Expression of pejerrey gonadotropin-releasing hormone in three orders of fish.

Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.

Vitellogenin-induced pathology in male summer flounder (Paralichthys dentatus).

Male summer flounder (Paralichthys dentatus) were given two injections (initially and 2 weeks later) of 17beta-estradiol (E2) totaling 0.2 (2 x 0.1), 2.0 (2 x 1.0) or 20.0 (2 x 10.0) mg E2/kg body weight. Blood and tissue samples were collected 4, 6 and 8 weeks after the initial injection in the (2 x 0.1) mg/kg treatment, 4, 6, 8, and 15 weeks after the first injection in the (2 x 1.0) mg/kg treatment and at 4 weeks only in the (2 x 10.0) mg/kg treatment. Five of the 12 fish injected twice with 10.0 mg/kg were moribund before the first sampling period. Circulating levels of vitellogenin (VTG) in the blood of all E2-injected fish from all treatments were comparable with those concentrations found in the blood of wild male carp (Cyprinus carpio) and walleye (Stezostedion vitreum) previously collected near a sewage treatment plant (0.1-10.0 mg VTG/ml plasma). Excessive hyalin material accumulated in the livers, kidneys and testes of the treated fish. A portion of that material was identified as VTG by immunohistochemistry. The accumulation of VTG, and possibly other estrogen-inducible proteins, resulted in hepatocyte hypertrophy, disruption of spermatogenesis, and obstruction or rupture of renal glomeruli.

Sexually dimorphic development and binding characteristics of NMDA receptors in the brain of the platyfish.

This study investigated age- and gender-specific variations in properties of the glutamate N-methyl-d-aspartate receptor (NMDAR) in a freshwater teleost, the platyfish (Xiphophorus maculatus). Prior localization of the immunoreactive (ir)-R1 subunit of the NMDAR protein (R1) in cells of the nucleus olfactoretinalis (NOR), a primary gonadotropin-releasing hormone (GnRH)-containing brain nucleus in the platyfish, suggests that NMDAR, as in mammals, is involved in modulation of the platyfish brain-pituitary-gonad (BPG) axis. The current study shows that the number of cells in the NOR displaying ir-R1 is significantly increased in pubescent and mature female platyfish when compared to immature and senescent animals. In males, there is no significant change in ir-R1 expression in the NOR at any time in their lifespan. The affinity of the noncompetitive antagonist ((3)H)MK-801 for the NMDAR is significantly increased in pubescent females while maximum binding of ((3)H)MK-801 to the receptor reaches a significant maximum in mature females. In males, both MK-801 affinity and maximum binding remain unchanged throughout development. This is the first report of gender differences in the association of NMDA receptors with neuroendocrine brain areas during development. It is also the first report to suggest NMDA receptor involvement in the development of the BPG axis in a nonmammalian vertebrate.

Novel laboratory approaches to multi-purpose aquatic bioregenerative closed-loop food production systems.

Based on the construction principle of the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) two novel combined animal-plant production systems were developed in laboratory scale the first of which is dedicated to mid-term operation in closed state up to two years. In principle both consist of the "classic" C.E.B.A.S. subcomponents: animal tank (Zoological Component), plant cultivators (Botanical Component), ammonia converting bacteria filter (Microbial Component) and data acquisition/control unit (Electronical Component). The innovative approach in the first system is the utilization of minimally three aquatic plant cultivators for different species. In this one the animal tank has a volume of about 160 liters and is constructed as an "endless-way system" surrounding a central unit containing the heat exchanger and the bacteria filter with volumes of about 1.5 liters each. A suspension plant cultivator (1 liter) for the edible duckweed Wolffia arrhiza is externally connected. The second plant cultivator is a meandric microalgal bioreactor for filamentous green algae. The third plant growth facility is a chamber with about 2.5 liters volume for cultivation of the "traditional" C.E.B.A.S. plant species, the rootless buoyant Ceratophyllum demersum. Both latter units are illuminated with 9 W fluorescent lamps. In the current experiment the animal tank contains the live-bearing teleost fish Xiphophorus helleri and the small pulmonate water snail Biomphalaria glabrata because their physiological adaptation to the closed system conditions is well known from many previous C.E.B.A.S. experiments. The water temperature is maintained at 25 degrees C and the oxygen level is regulated between 4 and 7 mg/l by switching on and off the plant cultivator illuminations according to a suitable pattern thus utilizing solely the oxygen produced by photosynthesis. The animals and the microorganisms of filter and biofilm provide the plants with a sufficient amount of carbon dioxide. Oxygen concentration, pH value, temperature and redox potential are on-line recorded. Ion concentrations and numbers of living germs in the system water are determined twice monthly in the laboratory from samples taken from a special "sample removal module"; the sample volume is automatically replaced from an reservoir container. A rotatory pump produces a water flow of about 38 l/min. For a similar smaller test system with approx. 10 l volume developed from the C.E.B.A.S.-MINI-MODULE a novel indirect solar energy supply is tested which has a buffer capacity to maintain the system for 7 days in darkness under central European climate conditions also in winter. It contains only a single plant cultivator which is operated with Wollfia arrhiza. This lemnacean plant is able to produce large amounts of plant biomass in a short time by vegetative reproduction via daughter fronds. This easy-to-handle apparatus is dedicated to be operative more than 4 month. The experimental animals and microorganisms are the same as in the large system. The paper provides detailed information on the system construction principles and the biological, physical and chemical data of the initial phase of the test runs of both systems with the main focus on the large one.

Developmental changes in NMDA receptor expression in the platyfish brain.

We have examined the distribution of the N-methyl-D-aspartate (NMDA) receptor in the brain of a freshwater teleost using an antibody against the R1 subunit of the receptor (NMDAR1). The primary site of localization was the nucleus olfactoretinalis (NOR), a significant gonadotropin releasing hormone (GnRH)-containing brain nucleus. The number of cells expressing NMDAR1 in this nucleus was dependent upon developmental stage, with pubescent and mature animals displaying significantly more stained cells than immature and senescent animals. This is the first reported observation of age- and maturity-related NMDA receptor association with GnRH-containing brain areas.

Animal protein production modules in biological life support systems: novel combined aquaculture techniques based on the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.).

Based on the experiences made with the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) which was primarily developed for long-term and multi-generation experiments with aquatic animals and plants in a space station highly effective fresh water recycling modules were elaborated utilizing a combination of ammonia oxidizing bacteria filters and higher plants. These exhibit a high effectivity to eliminate phosphate and anorganic nitrogen compounds and are, in addition, able to contribute to the oxygen supply of the aquatic animals. The C.E.B.A.S. filter system is able to keep a closed artificial aquatic ecosystem containing teleost fishes and water snails biologically stable for several month and to eliminate waste products deriving from degraded dead fishes without a decrease of the oxygen concentration down to less than 3.5 mg/l at 25 degrees C. More advanced C.E.B.A.S. filter systems, the BIOCURE filters, were also developed for utilization in semiintensive and intensive aquaculture systems for fishes. In fact such combined animal-plant aquaculture systems represent highly effective productions sites for human food if proper plant and fish species are selected. The present papers elucidates ways to novel aquaculture systems in which herbivorous fishes are raised by feeding them with plant biomass produced in the BIOCURE filters and presents the scheme of a modification which utilizes a plant species suitable also for human nutrition. Special attention is paid to the benefits of closed aquaculture system modules which may be integrated into bioregenerative life support systems of a higher complexity for, e.g., lunar or planetary bases including some psychological aspects of the introduction of animal protein production into plant-based life support systems. Moreover, the basic reproductive biological problems of aquatic animal breeding under reduced gravity are explained leading to a disposition of essential research programs in this context.

A controlled aquatic ecological life support system (CAELSS) for combined production of fish and higher plant biomass suitable for integration into a lunar or planetary base.

Based on the construction principle of the already operative Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) the concept of an aquaculture system for combined production of animal and plant biomass was developed. It consists of a tank for intensive fish culture which is equipped with a feeding lock representing also a trap for biomass removal followed by a water recycling system. This is an optimized version of the original C.E.B.A.S. filters adapted to higher water pollutions. It operates in a fully biological mode and is able to convert the high ammonia ion concentrations excreted by the fish gills into nitrite ions. The second biomass production site is a higher plant cultivator with an internal fiber optics light distributor which may utilize of solar energy. The selected water plant is a tropical rootless duckweed of the genus Wolffia which possesses a high capacity in nitrate elimination and is terrestrially cultured as a vegetable for human nutrition in Southeast Asia. It is produced in an improved suspension culture which allows the removal of excess biomass by tangential centrifugation. The plant cultivator is able to supply the whole system with oxygen for respiration and eliminates vice versa the carbon dioxide exhaled by the fish via photosynthesis. A gas exchanger may be used for emergency purposes or to deliver excess oxygen into the environment and may be implemented into the air regeneration system of a closed environment of higher order. The plant biomass is fed into a biomass processor which delivers condensed fresh and dried biomass as pellets. The recovered water is fed back into the aquaculture loop. The fresh plants can be used for human nutrition immediately or can be stored after sterilization in an adequate packing. The dried Wolffia pellets are collected and brought into the fish tank by an automated feeder. In parallel the water from the plant cultivator is driven back to the animal tank by a pump. The special feature of the system described is, however, the used fish species. It is the herbivorous teleost Ctenopharyngodon idellus (Chinese Grass Carp) which can be raised solely with plant biomass. In this case, moreover, it can be useful for the bioregeneration of plant biomass inedible for humans which can be used easily as additional food for the fishes thus resulting in an intensivation of animal protein production. The resupply of removed fish biomass has to be guaranteed by a separate hatchery.

Distribution of variant forms of immunoreactive gonadotropin-releasing hormone and beta-gonadotropins I and II in the platyfish, Xiphophorus maculatus, from birth to sexual maturity.

Immunoreactive (ir) lamprey (l), mammalian (m), chicken II (chII), and salmon (s) gonadotropin-releasing hormone (GnRH) and ir-coho salmon beta-gonadotropins I and II (GTH I and GTH II) have been localized in the brain and pituitary gland of Xiphophorus maculatus, the platyfish, at various stages of development from birth to sexual maturity. Ir-GTH I was found in the pituitary gland at all stages examined; ir-GTH II was found only in animals in the pubertal and mature stages. Ir-sGnRH was seen only in the pituitary gland of neonatal and immature animals. In pubertal fish, it was found in the nucleus olfactoretinalis (NOR) of the brain as well as in the pituitary gland. In older animals it was found in the nucleus preopticus periventricularis (NPP) and the nucleus lateralis tuberis (NLT), as well as in the NOR and the pituitary gland. Ir-mGnRH was seen in the pituitary of neonatal and immature fish. It was restricted to the NOR and pituitary gland of pubertal animals. In mature animals, it had a similar, but not identical, distribution as that of ir-sGnRH. Antisera to mGnRH and sGnRH appeared to stain the same cells in the brain and pituitary gland. Ir-lGnRH was found only in the pituitary gland of animals of all ages where it stained the same cells as ir-mGnRH and ir-sGnRH. Ir-chIIGnRH was absent in all immature stages. In animals that were sexually mature it was seen in tracts in the NPP, nucleus preopticus (NPO), NLT, and pituitary gland. Ir-chIIGnRH seemed to colocalize with ir-sGnRH in the pituitary but there was no apparent colocalization with any other form of ir-GnRH in the brain. These results demonstrate that variant forms of GnRH and GTH are present at defined stages of development in specific regions of the brain and pituitary gland and suggest that different forms of GnRH and GTH regulate different aspects of reproductive system development and physiology.

The distribution of immunoreactive FMRF-amide, neurotensin, and galanin in the brain and pituitary gland of three species of Xiphophorus from birth to sexual maturity.

This report seeks to extend the existing information on the relationship of regulatory neuropeptides to neuroendocrine and pituitary function through a longitudinal study of the distribution of FMRF-amide, galanin (GAL), and neurotensin (NT) in the brain and pituitary gland of three species of Xiphophorus from birth to sexual maturity. In the pituitary gland, immunoreactive (ir)-NT and -GAL were localized in the three regions of the adenohypophysis; ir-FMRF-amide was found in the neurohypophsis and in cells of the rostral pars distalis, but the immune reaction to this antisera in the pituitary was of very low intensity. Ir-GAL was found to colocalize with growth hormone, prolactin, and somatolactin in pituitary cells. In the brain, ir-GAL was found in the posterior nucleus lateralis tuberis (NLT), nucleus preopticus (NPO), and in the nucleus preopticus periventricularis (NPP). Ir-NT was localized in the anterior NLT. Ir-FMRF-amide was localized in the nucleus olfactoretinalis (NOR) where it was colocalized with gonadotropin releasing hormone, as well as in tracts that appeared to extend from the NOR, through the NPO and NPP, to the NLT. The NLT and the NOR have been previously implicated in the pituitary regulation of reproductive function. The above-cited localizations suggest that these peptides are involved in the neuroendocrine regulation of growth and reproduction in this genus.

The distribution of neuropeptide Y and dynorphin immunoreactivity in the brain and pituitary gland of the platyfish, Xiphophorus maculatus, from birth to sexual maturity.

Immunoreactive neuropeptide Y and dynorphin have been localized in the brain and pituitary gland of the platyfish, Xiphophorus maculatus, at different ages and stages of development from birth to sexual maturity. Immunoreactive neuropeptide Y was found in perikarya and tracts of the nucleus olfactoretinalis, telencephalon, ventral tegmentum and in the neurohypophysis and in the three regions of the adenohypophysis. Immunoreactive dynorphin was found in nerve tracts in the olfactory bulb and in cells of the pars intermedia and the rostral pars distalis of the pituitary gland.

Hypophysiotropic neurons in the hypothalamus of the catfish Clarias batrachus: a cobaltous lysine and HRP study.

Seven hypothalamic nuclei and several isolated perikarya that send projections to the pituitary gland were identified following administration of cobaltous lysine or horseradish peroxidase (HRP) to severed hypophysial stalks of previously hypophysectomized catfish, Clarias batrachus. Retrogradely labelled neurons were identified in the nucleus preopticus periventricularis, suprachiasmatic nucleus, paraventricular and supraoptic divisions of the magnocellular nucleus preopticus (NPO) and nucleus lateralis tuberis. A few neurons in the paraventricular subdivision of the NPO, however, remained unfilled; these may project to extrahypophysial sites. Three other nuclei contributing to the innervation of the pituitary gland include the paraventricular organ, nucleus recessus lateralis (NRL) and nucleus recessus posterioris (NRP), all of which contain cerebrospinal fluid-contacting aminergic neurons. These three neuronal aggregations were retrogradely labelled with cobaltous lysine but not with HRP. Isolated neurons displaying hypophysial connections were identified in the organon vasculosum laminae terminalis area, in the nucleus hypothalamicus ventromedialis, and in the vicinity of the NRL and NRP. Thus the present study reveals that hypothalamic projections to the pituitary gland of catfish have their origin in several peptidergic and aminergic nuclei and perikarya hitherto unreported.

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.

Genetic influences on reproductive system development and function: A review.

This paper presents a current view of the genomic and neuroendocrine interaction based on our studies of the reproductive system in the platyfish (Xiphophorus maculatus). It also presents observations from basic research and applied biologists on natural and artificially reared fishes and indicates that there is a direct genetic involvement in the control of spawning, growth rates, size and age at maturation and final body size, similar to that described in platyfish. The past, present and future association of aquaculture and basic science, especially DNA technology, is discussed and potential directions for future research are presented.

Sexually dimorphic age-related differences in the immunocytochemical distribution of somatostatin in the platyfish.

Somatostatin (SRIF) was localized by immunocytochemistry in the brains and pituitary glands of male and female platyfish (Xiphophorus maculatus) between the ages of 8 and 30 months (average life span is 30 months). In the brain, immunoreactive (ir-) SRIF is found in the perikarya of the ventrobasal hypothalamus (nucleus lateralis tuberis, nucleus anterior tuberis), dorsomedial hypothalamus, dorsal thalamus, ventral tegmentum and rhombencephalic basal plate; in the pituitary it is localized surrounding the ir-growth hormone containing cells of the caudal pars distalis, and in older fish it is also found within certain cells of the pars intermedia. We have demonstrated that there are consistent, sexually dimorphic, age-related changes in the intensity and/or distribution of ir-SRIF. The sexual dimorphism of the changes in SRIF immunoreactivity during the aging of platyfish may be related to the different patterns of growth observed in males and females.

Differences between early- and late-maturing genotypes of the platyfish (Xiphophorus maculatus) in the morphometry of their immunoreactive luteinizing hormone releasing hormone-containing cells: a developmental study.

Immunoreactive luteinizing hormone releasing hormone (ir-LHRH) containing perikarya, brain nuclei, and pituitary cells were studied during the postnatal development of male sibling platyfish (Xiphophorus maculatus) genetically determined to reach puberty at two different ages using immunocytochemical, cytological, and morphometric methods. Ir-LHRH-containing perikarya first appear in the nucleus olfactoretinalis (NOR) at 5 weeks of age in early maturers and at 11 weeks in late maturers at a maximum number which is similar in both genotypes and remains constant into adulthood. The dimensions of the NOR and its perikarya increase up to the initiation of sexual maturation (gonopodial stage 2) in both genotypes. In the nucleus preopticus periventricularis (NPP), ir-perikarya appear at stage 2 and are maximum in number at maturity (stage 6) when early maturers have 50% more than late maturers. Measurements for the NPP increase between 1 week and stage 6 in both genotypes. In the nucleus lateralis tuberis (NLT), ir-perikarya appear soon after stage 2 in early maturers but are never seen in late maturers. Late maturers also have fewer ir-LHRH containing pituitary cells than early maturers. In both genotypes, measurements for the NLT increase to stage 2 and then decrease to stage 6. During sexual development there are differences between early- and late-maturing genotypes in the morphometry of their LHRH-containing brain centers. The timing of sexual development creates significant differences in the cytological and cytometric characteristics of the three ir-LHRH-containing brain nuclei in fish of the same age but different genotype. Our results also show that for both genotypes there is a positive correlation between the total number of ir-LHRH brain neurons and the total number of ir-pituitary cells and both are lower for late maturers at every age and stage.

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.

The influence of androgen administration on the structure and function of the brain-pituitary-gonad axis of sexually immature platyfish, Xiphophorus maculatus.

This report demonstrates that the administration of testosterone (T) or 11-ketotestosterone (11-KT) to sexually immature (8 wks old) male platyfish (Xiphophorus maculatus) of early- and late-maturing genotypes affects the synthesis and/or release of luteinizing hormone-releasing hormone (LHRH), as assessed by immunocytochemical evaluation, increases the number and activity of pituitary gonadotropes, stimulates the production of sperm and, thus, advances the age of sexual maturation over that dictated by the genome. We also show that 11-KT and T affect different LHRH-containing centers in the brain and have differential effects on rate and degree of sexual maturation, regardless of whether the hormones are administered to early or late-maturing genotypes.