Benzodiazepine receptors in fish brain: [3H]-flunitrazepam binding and modulatory effects of GABA in rainbow trout.

We have identified and partially characterised benzodiazepine binding sites in whole brain membranes of male rainbow trout. In terms of Bmax and KD values trout brain receptors are remarkably similar to those in rat and human brain. The Hill coefficient was 0.98, indicating a single binding site. GABA (10(-4) M) was able to significantly elevate binding of [3H]-FNZ through a change in KD rather than Bmax. This effect was prevented by the GABA receptor antagonist bicuculline methiodide.

Manipulation of the hypothalamo-pituitary gonadal axis in the juvenile rainbow trout: influence of pituitary transplantation and LHRH analogue treatment.

The effect of implanting an extra pituitary containing large amounts of gonadotropic hormone (GtH), combined or not with a luteinizing hormone releasing-hormone analogue (LHRHa) treatment, on GtH levels and gonadal development was investigated in juvenile host fish. The extrapituitaries were collected from mature spermiating fish or from immature fish treated with testosterone. In recipient males and females circulating plasma GtH levels increased following transplantation of both types of pituitaries. Elevated GtH levels presumably triggered steroid synthesis by the immature male gonad since pituitary GtH content was observed to accumulate in recipient males and not in females. However, the potency of the two kinds of pituitaries seemed different since spermatogenesis was stimulated only in some recipient males bearing a mature adult pituitary. This divergence could be due to a different sensitivity to endogenous gonadotropin-releasing hormone (GnRH). Only mature extrapituitaries might be highly sensitive to GnRH, as suggested by results obtained in juvenile host fish after LHRHa treatment. At the end of the 6-week experimental period, this LHRHa treatment stimulated spermatogenesis and induced a significant increase in pituitary GtH content only in juvenile hosts transplanted with a mature pituitary. Such a result was not observed in juvenile hosts submitted to a LHRHa treatment combined or not with the transplantation of juvenile testosterone-treated pituitary. However, previous works have shown that pituitaries collected from immature testosterone-treated fish are sensitive to GnRH. In the present experiment, the amount of GnRH-induced GtH release might have been too low to initiate spermatogenesis during the 6-week experimental period.

Influence of gonadotropic hormone-releasing hormone analog (GnRH-A) on plasma sex steroid profiles and milt production in male winter flounder, Pseudopleuronectes americanus (Walbaum).

The effects of gonadotropic hormone-releasing hormone analog (GnRH-A) treatment on the onset and duration of increases in plasma sex steroids and milt production (milt volume and number of spermatozoa) were investigated in prespawning male winter flounder. After treatment of maturing males during the winter with a single injection of either 20 or 200 µg/kg [D-Ala(6), Pro(9)-NHEt]LHRH (GnRH-A), plasma levels of testosterone and 11-ketotestosterone were increased within 12h and the steroid hormone levels remained elevated for long periods lasting several days. The androgenic steroid response of males was delayed after the administration of a lower dose of GnRH-A (2 µg/kg). Although a single GnRH-A injection in December or January advanced the onset of spermiation in some males, only small amounts (<50 µl) of milt could be collected. By March, all males were in spermiating condition following GnRH-A treatment; however, significant increases in sperm production, particularly increases in milt volume, occurred in fish twice treated with GnRH-A.

Influence of testosterone and/or luteinizing hormone releasing hormone analogue on precocious sexual development in the juvenile rainbow trout.

The influence of testosterone, luteinizing hormone releasing hormone (LHRH) agonist and combinations of these hormones on gonadotropic hormone (GtH) levels in the sexually immature trout was investigated. Both the steroid and releasing hormone preparations, testosterone in Silastic capsules and cholesterol-pelleted LHRH-A, were formulated for sustained release and long-term biological action following a single hormone implantation. Marked increases in pituitary GtH followed testosterone and/or testosterone and LHRH analogue treatment combined, but the low pituitary GtH level in controls remained unchanged after LHRH analogue administration alone. Plasma GtH titers increased with time after testosterone treatment, indicating a positive steroid feedback effect by androgen on GtH in the juvenile rainbow trout. When combined with testosterone treatment, LHRH analogue augmented plasma GtH levels compared to fish receiving testosterone treatment alone. In males the elevated plasma GtH levels were associated with testes stimulation and onset of spermatogenesis; in females, however, no significant stimulation of the ovaries was observed. It can be concluded from these studies that the testosterone stimulus is sufficient to induce onset of sexual development in immature males but not females. Whereas LHRH analogue releases GtH from the testosterone-primed trout pituitary, LHRH treatment alone under these conditions fails to stimulate the juvenile trout reproductive system.

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 varieties 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 of fish, 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,Pro9-NHEt]LHRH, [D-Nal(2)6-AzaGly10]LHRH, [D-Ala6,Pro9-NHEt]-LHRH, and the fish LHRH analogs, [D-Arg6,Trp7,Leu8,Pro9-NHEt]LHRH, [D-hArg(Et2(6),Trp7,Leu8,Pro9-NHEt]LHRH.

Monoaminergic substances in the teleost brain: Catecholamine levels in male and female winter flounder,Pseudopleuronectes americanus Walbaum, associated with gonadal recrudescence.

High performance liquid chromatography with electrochemical detection (HPLC-EC) was used to quantitate catecholamine (CA) levels in the winter flounder brain following perchloric acid extraction/alumina purification of CNS tissues. Greater concentrations of norepinephrine (NE) and dopamine (DA) were present in the hypothalamus compared with the CA levels in whole brain. A seasonal study of CA brain levels in reproductively active male and female flounder demonstrated that monoamine levels reach their maxima in October in association with the rapid increases in gonadosomatic index. When perchloric acid extracts of the teleost and rat hypothalamus were submitted to direct HPLC-EC analysis, without alumina purification of CA neurotransmitters, similar hypothalamic profiles were obtained indicating the presence of identifiable biogenic amine neurotransmitters substances including NE, DA and serotonin (5-HT).

Effects of dopamine and apomorphine on gonadotropin release from the transplanted pars distalis in goldfish.

Male goldfish bearing pars distalis transplants from other male goldfish have increased serum gonadotropin (GtH) levels, due to the high spontaneous release rate of GtH by the transplants. Intraperitoneal injection of dopamine or its agonist, apomorphine, each reduced the elevated serum GtH levels caused by the release from the transplanted pars distalis. These results suggest that dopamine has GtH-release-inhibitory activity and acts directly on gonadotrophs to inhibit spontaneous secretion of GtH.

A stereotaxic atlas and implantation technique for nuclei of the diencephalon of Atlantic salmon (Salmo salar) parr.

A stereotaxic apparatus and technique for its implantation in diencephalic nuclei of Atlantic salmon parr of 20 to 30 g body weight is described. An atlas of nuclei in the diencephalon is also presented.

Expression of histone and tubulin genes during spermatogenesis. Evidence of post-meiotic transcription.

The synchrony of spermatogenesis in the winter flounder has enabled us to examine the population of mRNAs expressed in each testis cell type, from spermatogonia to spermatids. Two of the most abundant sets of mRNAs in this tissue were those coding for histones and tubulins. The levels of histone mRNAs rose sharply at the onset of spermatogenesis, declined rapidly after the 1 degree spermatocyte stage, and were barely detectable in early spermatids. Histone genes were expressed again briefly in mid-spermatids, along with a spermatid-specific H3 mRNA-like transcript which was more than twice the length (1 100 nucleotides) of the H3 mRNA. Whereas the first and major round of histone mRNA synthesis appeared to be coupled to DNA replication, the second round of synthesis occurred after meiosis and coincided with the major reorganization of chromatin structure that takes place during the mid-spermatid stage of spermatogenesis. Levels of alpha- and beta-tubulin mRNAs increased 25-fold around the time of transition between spermatocytes and spermatids when sperm tail synthesis is initiated. These mRNAs appear to be utilized right away rather than stored, since the percentage of tubulin mRNA in the polysome fraction also increased at that juncture.

Ultrastructure of the spermatozoa and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine fish.

Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid-piece (one to two times the sperm head length) containing numerous well-developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 +/- 2) of microtubules. This long mid-piece and the biflagellate nature of the sperm appear to be associated with the long life-span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertility in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water.

Gonadotropin release from the pars distalis of goldfish, Carassius auratus, transplanted beside the brain or into the brain ventricles: additional evidence for gonadotropin-release-inhibitory factor.

The pars distalis of the pituitary was transplanted from one goldfish to another, of matched gonadal condition and body size, either beside the brain ("juxta" location) or into the brain ventricle in the preoptic region or under the optic tectum. Recipients of a pars distalis had significantly higher serum gonadotropin (GtH) levels than sham-operated controls without transplants; following removal of the transplants, serum GtH levels decreased to levels found in controls within 24 hr. This demonstrates that the pars distalis releases GtH spontaneously when removed from its normal connections with the hypothalamus providing evidence for tonic inhibition of GtH release by a GtH-release-inhibitory factor (GRIF). The juxta-transplanted pars distalis of sexually recrudescing and mature (= completed recrudescence) females released more GtH than the pars distalis of sexually regressed females. The juxta-transplanted pars distalis of sexually mature males released more GtH than the pars distalis of males in early stages of testicular recrudescence. These results demonstrate a seasonal variation in the ability to release GtH independent of GRIF. This may be due to the greater content of GtH in the pituitary, and a greater ability to synthesize GtH by the pituitary in sexually mature and recrudescing goldfish compared to sexually regressed fish. In sexually recrudescing and mature males and females, transplantation of the pars distalis into the brain ventricle in either the preoptic region or ventral to the optic tectum resulted in significantly lower serum GtH levels in recipients than transplantation beside the brain. This demonstrates the presence of some factor in the brain that inhibits GtH release, supporting the idea of GRIF. In sexually regressed female goldfish, GtH release from the transplanted pars distalis was not influenced by location in the brain ventricles versus beside the brain. This suggests a seasonal variation in GRIF activity in the brain, with reduced activity in sexually regressed females coincident with less ability to release GtH by the pars distalis.

A study of LH-RH receptors in the pituitary gland of the winter flounder (Pseudopleuronectes americanus Walbaum).

A study using an iodinated [D-Ser(tBu)6,Pro9-NHEt]LH-RH (Buserelin), demonstrated the presence of a single class of high-affinity (KD = 2.90 nM), high-capacity LH-RH binding sites in pituitaries obtained from sexually mature male and female winter flounder. Displacement curves for unlabeled Buserelin and other preparations of mammalian and fish LH-RH, but a lack of competition for structurally unrelated peptide hormones, indicated the hormone specific nature of the fish pituitary LH-RH receptor preparation. Compared with native mammalian LH-RH and salmon LH-RH, Buserelin and an analog of salmon LH-RH, [D-Arg6,Trp7,Leu8,Pro9-NHEt]LH-RH, had significantly higher binding affinities for the flounder pituitary receptor correlating with results of previous studies demonstrating the superagonist biological activity of LH-RH analogs in trout and goldfish.

Use of pituitary cells in primary culture to study the regulation of gonadotropin hormone (GtH) secretion in rainbow trout: setting up and validating the system as assessed by its responsiveness to mammalian and salmon gonadotropin releasing hormone.

To study the regulation of gonadotropin secretion in rainbow trout in vitro, a method for preparing primary cultures of dispersed pituitary cells is described. Cells were dispersed by collagenase 0.1% in Hank's saline solution for 20 hr at 12 degrees and a high yield of viable cells was obtained. Attempts to improve cell functioning were made by varying culture conditions (density of cells initially plated, age of the culture). Cell functioning was assessed by their ability to respond to increasing doses of mammalian and salmon GnRH. Pituitaries were collected from spermiating males whose pituitaries are known to be sensitive to mammalian GnRH in vivo. Using 96-well plates, optimal conditions for good biological activity, are initial plating with 6.2 X 10(4) cells, incubation with GnRH for 24 hr on the third day after plating. In these conditions mammalian analog and salmon GnRH induced an increase in GtH release for doses ranging from 10(-9) to 10(-6) M. The GtH released during the GnRH incubation period does not decrease the sensitivity of the system since addition of 20 ng of GtH at the beginning of incubation does not modify the response profile.

17 alpha,20 beta-dihydroxy-4-pregnen-3-one: plasma levels during sexual maturation and in vitro production by the testes of amago salmon (Oncorhynchus rhodurus) and rainbow trout (Salmo gairdneri).

The present study investigated seasonal changes in plasma 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) levels in precociously mature and fully grown mature male amago salmon (Oncorhynchus rhodurus) and in vitro 17 alpha,20 beta-diOHprog production by the testes of amago salmon and rainbow trout (Salmo gairdneri) in response to chum salmon gonadotropin. Plasma 17 alpha,20 beta-diOHprog levels were very low from June to September, and rapidly increased in October at the beginning of the spawning season in both precociously mature and fully grown mature male amago salmon. High levels of 17 alpha,20 beta-diOHprog were maintained during the period of active spermiation in precociously mature males, and quickly declined in mid-November. Incubation of testicular fragments from spermiating rainbow trout with chum salmon gonadotropin or 17 alpha-hydroxyprogesterone resulted in a highly significant increase in 17 alpha,20 beta-diOHprog levels in the incubation medium. The chum salmon gonadotropin was also effective in stimulating 17 alpha,20 beta-diOHprog production by testicular fragments obtained from amago salmon after the breeding season. These findings are discussed in relation to the possible involvement of 17 alpha,20 beta-diOHprog in spermiation of salmonids.

Plasma 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one levels during sexual maturation of amago salmon (Oncorhynchus rhodurus): correlation with plasma gonadotropin and in vitro production by ovarian follicles.

Plasma gonadotropin (GtH) and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-diOH-prog) levels were assessed by radioimmunoassay during the sexual maturation of female amago salmon (Oncorhynchus rhodurus). Both GtH and 17 alpha, 20 beta-diOHprog levels were low in vitellogenic females (June to September) and in those with full-grown immature oocytes and were elevated in mature and ovulated females to 40 ng/ml for GtH and 50-70 ng/ml for 17 alpha, 20 beta-diOHprog. In vitro production of 17 alpha, 20 beta-diOHprog by ovarian follicles and its stimulation by partially purified chinook salmon gonadotropin (SG-G100) was examined monthly using 18 hr incubations. In June and July, levels were too low to detect (less than 30 pg/ml) in media from all treatment groups. In August and September, SG-G100 at a concentration of 1 microgram/ml stimulated low levels of production (0.2-0.3 ng/ml). Full-grown, immature follicles in October showed a dose-response production of 17 alpha, 20 beta-diOHprog, averaging over 10 ng/ml when incubated with 1 microgram/ml SG-G100. Postovulatory follicles (1-2 days after ovulation) produced large amounts of 17 alpha, 20 beta-diOHprog, averaging over 100 ng/ml with 1 microgram/ml SG-G100. These results are discussed in relation to previous work on amago salmon and other species and together indicate that 17 alpha, 20 beta-diOHprog is the major steroid responsible for oocyte maturation in amago salmon, produced as the follicular mediator of gonadotropin.

Molecular cloning and sequencing of salmon gonadotropin beta subunit.

Gonadotropin (GTH) was purified from the pituitaries of the Pacific chinook salmon using a combination of stepwise ethanol precipitation and concanavalin-A affinity chromatography. The alpha and beta subunits were dissociated and fractionated by C-18 reverse-phase high-performance liquid chromatography with a 0.1% trifluoroacetic acid/acetonitrile gradient. An enriched cDNA library was screened for the beta-GTH gene(s) using two synthetic oligonucleotides based on partial protein data. A positive, full-size clone (E3) was identified and sequenced. It contains 657 base pairs and codes for a 142-amino-acid precursor protein. The mature salmon beta-GTH (119 amino acids) is structurally homologous to human luteinizing hormone and chorionic gonadotropin. The effect of testosterone implantation on pituitary GTH and beta-GTH mRNA was examined with radioimmunoassay and Northern blot analysis. There was a corresponding increase in both the pituitary GTH and mRNA levels.

Evidence of GnRH receptors in cultured pituitary cells of the winter flounder (Pseudopleuronectes americanus W.).

For continued studies of GnRH receptor regulation in the winter flounder, we have developed an in vitro system consisting of cultured pituitary cells dissociated by collagenase. Using immunocytochemical staining methods for gonadotropin, growth hormone, and prolactin, these cell types were represented at the levels of 25, 20, and 19.5% of total pituitary cell population, respectively. Receptors for GnRH were characterized in intact monolayered attached pituitary cells, maintained in RPMI culture medium. The cell GnRH receptor characteristics were compared with those previously described using pituitary homogenates. The cells were capable of binding GnRH in a similar manner on Day 2 or Day 3 of culture, indicating the integrity of GnRH receptors. The specificity of binding was demonstrated since only high doses of cold GnRHa competed with 125I-GnRHa uptake, different peptides being without effect. The specific binding is saturable and the data suggest the presence of a single class of high-affinity (apparent Ka = 1.50 x 10(9) M-1), high-capacity sites (binding capacity = 25.03 fmol/2.5 x 10(5) cells or 242.23 x 10(3) sites/gonadotroph) which is in accordance with the characteristics of GnRH receptors present in homogenates of pooled male and female pituitary glands. All these observations suggest that such an in vitro pituitary cell system would be appropriate for studying GnRH receptor characteristics under different physiological conditions.