel pituitary mRNA using antisera to either denatured bovine LH beta or ovine FSH beta. Our data suggest that E2, depending on vertebrate group and probably on sexual status, may exert either positive or negative control on gonadotropin synthesis by opposite effects on the levels of specific mRNA.     

Conversion of human choriogonadotropin into a follitropin by protein engineering.

Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the alpha subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their beta subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG beta-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH beta and hFSH beta in receptor recognition and activation. Since the beta subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH beta subunits, coexpressed them with the human alpha subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH beta residues 33-52 for hCG beta residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG beta residues 108-145 or substitution of residues in the "determinant loop" located between hCG beta residues 93 and 100.     

Effects of ghrelin upon gonadotropin-releasing hormone and gonadotropin secretion in adult female rats: in vivo and in vitro studies

A reproductive facet of ghrelin, a stomach-derived orexigenic peptide involved in energy homeostasis, has been recently suggested, and predominantly inhibitory effects of ghrelin upon luteinizing hormone (LH) secretion have been demonstrated in rat models. Yet, the modulatory actions of ghrelin on the gonadotropic axis remain scarcely evaluated. We report herein a detailed analysis of the effects of ghrelin upon LH and follicle-stimulating hormone (FSH) secretion in the female rat, using a combination of in vivo and in vitro approaches. Intracerebroventricular administration of ghrelin (3 nmol/rat) evoked a significant inhibition of LH secretion in cyclic female rats throughout the estrous cycle (proestrus afternoon, estrus, metestrus), as well as in ovariectomized females. In good agreement, gonadotropin-releasing hormone (GnRH) secretion by hypothalamic fragments from ovariectomized females was significantly inhibited by ghrelin. In contrast, ghrelin dose-dependently stimulated basal LH and FSH secretion by pituitary tissue in vitro; a phenomenon that was proven dependent on the phase of estrous cycle, as it was neither detected at estrus nor observed after ovariectomy. Conversely, GnRH-stimulated LH secretion in vitro was persistently inhibited by ghrelin regardless of the stage of the cycle, whereas stimulated FSH secretion was only inhibited by ghrelin at estrus. In addition, cyclic fluctuations in mRNA levels of growth hormone secretagogue receptor (GHS-R)1a, i.e. the functional ghrelin receptor, were observed in the pituitary, with low values at estrus and metestrus. GHS-R1a mRNA levels, however, remained unchanged after ovariectomy. In summary, our data illustrate a complex mode of action of ghrelin upon the gonadotropic axis, with predominant inhibitory effects at central (hypothalamic) levels and upon GnRH-induced gonadotropin secretion, but direct stimulatory actions on basal LH and FSH secretion. Overall, our results further document the reproductive role of ghrelin, which might be relevant for the integrated control of energy balance and reproduction.     

Luteinizing hormone receptor knockout (LuRKO) mice and transgenic human chorionic gonadotropin (hCG)-overexpressing mice (hCG alphabeta+) have bone phenotypes

Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG), within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. Western blotting, immunolocalization, and RT-PCR supported the presence of osteoblast LH receptors. However, osteoblast cells failed to bind [(125)I]hCG and treatment with hCG failed to generate either cAMP or phosphorylated ERK 1/2. Bone mineral density (BMD) and bone histomorphometry were examined in the following models: 1) LH receptor null mutant (LuRKO) mice; 2) transgenic mice overexpressing hCG (hCG alphabeta+); and 3) ovariectomized (OVX) hCG alphabeta+ model. Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9- to 10-wk-old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation or an increase in bone resorption. By contrast, there were significant increases in BMD and histomorphometric indices for female, but not male, hCG alphabeta+ mice, indicating that chronic exposure to hCG results in bone formation or a decrease in bone resorption. However, OVX of the hCG alphabeta+ mice resulted in a significant reduction in BMD comparable to OVX WT controls. Although gonadotropin levels are tightly linked to sex steroid titers, it appears that their effects on the skeleton are indirect.     

The molecular regulation of Chinook salmon gonadotropin β-subunit gene transcription

Expression of the gonadotropin β-subunit genes is tightly regulated both cell-specifically and by the regulatory hormones to achieve the appropriate gonadotropic hormone levels required for reproductive development and function. Although the cDNA sequences of these genes are highly conserved across species, their promoter sequences are not and few functional studies have been carried out to understand the molecular mechanisms through which their expression is regulated. We and others have carried out several studies on the LHβ gene promoter of Chinook salmon (Oncorhynchus tschawytscha), and also isolated the FSHβ gene from the same species. We present here a review of these studies and also novel data pertaining to both genes, in an attempt to collate the current understanding of the molecular regulation of the gonadotropin β-subunit genes in these fish.     

The pituitary-gonadal axis in women with benign or malignant ovarian tumors.

The serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), estradiol, progesterone, androstenedione, testosterone (total and free) and dehydroepiandrosterone sulphate (DHEAS) were investigated prior to surgery in 24 postmenopausal women with benign and 28 postmenopausal women with malignant epithelial ovarian tumors. The serum concentrations of hormones were compared with those of 28 healthy, postmenopausal, age-matched controls. Significantly lower serum FSH levels were demonstrated in women with malignant tumors. No significant differences were found between the groups regarding the serum LH levels. The hCG levels were low in all groups. Regarding progesterone and estradiol levels, low postmenopausal steroid levels were found in all groups examined and no significant differences were demonstrated within the groups. No significant correlations between the levels of estradiol and FSH or progesterone and LH were demonstrated. To exclude a central depression of gonadotropin release mediated by the dopaminergic system we examined the thyroid stimulating hormone (TSH) and prolactin. No differences were found between the groups regarding TSH and prolactin levels. A possible relationship between other hormones/factors produced by the tumor and exerting a negative feedback, either centrally or directly, on the gonadotropin release remains to be investigated. A change in biological activity in the gonadotropins might explain the present findings.     

Hormonal regulation of gonadotropin receptor mRNA in rat ovary during follicular growth and luteinization.

To investigate the regulation of the follicle-stimulating hormone (FSH) and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor genes by gonadotropins, we examined the effect of pregnant mare's serum gonadotropin (PMSG) or PMSG-hCG on the expression of FSH and LH/hCG receptors in rat ovaries. After administration of PMSG, Northern blot analysis using the FSH receptor cDNA probe revealed that a major band of 2400 nucleotides was detected which reached the maximal level on day 3. On the other hand, the level of LH/hCG receptor mRNA, a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300 and 1200 nucleotides, increased progressively during 4 days. Treatment with hCG resulted in a decrease of FSH and LH/hCG receptor mRNA levels, and the level of FSH receptor mRNA was completely suppressed. Although the level of LH/hCG receptor mRNA was also suppressed from 3 h to an almost undetectable level at 24 h after hCG injection, it recovered to the control level by 48 h and exceeded this level several fold by 72 h. The reappearance of LH/hCG receptors following desensitization was preceded by an increase in mRNA levels. These studies demonstrate that hormonal regulation of gonadotropin receptor mRNAs on rat ovary reflects the changes in gonadotropin receptor levels.     

Steroid feedback on gonadotropin release and pituitary gonadotropin subunit mRNA in mice lacking a functional estrogen receptor alpha

Steroid hormones regulate levels of gonadotropin mRNA in the pituitary, and gonadotropic hormones in plasma. To determine whether estrogen receptor a (ERα) mediates steroid negative feedback, wild type (WT) and estrogen receptor a knockout (ERαKO) mice of both sexes were gonadectomized and implanted with a Silastic capsule containing either estradiol (E₂), dihydrotestosterone (DHT), testosterone, or a blank capsule. Ten days later, plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured. Pituitary mRNA levels of gonadotro-pin subunit (α, LHβ, FSHβ) and prolactin (PRL) were quantified. LH levels in gonad-intact ERαKO females were elevated, similar to values seen following gona-dectomy. By contrast, serum LH concentrations in gonad-intact ER a KO males were low and rose follow-ing gonadectomy, suggesting androgen feedback. Estradiol treatment significantly decreased plasma LH in WT animals, but not in ER a KOs. In fact, in female ER a KOs, our dose of E₂ increased plasma levels of LH as compared with untreated, ovariectomized ER a KOs. All the steroid treatments suppressed LH in WT ani-mals whereas only DHT consistently suppressed LH concentrations in ER a KO mice. The postgonadectomy rise in plasma FSH was prevented by steroid treatments in WT females, but not in any of the other groups. Gonadotropin subunit and PRL mRNA responses to E₂ treatment (both inhibitory and stimulatory) were absent in ER a KO mice, suggesting a critical role for ERα. Although E₂ can exert negative feedback effects on LH release in both males and females by actions at the ERα, the androgen receptor plays the primary physiological role in the male mouse.     

Development of a homologous radioimmunoassay for eel prolactin

A highly specific and homologous radioimmunoassay (RIA) for the measurement of prolactin (PRL) in the plasma and the pituitary of the eel was developed using a rabbit antiserum to eel PRL. PRL was purified from the pituitary of Japanese eel (Anguilla japonica). Pituitary extracts and plasma from the Japanese and European eels exhibited displacement curves parallel to the eel PRL standard. Plasma and pituitary extracts from chum salmon, rainbow trout, Japanese charr, tilapia, goldfish, and carp, as well as plasma from hypophysectomized eel, showed negligible cross-reactivity. PRL and growth hormone (GH) preparations from chum salmon, tilapia, and sheep, carp PRL, and eel GH did not cross-react with the antibody. The RIA sensitivity was less than 0.1 ng eel PRL per milliliter. Intra- and interassay coefficients of variations were 2.4 and 11.8%, respectively. The immunoreactive PRL levels in plasma and pituitary of the eel adapted to 50% seawater were significantly lower than those of the eel in fresh water. Plasma PRL levels increased maximally 2 days after transfer from seawater to fresh water, as would be expected from the well-established role of PRL in freshwater adaptation in several euryhaline teleosts.