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GnRH immunoreactivity appeared in the medial olfactory placode very early in the development of Cirrhinus mrigala. The immunoreactive elements were divisible into distinct migratory and non-migratory components. The migratory component appeared as a patch of intensely immunoreactive cells located close to the olfactory epithelium in day 6 post-fertilization larvae. Subsequently, these neurons migrate caudally along the ventromedial aspect of the developing forebrain and enroute give rise to GnRH immunoreactive neurons in the (1) nervus terminalis located in ventral and caudal part of the olfactory bulb (day 8), and (2) basal telencephalon (day 9). The non-migratory GnRH immunoreactive component appeared in the olfactory placode of day 1 post-fertilization larvae. It consisted of few olfactory receptor neuron (ORN)-like cells with distinct flask-shaped somata, dendrites that communicate with the periphery and a single axon on the basal side; GnRH immunoreactivity was seen throughout the neuron. Considerable increase in the number of immunoreactive ORNs was encountered in day 2 post-fertilization larvae. On day 3, the dendrites of ORNs sprout bunches of apical cilia, while on the basal side the axonal outgrowths can be traced to the olfactory bulb. GnRH immunoreactive fibers were distributed in the olfactory nerve layer in the periphery of the bulb and glomeruli-like innervation was clearly established in 5 days old larvae. The innervation to the olfactory bulb showed a considerable increase in GnRH immunoreactivity in 9 and 19 days old larvae. However, GnRH immunoreactivity in non-migratory as well as migratory components gradually diminished and disappeared altogether by the age of 68 days. Results of the present study suggest that GnRH may serve a neurotransmitter role in the ORNs during early stages of development in C. mrigala.

This report focuses on the impact of low concentrations of two purported endocrine-disrupting xenobiotic compounds, nonylphenol and methoxychlor, on two freshwater teleosts of the genus Xiphophorus , X. maculatus , commonly known as the platyfish, and X. helleri , commonly known as the swordtail. Nonylphenol exposure increased mortality rates, elicited stress behaviors, decreased body weight and significantly hampered gonadal development and reproductive function. Methoxychlor exposure elicited stress behaviors, decreased body weight and significantly retarded sexual development. This report demonstrates that, even at “acceptable” concentrations, these compounds pose a serious threat to our aquatic ecosystems.

The N-methyl-D-aspartate glutamate receptor (NMDAR) is found in hypothalamic nuclei involved in the regulation of reproduction in several species of mammals and fishes. NMDAR is believed to affect reproductive development and function by regulating gonadotropin releasing hormone (GnRH)-producing cells. These pathways are likely to be sexually dimorphic, as are several other neurotransmitter systems involved in reproductive function. In this report, male and female platyfish received intraperitoneal injections of 0, 5, 10, 20, 40 or 60 microg/g body wt. of the non-competitive NMDAR antagonist MK-801. Injections began at 6 weeks of age and continued thrice weekly until control animals reached puberty, as evidenced by anal fin maturation. The percent of pubescent animals was significantly affected by sex and treatment, with fewer MK-801-injected females in puberty than control females at each dose (P

The widely used herbicide atrazine (ATR) may have endocrine-associated adverse effects, including on behavior. In this study, 120 adult freshwater mussels, Elliptio complanata, were exposed to ATR at the environmentally-relevant concentrations of 1.5, 15, or 150 μg/L. Burrowing depth was evaluated hourly for 6 hr and at sacrifice animals were sexed by gonad smear. Female controls burrowed overall approximately 30% less than males, the first report of sexual dimorphism in this behavior. Atrazine at 15 μg/L feminized burrowing in both sexes in that exposed animals burrowed 20% less than their same sex controls. Males treated with 1.5 μg /L ATR displayed approximately 20-fold higher vitellogenin (VTG) levels than same sex controls. Higher concentrations of ATR were not associated with increasing effects. A scatterplot showed a weak binomial curve associating low burrowing with high VTG levels. Taken together, these data suggest a non-linear dose-response in behavioral and physiological feminization produced by ATR and support the need to reconsider the widespread use of this compound.

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. Copyright 1999 Academic Press.

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 elemination 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 guarantueed by a separate hatchery.

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.

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.

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.

The reproductive biology of the Indian major carp Cirrhinus mrigala is tightly synchronized with the seasonal changes in the environment. While the ovaries show growth from February through June, the fish spawn in July-August to coincide with the monsoon; thereafter the fish pass into the postspawning and resting phases. We investigated the pattern of GnRH immunoreactivity in the olfactory system at regular intervals extending over a period of 35 months. Although no signal was detected in the olfactory organ of fish collected from April through February following year, distinct GnRH-like immunoreactivity appeared in the fish collected in March. Intense immunoreactivity was noticed in several olfactory receptor neurons (ORNs) and their axonal fibers as they extend over the olfactory nerve, spread in the periphery of the olfactory bulb (OB), and terminate in the glomerular layer. Strong immunoreactivity was seen in some fascicles of the medial olfactory tracts extending from the OB to the telencephalon. Some neurons of the ganglion cells of nervus terminalis showed GnRH immunostaining during March; no immunoreactivity was detected at other times of the year. Plexus of GnRH immunoreactive fibers extending throughout the bulb represented a different component of the olfactory system; the fiber density showed a seasonal pattern that could be related to the status of gonadal maturity. While it was highest in the prespawning phase, significant reduction in the fiber density was noticed in the fish of spawning and the following regressive phases. Taken together the data suggest that the GnRH in the olfactory system of C. mrigala may play a major role in translation of the environmental cues and influence the downstream signals leading to the stimulation of the brain-pituitary-ovary axis.

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” surronding 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 facilitiy 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 snailBiomphalaria 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 °C and the oxygen level is regulated between 4 and 7 mg/1 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 micoorganisms of filter and bioflim 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 I 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 Wollfla 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 apparartus 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.

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.

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.

Immunoreactive (ir) lamprey (l), mammalian (m), chicken II (chII), and salmon (s) gonadotropin-releasing hormone (GnRH) and ir-coho salmon β-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.

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.

Sediments are evaluated for toxicity by measuring mortality in a single cohort of amphipods in either acute (10-d) or chronic (28-d) bioassays. This investigation differed from conventional bioassays in four ways: Sublethal effects (fecundity) were estimated; the testing period was 60+ d; two successive generations were examined; and Elasmopus laevis Smith, 1873, amphipods were employed. Four test sediments were created between 58 and 424 microg/g of lead using the 30-microg/g whole-sediment as the control. Bioaccumulated lead at 60 d varied as a linear function of lead concentration in the sediments. Fecundity, as estimated by offspring-per-chamber and/or percent reproductive success, was reduced as sediment lead concentrations increased and reproduction was delayed compared with the control. The reduction in offspring production per test chamber varied significantly as an inverse function of lead sediment concentration, best described by a curvilinear exponential equation. It was concluded that E. laevis exposed to 118 microg/g and higher could not maintain a population as large as that in the control. Although the current sediment quality guideline for lead stipulates that adverse biological effects likely will occur above 218 microg/g, this study revealed a statistically significant negative reproductive response at 118 microg/g lead, and suggests that the current regulatory guideline for lead, based on lethality, should be reconsidered.

Male platyfish, Xiphophorus maculatus, ranging in age from 12 to 54 months (normal life span is 30 months), were examined for age-related changes in the distribution of immunoreactive gonadotropin (ir-GTH) and luteinizing hormone releasing hormone (ir-LHRH) in their pituitary glands, and for changes in the histology of their testes. Males sacrificed at 13 and 16 months of age served as controls. Immunocytochemical methods demonstrate that even in the oldest fish, some almost twice the average platyfish life span, ir-GTH and ir-LHRH are localized in the same pituitary cell types in the caudal pars distalis and pars intermedia as in younger sexually mature fish. The testes of old fish continue to contain all stages of spermatogenesis; however, there are age-related increases in the amount of intertubular connective tissue and the relative number of spermatozeugmata, and distortions in the organization of acini. Our observations indicate that GTH and LHRH production and spermatogenesis continue, and thus a potential for reproductive capability persists, in male platyfish up to the time of their death resulting from old age.

For the first time immunoreactive luteinizing hormone-releasing hormone (LH-RH) is demonstrated in both the brain and pituitary gland of a teleost (Xiphophorus maculatus) using an immunoperoxidase procedure. It is specifically localized in the perikarya and their axons of the ventral telencephalon and nucleus lateralis tuberis and within and between the gonadotrops and within some cells of the pars intermedia. These immuno-reactions are extinguished when antiserum to LH-RH is preincubated with LH-RH antigen but not with neurohypophysial hormones.

The gonads of 3-day- to 7-month-old male and female platyfish (Xiphophorus maculatus) were examined for the presence of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and glucose-6-phosphate dehydrogenase (G6PD) by histochemical means. In 3-day-old males a positive response for both enzymes is localized in the Leydig cells. With subsequent testicular development, these cells increase in number and display greater activity at the periphery of the testis and around the efferent ducts. In 3-day-old females 3 beta-HSD and G6PD are localized in the stromal cells of the ovary. These cells increase in number and activity as the animals become sexually mature. Sertoli cells, efferent duct epithelium, and ovarian granulosa cells are negative at all stages of development examined. Our findings suggest that the gonads of neonatal fish possess the potential for steroidogenesis. The role played by sex-steroid hormones in the maturation of the brain-pituitary-gonad axis is discussed.

Sexually dimorphic, age-related changes occur in the distribution of pituitary gonadotrops in platyfish. In females, shortly after reaching sexual maturity, cells containing immunoreactive gonadotropin begin to appear among the prolactin cells of the rostral pars distalis and they become more numerous with advancing age. In males, if these cells do appear it is almost a year later and they are much fewer in number than in females. Evidence is presented which suggests that the gonadotropin containing cells in the rostral pars distalis are true gonadotrops rather than gonadotropin-secreting prolactin cells.

We have used the unlabeled antibody peroxidase-antiperoxidase (PAP) immunocytochemical method to demonstrate that antisera generated against rat neurophysin (anti-rNP) and arginine vasotocin (anti-AVT) react with material in the pars parvocellularis and pars magnocellularis of the nucleus preopticus (NPO) and in the neurohypophysis of sexually mature platyfish ( Xiphophorus maculatus ). With anti-rNP and anti-AVT, long beaded axons were seen to extend between the NPO and the neurohypophysis. Neurophysin and arginine vasotocin are immunologically distinct entities as shown by control absorption procedures. The two appear to be elaborated and stored in similar locations and follow comparable routes of transport between the NPO and the pituitary gland.

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.

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.

An immunoperoxidase method has been used to localize luteinizing hormone releasing hormone (LHRH) and gonadotropin (GTH) in neonatal (1-, 3-, 6-, and 9-day-old) platyfish ( Xiphophorus maculatus ). Immunoreactive (ir-) GTHβ and ir-LHRH are found in cells of the two extreme lateral regions of the caudal pars distalis (CPD), in a small number of the chromophobes comprising the single cell layer of the ventral CPD, and in many PAS+ cells of the pars intermedia (PI). Antiserum to the alpha subunit of GTH stains the same cells in the CPD but none in the PI. Neither ir-GTH nor ir-LHRH is found in the brain. Evidence that suggests that the PI is the source of a GTH which is not identical to the one in the CPD and which may function early in postnatal development is presented and discussed. The speculation that the cells of the lateral CPD give rise to the ventral GTH zone of the CPD which is characteristic of sexually mature fish is also presented.

In this communication we describe the immunocytochemical cross-reactivity between antisera to various human pituitary hormones and specific hormone producing cell types in the pituitary gland of sexually mature male platyfish (Xiphophorus maculatus). Antisera to human pituitary hormones cross-reacted either with cells known to produce corresponding hormones (or hormone subunits) in the platyfish (e.g., ACTH, prolactin, TSH beta, LH alpha, FSH alpha, TSH alpha) or with no pituitary cells at all (e.g., HL beta, FSH beta). The one exception was antiserum to human growth hormone which cross-reacted with MSH and ACTH producing cells. The platyfish pituitary is proposed as a test system for immunocytochemically screening antisera for purity and specificity in order to determine their applicability in particular studies.

Ten species of teleosts belonging to three families of Atheriniformes (Poeciliidae, Cyprinodontidae, Goodeidae) have been tested for their ability to survive in fresh water without a pituitary gland. The fish of the current report respond similarly to Atheriniformes previously investigated. Hypophysectomized fish enter into osmotic shock and generally die within three weeks when placed into fresh water but they will survive in dilute sea water (1.2% solution of commercial sea salts). Fish belonging to Poecilia survive for the shortest period. By comparison the mean survival time for Xiphophorus is longer and more variable. No correlation exists between the number of days hypophysectomized fish are held in one third sea water prior to testing and the length of time they survive in fresh water. This is also true for fish challenged a second time after recovering from osmotic shock. Presumably, osmotic difficulty commences upon exposure to fresh water but can be alleviated when fish are returned to dilute sea water. Five hypophysectomized fish survived in fresh water. Each of these exceptional animals had suffered brain damage during surgery. The significance of these observations is discussed as it is related to the natural habitat and salt tolerances of the fish studied.

Tetanic seizures and hypocalcemia were observed in hypophysectomized Fundulus diaphanus maintained in fresh water. Serum sodium and chloride levels did not decrease in proportion to the decreases in serum calcium levels. It is suggested that the pituitary influence on serum calcium might be distinct from its effect on serum sodium or chloride.

X. maculatus is polymorphic for sex-linked macromelanophore patterns. Fish of different river systems exhibit identical markings, but they have a different genetic basis. The macromelanophore factors give rise to a population or species-specific pattern only within their own gene pool. Crosses involving XsdYsr males of the Jamapa stock (Jp) gave rise to 3305 progeny — all but one either Sd or Sr. In the exceptional male Sd (spotted-dorsal) became linked to Sr (stripe-sided), SrSd. The dorsal fin of this fish and all of its SrSd descendants (5 generations) was almost solid black and heavy spotting was present in the caudal fin. There was no change in expression of Sr. Jp fish with Xsd Ysr, by comparison, possess from one to six well defined spots in the dorsal fin and a clear caudal fin. After introduction into the Hondo stock of X. maculatus Sd of Jp exhibits zero penetrance; the expression of Sd linked to Sr becomes greatly reduced. Thus the changes in gene expression go in the same direction. However, Sd and SrSd behave differently after introgression into X. couchianus. Sd is completely suppressed while Sd linked to Sr gives rise to melanotic and melanomatous dorsal and caudal fins. A similar alteration in gene expression towards a more extreme phenotype has also been observed among the offspring of a Jp Xsp Ysr male in which Sp and Sr became apparently linked. It is postulated that a modifier is closely linked to the macromelanophore factor that regulates its expression. The new phenotype is due to the separation of the two genetic elements. According to this view the phenotype that is expressed when a macromelanophore gene is introduced into a foreign genotype, is primarily determined by the modifier.

The teleosts Xiphophorus maculatus, Xiphophorus hellerii, Xiphophorus milleri , and Xiphophorus variatus xiphidium are native to freshwater streams. Following hypophysectomy their ability to survive in fresh water is abolished and they must be maintained in 1/3 sea water. When hypophysectomized fish are returned to fresh water they succumb within 16 days. The mean survival time of these fish in fresh water is: X. maculatus , 8.7 days; X. hellerii , 8.6 days; X. milleri , 5.5 days; X. v. xiphidium , 4.3 days. Fish in osmotic shock, when returned to dilute sea water, generally show gradual recovery. Injections of saline, neurohypophysial hormones (vasopressin and oxytocin), growth hormone, TSH, and ACTH failed to maintain hypophysectomized X. maculatus in fresh water. Only prolactin (ovine) was effective. Platyfish, injected with 10 μ g of prolactin per gram weight of fish, survived the duration of hormone administration. However, following the withdrawal of hormone, all completely hypophysectomized fish died within 16 days. Prolactin in quantities of less than 1.0 μ g is totally unsuccessful in promoting freshwater survival of fish without their pituitary glands. Compared with uninjected controls, the administration of saline or ineffective hormones considerably shortens the mean survival time of hypophysectomized fish in fresh water. In a few fish, hypophysial tissue in amounts sufficient to support them in fresh water, regenerated in four to six weeks. In some of these individuals the glands regenerated without connection to the hypothalamus.

A sex-linked gene controlling the age at which the gonadotropic zone of the adenohypophysis differentiates and becomes physiologically active has been identified in the teleost Xiphophorus maculatus . Within the Belize stock, the factor P e , for early differentiation, is linked to a pigment gene Ir (red iris) whereas P l , for late differentiation, is linked to Br (red body). Males homozygous for Ir (Y- Ir Y- Ir ) become sexually mature between 10 and 16 weeks and heterozygous males (Y- Ir Y- Br ) between 14 and 25 weeks. No overlap in maturation times is found when the two kinds of males are raised under identical conditions. A cross of a heterozygous Belize male, Y- Ir Y- Br , with an XX female of the Jamapa stock homozygous for early maturation, yielded 13 Ir males that matured between 12 and 15 weeks and 15 Br males that differentiated sexually between 18 and 24 weeks. At 60 days the gonadotropic zone of the pituitary gland of males homozygous for Ir is well developed while that of Ir Br male heterozygotes is composed of only a few chromophobes. No other differences in pituitary structure were detected. At 105 days the gonadotropic zone of Ir Br males is fully formed. Presence or absence of the gonadotropic zone is well correlated with the differentiation of the testis. Androgen from the developing gonad, in turn, controls the metamorphosis of the anal fin into a gonopodium. Because the growth rate of males decreases markedly at the time of sexual maturity, an effect also mediated by androgenic hormone, early-maturing males (Ir) are significantly smaller than late-maturing ones (Br). Up to that time the growth rate of the two kinds of males is the same. Size is thus the ultimate permanent record of the differences between the two kinds of males. The action of the P genes in females has not yet been investigated. Preliminary evidence shows that males homozygous for P l mature between 20 and 32 weeks and grow correspondingly larger. the polymorphism effecting gonadotrop differentiation is a natural component of wild populations and of laboratory stocks derived from them.

Immunoreactive tyrosine hydroxylase (ir-TH), the rate-limiting enzyme for the synthesis of dopamine and other catecholamines, was localized in the brain and pituitary gland of sexually mature platyfish (Xiphophorus maculatus). This is the first report of ir-TH in the nucleus olfactoretinalis, an LHRH-containing nucleus in the brain which plays an important role in the development and functioning of the reproductive system in platyfish. Ir-TH was also localized in the nucleus preopticus and paraventricular organ. In the pituitary gland ir-TH is found in the prolactin cells and in some fish, in some of the gonadotropin-containing cells of the pars intermedia, but not in the gonadotrops of the pars distalis. The localization of ir-TH in brain centers and pituitary cells associated with reproductive system regulation is discussed in the context of the interaction of monamines, neuropeptides and pituitary hormones during the maturation and operation of the brain-pituitary-gonadal axis.

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.

A sex-linked gene controls the age at which the gonadotropic zone of the pituitary gland develops and becomes physiologically active. The alleles for early (Pe) and late(Pl) differentiation are linked to pigment genes that serve as genetic markers. Pe may be marked by Sp, Dr, Sr, Ir and + and Pl by Br and N. Ir females (PePe) matured between 10 to 15 weeks of age and Br (PePl) females between 16 to 20 weeks. Dr (PePe) females had ripe ovaries six to eight weeks earlier than NDr (PePl) females (8 to 12 weeks versus 16 to 20 weeks). The average age of sexual maturation was 12.5 weeks for IrIr (PePe) males, 20 weeks for IrBr (PePl) and 26.5 weeks for BrBr (PlPl) males. DrSr (PePe) males reached maturity at 13.5 weeks and NSr (PePl) males at 27.5 weeks. The transformation of the anal fin into a gonopodium which is under androgenic control, takes longest in the late maturing fish (IrIr, 3.8 weeks; IrBr, 4.8; BrBr, 6.5; DrSr, 4.2; NSr, 6.4). Similarly, the rate of development of the gonadotropic zone and of the gonad is protracted in the late genotypes. Eventually, the gonadotropic zone of early and late maturing fish are indistinguishable. The Pl factor linked to N delays sexual maturity to a greater extent than the one linked to Br suggesting that not all Pl factors are identical and that important allelic interactions may exist. The growth rate of immature males and females is the same, however it declines sharply in males (but not females) at the time of sexual maturity. This leads to significant differences in adult size between male genotypes. Female genotypes do not exhibit size differences and eventually become larger than all males. The development of the gonadotropic zone precedes, and is essential for, complete gonadal maturation. In the absence of a functional gonadotropic zone, oocytes developed through the oil droplet stage and were surrounded by active follicle cells and a prominent zona pellucida. Spermatogenesis proceeded up to the spermatocyte stage and the cells lining the efferent ducts displayed a modicum of activity. Some of our data suggest that low levels of androgens are present in all males as early as five weeks of age prior to the development of gonadotrops. The genes regulating the development of the pituitary-gonadal axis may be an important factor in explaining the evolution of poeciliid fish.

Radioimmunoassay (RIA) and immunocytochemistry (ICC) were used to study the effect of hypophysectomy (H) on the distribution and quantity of luteinizing hormone-releasing hormone (LH-RH) in the brains of sham-operated-saline-injected, H-saline-injected and H-gonadotropin (GTH)-injected mature female platyfish (Xiphophorus maculatus). In fish hypophysectomized for 5 weeks, there was a decrease in the LH-RH content of the brain (mean +/- S.E. = 446 +/- 31 pg, H-saline-injected; 649 +/- 28 pg, sham-operated-saline-injected). The administration of five 10-micrograms injections of salmon GTH on alternate days beginning in the fifth week of H resulted in a small but significant (P less than 0.05) decrease in LH-RH beyond H-saline-injected group levels (362 +/- 36 pg). The ICC analysis of H fish indicated a decrease in immunoreactive (ir)-LH-RH in the nucleus preopticus periventricularis (NPP) and nucleus lateralis tuberis pars posterioris (NLT), but a marked increase in the immunoreactivity of the neurons in the nucleus olfactoretinalis (NOR). The H fish which received GTH showed a decrease in ir-LH-RH in the NOR, but a slight increase in the NPP and NLT. The results demonstrate that although there is a general decrease in the LH-RH content of brains in H fish as determined by RIA and ICC, one of 3 ir-LH-RH-containing centers of the brain, the NOR, shows a marked increase in ICC-demonstrable LH-RH, indicating that these 3 regions may differ in their roles in the regulation of the reproductive system. The drop in RIA-measurable LH-RH in the brain, along with the enlarged perikarya observed in all 3 regions, suggests that an increase in the synthesis and release of LH-RH occurs in response to removal of the pituitary. The functional significance of the NOR and the feedback mechanisms between brain, pituitary and gonads are discussed.

The structural and functional link between olfactory reproductive systems in male and female platyfish (Xiphophorus maculatus) is demonstrated by the connection of receptors in the nasal epithelium to a center in the brain that has a primary role in the development and functioning of the reproductive system. Profound morphological changes occur in the nasal epithelium, and LH-RH content increases in tracts of the olfactory lobe, at specific stages of sexual maturation and not according to chronological age. Our study provides new insight into the development of the mechanisms by which chemical environmental cues are received.

Hypophysectomy produced hypocalcemia in male killifish, Fundulus heteroclitus , adapted to calcium-deficient sea water. Such changes in serum calcium levels were corrected by intraperitoneal injections of a homogenate of whole pituitary glands or by transplantation of the gland to the musculature of the caudal peduncle. Injections of homogenates of the anterior or posterior parts of the pituitary gland were also effective while the homogenate of the middle part of the gland produced a smaller change. Injections with prolactin at the doses of 5, 2.5, and 0.5 μg per gram body weight per injection significantly increased the serum calcium levels of the hypocalcemic hypophysectomized fish. Cortisol (2.5 μg per gram body weight per injection) and ACTH (0.05 IU per gram body weight) were also hypercalcemic, but lower doses of ACTH (0.02 and 0.005 IU per gram body weight) and two doses of MSH (2.5 and 5 μg per gram body weight) were without effect. It is suggested that either ACTH, prolactin or both may have hypercalcemic effects on fish in calcium-deficient environments.

An immunoperoxidase method has permitted the identification of gonadotropin (GtH)- and thyrotropin (TSH)-producing (containing) cells in the caudal pars distalis of the pituitary gland of mature platyfish ( Xiphophorus maculatus ). The results corroborate earlier reports where the locations of these cells were determined by the use of basic staining methods. The immunoreactivity of antisera generated against a number of teleostean gonadotropins and their subunits and against human TSH was examined. To verify the specificity of the response, controls were employed which included the substitution of normal rabbit serum for the specific antiserum and/or the absorption of the final dilution of antiserum with corresponding homologous or heterologous antigens. Antiserum to carp GtH (anti-cGtH) displayed the highest degree of specificity when compared to other preparations (trout and salmon) produced against the intact GtH molecule. Antiserum against the α subunit of carp GtH (anti-cGtH-α) cross-reacted with both thyrotrops and gonadotrops whereas the β subunit antiserum (anti-cGtH-β) displayed immunoreactivity with gonadotrops but not with thyrotrops. Antiserum against human TSH (anti-hTSH) cross-reacted only with thyrotrops. Our results indicate that the use of suitable antisera permits the localization of thyrotrops and gonadotrops. This report is presumably the first to show that the α subunit of carp GtH is a chain common to both TSH and GtH in fish. It is also the first demonstration of antigenic similarity between human and fish TSH.

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.

Specific binding sites for estrogen, testosterone, and progesterone have been demonstrated in the pituitary gland of mature male and female platyfish (Xiphophorus maculatus). With a histochemical procedure, fluorescent-steroid-hormone conjugates were localized in the cytoplasm and nucleus of the gonadotrops of the caudal pars distalis (CPD) and in cells of the pars intermedia (PI) previously demonstrated to contain immunoreactive gonadotropin. The specificity of the response was confirmed by means of competitive binding analyses and by using fluoresceinated BSA not linked to steroids. The physiological significance of steroid binding in the PI, as well as in the CPD, is discussed in the light of other recent studies on the pituitary gland of the platyfish.

Hypophysectomized killifish ( Fundulus heteroclitus ) adapted to calcium-deficient seawater showed significant decreases in plasma calcium levels 8 days after the operation. At Day 16, plasma calcium levels were no longer significantly different from those of operated control fish. Plasma sodium and potassium levels were unaffected at all time periods. Injections of whole carp ( Cyprinus carpio ) pituitary homogenate corrected the hypocalcemia seen in the hypophysectomized killifish. When the carp glands were divided into “anterior”, “middle”, and “posterior” portions and tested in the hypocalcemic fish, the “middle” portion had the greatest corrective effect. Histological studies demonstrated that this portion probably contains the largest number of prolactin cells. In all cases, plasma sodium levels were elevated. In one experiment, a prolactin cell-free “posterior” portion had no hypercalcemic or hypernatremic effect. Injections of prolactin produced a log-dose-related hypercalcemia and hypernatremia in the hypophysectomized killifish. The highest dose of prolactin used (10 μg/g of body wt per injection) had a reduced hypercalcemic effect. Based on all these data, we suggest that prolactin may be the pituitary hypercalcemic principle in the killifish.

Immunoreactive serotonin (ir-5HT) containing cells were localized in the brain and pituitary gland of the platyfish by use of immunoperoxidase procedures. In the brain, ir-neurons were found lining the wall of the third ventricle and in its lateral and posterior recesses. More caudally, ir-perikarya were found in the valvular portion of the cerebellum and in the raphe region. Ir-5HT was also localized within the pineal gland in fish that had been sacrificed before 1:00 p.m. Within the pituitary gland, ir-5HT was localized in periodic acid Schiff-positive cells of the pars intermedia of all fish while, in only a few animals, less intense immunoreactivity was also present in gonadotrophs of the caudal pars distalis.

We have traced the development from birth to puberty of the ir-LHRH producing centers, the nucleus olfactoretinalis (NOR), nucleus praeopticus periventricularis (NPP) and nucleus lateralis tuberis pars posterior (NLT), in male sibling platyfish genetically determined by differing P alleles to mature at two different ages. In sexually immature fish of both genotypes ir-LHRH is first localized in the NOR in perikarya and neuronal fibers and in the NPP where it is limited to the fibers. At the onset of puberty in early maturing fish (11 weeks), ir-LHRH accumulates in perikarya and fibers of the NPP and NLT. Late maturing siblings first have ir-perikarya in the NPP and a few ir-fibers in the NLT at the same developmental stage but at a later age (25–26 weeks). Ir-perikarya are still not seen in the NLT in late-maturing fish even when maturation is complete. This pattern of a sequential development of the three ir-LHRH containing areas of the brain, which we refer to as a “cascade effect”, is similar in early and late genotypes. It is directly correlated to stage of development and not to chronological age. It is clear that the appearance of LHRH producing brain centers precedes, and is presumably essential for, the completion of the development of pituitary gonadotrops and the subsequent maturation of the gonads. The functional significance of the NOR in puberty and reproduction and as a possible site of action for the P gene is discussed.

Removal of the corpuscles of Stannius produced a hypercalcemia in male killifish, Fundulus heteroclitus , which were maintained in sea water with normal calcium content (10 mmoles/liter). When the corpuscles of Stannius were transplanted to the caudal peduncle at the time of stanniectomy, the elevation of serum calcium levels was prevented. After 2 wk in their ectopic position, the transplanted glands were well vascularized and their histological profile was generally typical in appearance. The structural integrity of the cells showed considerable variability. Within the same gland, cells ranged from “degenerating” to “active” in their appearance. The significance of the structure of these transplanted organs in the light of the physiology of the fish is discussed.

Immunoreactive luteinizing hormone releasing hormone (irLHRH) and immunoreactive gonadotropin (irGtH) are demonstrated in the pituitary gland of a teleost fish (Xiphophorus maculatus, the platyfish) at the ultrastructural level. Using an immunoperoxidase procedure, irGtH and irLHRH are specifically localized over the secretory granules of both gonadotropes and some cells of the pars intermedia (PI). All other pituitary cell types lack immunoreactive material. A concept of LHRH receptors on gonadotrope granules is discussed and a new function for a class of PI cells is suggested.

In an investigation of six stocks of platyfish (Xiphophorus maculatus) in the Genetics Laboratory of the New York Zoological Society, only one exhibited pituitary cysts. Cysts were discovered in 58 of 76 (76%) of the fish of the Gp (Grijalva) strain that were one month or older. This strain originated from the Rio Grijalva, Mexico, and has been inbred for many generations in this laboratory. Cysts were first seen shortly after birth and they increased in size with age. This enlargement results from an increase in the number of typical goblet cells that form the wall of the cyst and by an accumulation of their PAS — positive secretion. More than one cyst rarely occurs in a gland. Cysts may be found in all portions of the adenohypophysis, most frequently in the meso-adenohypophysis, but they never occur in the neurohypophysis. Hypophysial cysts were not found in Gp embryos, in offspring of crosses between Gp females and males from other platyfish stocks and in wild-caught fish from the Rio Grijalva. Fish of the Gp strain that showed abnormal gonadal development always possessed a pituitary cyst, but not all fish with pituitary cysts were hypogonadal. The information presented points to the fact that the origin of cysts has a genetic basis and perhaps arose as a result of inbreeding. It is suggested that cysts result when cells of the adenohypophysial anlage fail to differentiate into pituitary cells but become goblet cells instead.

1. 1. Marked and consistent increases in hepatic activities of glucose-6-phosphate dehydrogenase were found in Xiphophorus maculatus exposed to 1 3 sea water. Prolonged treatment tended to increase the response. 2. 2. In the three diverse species of fish examined here, the second enzyme of the pentose shunt, 6-phosphogluconate dehydrogenase, was invariably present but always lower in activity than the first enzyme, glucose-6-phosphate dehydrogenase. 3. 3. An intact pentose shunt would appear to be a general feature of teleost liver.