Hemiclonal reproduction slows down the speed of Muller's ratchet in the hybridogenetic frog Rana esculenta

Rare recombination in otherwise asexually reproducing organisms is known to beneficially influence the fitness in small populations. In most of the investigated organisms, asexual and rare sexual generations with recombination follow each other sequentially. Here we present a case where clonal reproduction and rare recombination occur simultaneously in the same population. The hybridogenetic water frog Rana esculenta (E), a hybrid between R. lessonae (L) and R. ridibunda (R) produces gametes that only contain the unaltered maternal R part of their genome. New generations of R. esculenta usually arise from E x L matings. Intraspecific E x E matings produce mostly inviable offspring, but in rare cases, female R. ridibunda arise from such matings which are capable of recombination. In the absence of conspecific males, these R females have to mate with E males, which results in further R females, or with L males, which produces new E lineages. This indirect mechanism reintroduces recombination into the otherwise clonally transmitted R genomes in R. esculenta populations. In this study, we show through Monte Carlo simulations that, in most cases, it is sufficient that only between 1 % and 10 % of mixed water frog populations consist of R females to prevent or significantly reduce the fixation and accumulation of deleterious mutations.     

Biogene Amine im Gehirn vom Frosch (Rana esculenta)

Zusammenfassung Mit Hilfe der Methode zur fluoreszenzmikroskopischen Lokalisation von Catechol- und Tryptaminen wurde die Verteilung dieser Stoffe im ZNS von Rana esculenta untersucht. Catecholamin- und serotoninhaltige Neurone liegen im Nucleus reticularis mesencephali. Außerdem finden sich catecholaminhaltige Nervenzellen im Organon vasculosum hypothalami und in der Area praeoptica. Diese aminproduzierenden Zellen entsenden Zellfortsätze durch die Ependymschicht in den Ventrikel. Über diese Ausläufer erfolgt möglicherweise eine Sekretion biogener Amine in den Liquor cerebrospinalis. Catecholamin- und serotoninhaltige Axone erreichen voneinander verschiedene Kerngebiete und Areale. Neben dem periventrikulären Zellager im Tuber cinereum und in der Area praeoptica werden vor allem der ventrolaterale Teil des lateralen Septumkerns, Striatum ventrale und Epistriatum von Endstrecken catecholaminhaltiger Axone durchdrungen. Serotoninhaltige Varicositäten finden sich dagegen vor allem in Kerngebieten, die in sensorische Bahnen eingeschaltet sind (Nucleus isthmi, corpus geniculatum laterale, Area praetectalis, Tectum opticum, Thalamus dorsalis, Neostriatum). Weitere Ausbreitungsgebiete 5-Hydroxytryptamin-haltiger Fasern sind die Habenula und der Nucleus interpeduncularis, Kerngebiete, über die Erregungen aus dem limbischen System auf vegetative Zentren der Medulla oblongata geleitet werden.

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Biogenic amines in the brain of the frog (Rana esculenta)

Summary The distribution of biogenic amines in the central nervous system of Rana esculenta was investigated by means of the fluorescence-microscopical detection of catecholand tryptamines. The nucleus reticularis mesencephali was found to contain numerous neurones rich in catechol- and tryptamines. Apart from this nucleus nerve cells in the organon vasculosum hypothalami and in the area praeoptica were found to contain catecholamines. The clublike processes of these neurones penetrate the ependymal layer and extend into the ventricle. These structures are presumably responsible for a secretion of biogenic amines into the cerebrospinal fluid. Catecholamine- and serotonin-containing axons terminate on different nuclei and areas. Besides the periventricular cellular layer of the tuber cinereum and the area praeoptica, the pars ventrolateralis of the nucleus septalis lateralis, striatum ventrale and epistriatum are pervaded by terminals of catecholamine-containing neurons. Serotonincontaining varicosities are mainly to be found in nuclei, which are intercalated in sensory pathways (nucleus isthmi, corpus geniculatum laterale, area praetectalis, tectum opticum, thalamus dorsalis, neostriatum). Further areas of distribution of 5-hydroxytryptamine-fibers are the habenula and the nucleus interpeduncularis, nuclei which coordinate impulses from the limbic system projecting them on visceral centers of the medulla oblongata.

Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

A voltage-dependent and pH-sensitive proton current in Rana esculenta oocytes

Voltage clamp technique was used to study macroscopic ionic currents in Rana esculenta oocytes. Depolarization steps led to the activation of a single type of outward current (I _out) when contaminant potassium and calcium-dependent chloride currents were pharmacologically inhibited. The voltage threshold of I _out activation was 10 mV and this current, which did not inactivate, presented a deactivation the time constant of 73±21 msec (n=26) corresponding to a membrane voltage of –60 mV. Its reversal potential (E _rev) was dependent on the magnitude of the depolarization and also on pulse duration. These changes in E _rev were thought to reflect intracellular ion depletion occurring during activation of the remaining outward current. Furthermore, the activation threshold of I _out was clearly affected by modifications in extracellular and intracellular H⁺ concentrations. Indeed, intracellular alkalinization (evoked by external application of ammonium chloride) or extracellular acidification induced a rightward shift in the activation threshold while intracellular acidification (evoked by external application of sodium acetate) or extracellular alkalinization shifted this threshold toward a more negative value. Lastly, I _out was dramatically reduced by divalent cations such as Cd²⁺, Ni²⁺ or Zn²⁺ and was strongly decreased by 4 Aminopyridine (4-AP), wellknown H⁺ current antagonists already described in many cell types. Therefore, it was suggested that the outward current was prominently carried by H⁺ ions, which may play a key role in the regulation of intracellular pH and subsequent pH dependent processes in Rana oocyte.     

Arterial supply of the choriocapillaris of anuran amphibians (Rana temporaria,Rana esculenta)

Summary The pattern of the vascular supply to the choroid of the frog eye was studied in toto with the use of the injection-replication-SEM technique. The choroid of anuran amphibians is composed mainly of the choriocapillaris. In both species studied (Rana temporaria, Rana esculenta), an independent arterial supply to the choriocapillaris supplemented that from the ciliary arteries. This additional vascular route arises from the optic artery, a separate branch of the arteria infundibularis superficialis. The optic artery, accompanied by its vein within the vascular sheath of the optic nerve, joins the rich arterial capillary network of the choriocapillaris and supplies the posterior pole of the ocular bulb. The superficial capillary network displays a dense collar around the entrance of the optic nerve into the eye and is composed of a circular meshwork of small capillaries, several layers deep. More peripherally, however, it becomes single layered. This capillary network, as a whole, establishes numerous connections with the adjacent choriocapillaris at the posterior pole of the ocular bulb. In anuran amphibians the complex arrangement of both arterial systems supporting the choriocapillaris may be regarded as a more complete equivalent of the short posterior ciliary arteries of mammals.     

The superficial vascular hyaloid system in the eye of the frogs,Rana temporaria and Rana esculenta

Summary The angioarchitecture of the superficial vascular hyaloid system (membrana vasculosa retinae) of the frog eye was studied by means of scanning electron microscopy of vascular corrosion casts. The terminal vessels form a single-layered sheath intimately adjacent to the vitreal side of the avascular retina. The hyaloid system is subdivided by the ventral venous trunk into three central areas: the dorsal, the temporo-ventral, and the naso-ventral area. Toward the ora serrata, the hyaloid system is bordered by an arterial ring, and by nasal and temporal venous branches forming more or less complete hemicircles. A vascular zone composed of several tongue-like sectors establishes an inter-connection between the peripheral vascular rings and the central areas of the fundus. The arterial blood is supplied from the arterial ring. The drainage of the hyaloid system is provided via two routes: (1) the Y-shaped ventral trunk collects blood from the central areas, (2) the two peripheral venous branches drain the tongue-like sectors. The vessels within the dorsal area follow preferentially a dorso-ventral meridional direction. This densely capillarized territory corresponds in localization to the area centralis retinae. The ultrastructure of microvessels of the hyaloid system is characterized by features typical for capillaries of the central nervous system.     

Cytological changes in the pars distalis of pituitary of the green frog,Rana esculenta L., during the reproductive cycle

Summary The cytological changes in the pars distalis of pituitary of male and female green frog, Rana esculenta during the reproductive cycle have been studied by examining the form, cytoplasmic granulation and numerical proportions of the different cell types. Some sex differences were recorded, particularly in relative total number of cells in a median sagittal section of the pars distalis. No extensive cytological changes occur during the hibernation period, but there are striking alterations at the time of spring awakening, during spawning months and during replenishment of the spent gonads i.e. during late summer and autumn months. Important changes are to be observed in the acidophils type one (A1) and basophils type two (B2) and three (B3). Changes in the A1 cells during breeding season have been speculated as being in support of the spawning activity of the animal. The B2 cells exhibit most profound changes which correlate precisely with the morphological changes in gonads, and these cells are considered as the FSH-gonadotrops. The functional significance of the B3 cells is discussed and it is suggested that they might be the source of an ICSH- or LH-like hormone.The work has been supported by grant from the National Research Council of Italy.The award of postdoctoral fellowship by the National Research Council of Italy is gratefully acknowledged.     

Patterns of protein synthesis in oocytes and early embryos of Rana esculenta complex

Summary We have used isotopic labelling and both one-and two-dimensional electrophoretic procedures to analyse the protien synthesis patterns in oocytes and early embryos of three phenotypes of the European green frogs. The results demonstrated that protein patterns of Rana ridibunda and R. esculenta are identical, but that they differ from those of R. lessonae. Progeny of the lethal cross R. esculenta × R. esculenta showed a distinct delay in the appearance of stage-specific proteins during early embryogenesis. The heat-shock response of R. ridibunda and R. esculenta oocytes was found to be identical, but different from that of Xenopus laevis. The implications of these findings, with respect to hybridogenesis in R. esculenta complex and variations in the regulations of heat shock genes in different amphibian species, are discussed.     

Inhibition of pituitary gonadotropic effects in the pars distalis-ectomized Rana esculenta by methallibure (ICI 33,828)

Summary Effects of Methallibure on the gonadotropic stimulation of the regressed gonads and secondary sex characters of the pars distalis-ectomized adult green frog, Rana esculenta, were studied. Regression of the gametogenetic activity and reduction of oviducts and thumb pads are the eventual consequences of the removal of pars distalis. Homoplastic pars distalis homogenates restimulate the gametogenesis and regressed secondary sex characters due to their gonadotropic hormone contents. Methallibure in this case blocks the stimulation of these organs by homoplastic pars distalis, which indicates that this compound inhibits the action of gonadotropic hormones upon their target tissues.Work supported by the Population Council (Grants M70.082 C & M71.0132 C) of New York, and National Research Council of Italy.     

Intermitochondrial junctions in the heart of the frog,Rana esculenta

Summary In muscle fibers of the frog heart, junctions between outer membranes of adjacent mitochondrial profiles are occasionally found. In thin sections of embedded tissue and of mitochondrial pellets, the intermitochondrial junctional space is 5.4±0.15 nm; the external leaflets of the membranes are joined by periodic structures separated from each other by 16.3±0.29 nm. There are 65.3±2 periodic structures per m of membrane measured on a section perpendicular to the junction. After cryofracture, the outer membrane is cleaved into two parts. Closely packed, parallel rows of large particles and furrows are found either on the P-, or on the E-faces. The rows of particles are 11±0.3 nm thick and are separated from each other by 16.5±0.46 nm, their density being 65±2.28 per m of the membrane. In junctional areas, rows of particles on one membrane correspond with the furrows on the other membrane. Intermitochondrial junctions appear to be real structures and not artifacts due to preparation procedures. The conditions of their occurrence are discussed.     

Influence of changes in composition of the cerebrospinal fluid on the secretory activity of the subcommissural organ in Rana esculenta

Summary Normally the lymphatic sinuses of the lymph node are loosely packed with lymphocytes and free macrophages as well as with macrophages adhering to the fibrocellular trabeculae. After immunization with SRBC cluster formation occurs in the medullary sinuses of rats between a central macrophage and peripherally located lymphocytes. These rosette-like clusters are nearly identical with the clusters found during primary and secondary immune response against SRBC in vitro and seem to be the in vivo equivalent for the same immune response.     

Somatostatin-immunoreactive cells in the gastrointestinal tract of the frog Rana esculenta

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.Fellow of the Alexander von Humboldt Foundation, Bonn, Germany     

Formation of desmosomes and other contact specializations in cultured skin of the frog (Rana esculenta)

Summary Small trypsinized explants from ventral skin of frogs (Rana esculenta) were maintained in culture for 4 days during which a newly formed epithelium differentiated along the cut edges of the dermis. During the first 6 h adjacent cells produced numerous interdigitating lamellipodia. After 2 days, epithelial polarity was restored by the formation of zonulae occludentes and the epithelial cells were joined by a few small newly formed desmosomes and by numerous interdigitations. Bipartite junctional complexes consisting of a zonula occludens, followed by a series of typical desmosomes, and characteristic of adult frog epidermis were formed only after 4 days. When cultured in the presence of an inhibitor of protein synthesis (cycloheximide) the trypsinized epidermis no longer formed desmosomes. Therefore pools of one or more crucial desmosomal proteins must be very low or non-existent. However, cycloheximide did not prevent the formation of cell contact specializations, consisting of a highly developed system of complex lamellar interdigitations, between adjacent cells.     

Degenerationsstudien am Nervus pinealis von Rana esculenta L. nach stirnorgannaher und -ferner Durchtrennung

Zusammenfassung Am Nervus pinealis von Rana esculenta wurde der Verlauf der Wallerschen Degeneration nach Durchtrennung des Nervenstrangs unterhalb des Stirnorgans studiert.30 Std nach der Läsion tritt im distalen Stumpf der markhaltigen Nervenfasern eine Anhäufung von Glykogen auf; granulär-vakuolige Veränderungen zeigen diese Faserabschnitte am 4. Tag. Veränderungen der Myelinscheide beginnen am 7. postoperativen Tag an den Innenlamellen und greifen dann auf die ganze Markscheide über. Die Desintegration der einzelnen markhaltigen Fasern eines Nervus pinealis verläuft verschieden schnell; nach 25 Tagen ist sie an den meisten Fasern abgeschlossen.Am 7. Tag sind die ersten Veränderungen an den marklosen Fasern zu beobachten. Dabei werden die peripher in der Hüllzelle liegenden Fasern durch Retraktion des Hüllzellzytoplasmas ins Interstitium ausgestoßen und gehen dort zugrunde. Die mehr zentral in den Hüllzellen lokalisierten Fasern bleiben hingegen vom Hüllplasma umgeben. Die Degeneration dieser marklosen Elemente ist nach 14 Tagen abgeschlossen; es resultiert ein mit homogenem Inhalt geringer Elektronendichte gefüllter Kanal, der auf Querschnittsbildern eine Vakuole vortäuscht.Weiterhin ist für den degenerierenden Nervus pinealis eine starke Fibrosierung sowie der Schwund der intra- und perineuralen Lymphspalten charakteristisch. Nach etwa 60 Tagen treten im distalen Nervenstumpf von Büngnerschen Bändern umgebene marklose Nervenfasern auf. Dieses Auswachsen von Nervenfasern bestätigt elektrophysiologische Ergebnisse von Morita (s. Dodt und Morita, 1967), daß im Nervus pinealis erregungsleitende Elemente in beide Richtungen ziehen (frontofugale und frontopetale Fasern).Bindegewebsstrukturen, Degenerationsablauf und Strukturbild der Hüllzellen lassen den Nervus pinealis als peripheren Nerven erscheinen.

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Degeneration pattern of the pineal nerve of rana esculenta after transection at different anatomical levels

Summary Wallerian degeneration was studied in the pineal nerve of Rana esculenta with the electron microscope after transection of the nerve beneath the frontal organ.30 hours after the operation an accumulation of glycogen was found in the distal stump of the myelinated axons. Four days after interruption these axons displayed signs of granular and vaculoar disintegration. Myelin sheath changes started at the seventh day after the operation at the level of the inner lamellae and were subsequently followed by changes in the outer lamellar systems. The velocity of disintegration varied for individual myelinated fibers. 25 days after transection of the pineal nerve most of its myelinated fibers were completely degenerated.Initial changes of the unmyelinated fibers were recognized at the seventh postoperative day. Fibers located peripherally in the satellite cell were exposed by retraction of the satellite cytoplasm and degenerated rapidly. On the other hand, unmyelinated fibers which were located more centrally remained within the satellite cell and completed their degeneration only after 14 days. The residual structure observed was a channel occupied by a homogeneous material of low electron density.The number of collagen fibers increased and the intra- and perineural lymph spaces were obliterated in the distal nerve stump. Approximately 60 days after the operation chains of satellite cells and unmyelinated nerve fibers reappeared in the distal nerve stump. This result confirms the electrophysiological data of Morita (cf. Dodt and Morita, 1967) that nerve fibers run in both directions within the pineal nerve (i.e. they are frontofugal and frontopetal with respect to the frontal organ).The properties of the connective tissue between the pineal nerve fibers, the pattern of degeneration and the fine structure of the satellite cells suggest that the pineal nerve of Rana esculenta has the structure of a peripheral nerve rather than that of a central nervous tract.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. Ein Druckkostenzuschuß wurde aus Institutsmitteln zur Verfügung gestellt.     

The effects of gonadectomy and testosterone treatment on the Harderian gland of the green frog,Rana esculenta

The effects of gonadectomy and testosterone treatment on the fine structure of the Harderian gland in male and female green frogs were investigated in different periods of the year. Gonadectomy, carried out when the glands are in the lowest secretory phase (September), causes degenerative changes consisting of a reduction of the rough endoplasmic reticulum, the appearance of autolysosomes, and an increase of nuclear heterochromatin. These effects can be prevented by testosterone treatment. No castration effects are found during the recovery (November) and enhancement (April-May) phases of secretory activity. The results suggest that the frog Harderian gland's sensitivity to testosterone changes during the annual cycle. The androgen dependence of the Harderian gland is correlated with the presence of androgen receptors in both male and female forgs.     

Adenylate cyclase and carbonic anhydrase in the semicircular canal epithelium of the frog Rana esculenta

Summary Because the secretion of endolymph has been localized in the ampullar part of the frog semicircular canal, we attempted to determine by cytochemical methods the ultrastructural localization of two enzymes that are assumed to play a role in endolymph secretion: carbonic anhydrase and adenylate cyclase. Functionally, the epithelium of the frog semicircular canal can be schematically divided into three areas: sensory (crista ampullaris), secretory (dark cells), and non-sensory and nonsecretory (transitional and undifferentiated cells) areas. Carbonic anhydrase activity was widely distributed in dark cells. Dark cell labeling disappeared in the presence of acetazolamide. The other cells of the canal did not show any carbonic anhydrase labeling except for the supporting cells of the sensory cells. Adenylate cyclase activity was found on the basolateral and apical membranes of dark cells, and on the apical membrane of sensory cells; weak labeling was also observed in the other epithelial cells. In the apical membrane of the dark cells, adenylate cyclase labeling was dependent on the presence of vasotocin, the frog antidiuretic hormone. The dark cells of the frog semicircular canal thus possess the enzyme equipment needed for the secretion of endolymph and its possible hormonal regulation.     

Ecology of the Hybridogenetic Rana esculenta Complex: Differential Oxygen Requirements of Tadpoles

Because of intrinsic demographic load induced by hybridogenesis (infertility of homotypic hybrid matings), the maintenance of hybrid lineages supposes that they present better performances (heterosis) than their host species which allows them to coexist on a long-term basis. However, this necessity of high fitness can be relaxed if a relative niche partitioning occurs between the taxa, each of them differing in their ecological optima. In the waterfrog hybridogenetic complex (Rana esculenta complex), recent studies have revealed that hybrids show intermediate distribution between parental species across a gradient of river influence (that is related to a gradient of oxygen levels), and intermediate performances of their tadpoles with regard to oxygen availability (hypoxia). In investigating oxygen consumption rates, survival time in anoxia, and metabolite contents in the three forms of the complex, the present study confirms intermediate characteristics of hybrid tadpoles (R. esculenta) when compared to both parental lineages (R. lessonae and R. ridibunda). Whereas R. ridibunda tadpoles were the most sensitive to anoxia, R. lessonae tadpoles were the most tolerant. Because oxygen requirements of the hybrid proved to be intermediate, no heterosis was detected. These results confirm the hypothesis of the intermediate niche hypothesis to explain the coexistence of R. lessonae and R. esculenta and the success of the hybridogens.     

Morphology of neurons and axon terminals associated with descending and ascending pathways of the lateral forebrain bundle in Rana esculenta

Summary The cells of origin of afferent and efferent pathways of the lateral forebrain bundle were studied with the aid of the cobalt-filling technique. Ascending afferents originated from the lateral thalamic nucleus, central thalamic nucleus, posterior tuberculum and the cerebellar nucleus. They terminated in the anterior entopeduncular nucleus, amygdala and the striatum. Telencephalic projection neurons, which are related to the lateral forebrain bundle, were located mainly in the ventral striatum and the anterior entopeduncular nucleus, but were not so numerous in the dorsal striatum. Irrespective of their location, most of the neurons projecting axons into the lateral forebrain bundle had piriform or pyramidal perikarya. Long apical dendrites usually arborized in a narrow space, whereas widely arborizing secondary dendrites originated from short dendritic trunks. The other neurons that contributed to the lateral forebrain bundle were fusiform or multipolar cells. Striatal efferents terminated in the pretectal area and in the anterodorsal, anteroventral and posteroventral tegmental nuclei.     

Genetic compatibility between sexual and clonal genomes in local populations of the hybridogeneticRana esculenta complex

Summary Hybridogenetic species possess a hybrid genome: half is clonally inherited (hemiclonal reproduction) while the other half is obtained each generation by sexual reproduction with a parental species. We addressed the question of whether different hemiclones of the hybridogenetic water frogRana esculenta are locally adapted for genetic compatibility with their sexual parental hostRana lessonae. We artificially crossedR. esculenta females of three hemiclones (GUT1, GUT2 and GUT3) from a pond near Gütighausen, Switzerland and one hemiclone (HEL1) from near Hellberg, Switzerland each toR. lessonae males from both populations. We also created primary hybrids by crossing the sameR. lessonae males from both populations toR. ridibunda females from Pozna, Poland (POZ). Tadpoles were then reared in the laboratory at two food levels to assess their performance related to early larval growth rate, body size at metamorphosis and length of the larval period. Tadpoles from hemiclones GUT1, GUT3 and POZ had higher growth rates than those from hemiclones GUT2 and HEL1 at the low food level, but at the high food level all growth rates were higher and diverged significantly between hemiclones GUT2 and HEL1. Tadpoles from the intrapopulational crosses GUT2 × GUT and HEL1 × HEL were larger at metamorphosis than those from the interpopulational crosses GUT2 × HEL and HEL1 × GUT. A high food level increased the size at metamorphosis in all tadpoles. A high food level also decreased the days to metamorphosis and tadpoles from GUT1, GUT3 and POZ had the shortest larval period whereas those from GUT2 and HEL1 had the longest. These results indicate that the differential compatibility of clonal genomes may play an important role in hybridogenetic species successfully using locally adapted sexual genomes of parental species and that interclonal selection is likely important in determining the distribution of hemiclones among local populations.     

Distribution of gonadotropin-releasing hormone-like peptides in the brain during development of juvenile male Rana esculenta

Summary The distribution and density of cell bodies and fibers immunoreactive to GnRH-like peptides were investigated in the brain of male juvenile frogs (Rana esculenta) during postmetamorphic development. An immunohistochemical technique was used, involving antisera raised against 4 variants of GnRH: mammalian GnRH, chicken GnRH-I, chicken GnRH-II and salmon GnRH. A comparison of the immunohistochemical distribution at 8 different developmental stages shows that the maximum density of immunoreactive-GnRH elements, and the full distributional complexity of this system, is attained at the completion of spermatogenesis. Immunoreactive-GnRH cell bodies first appear in the anterior preoptic area during the metamorphic climax, and then appear sequentially in the medial septal area, tegmentum and, lastly, in the retrochiasmatic area and olfactory bulb when immunoreactive-fibers also reach the cerebellum. The GnRH system reacts positively to antisera for all 4 GnRH variants, but immunoreactivity for chicken GnRH-I is the weakest.