Mammalian and chicken I forms of gonadotropin-releasing hormone in the gonads of a protochordate, Ciona intestinalis.

Two forms of gonadotropin-releasing hormone (GnRH) were isolated from the gonads of the tunicate, Ciona intestinalis. The primary structure of the purified peptides was determined by MS and chemical sequence analysis. Both GnRH forms have blocked NH(2) and COOH termini, and their primary structures are identical to mammalian (mGnRH) and chicken I (cGnRH-I) forms reported previously in vertebrates. A total of 1.2 mg of purified cGnRH-I and 0.98 mg of mGnRH was obtained from 100 g of Ciona gonads. The physiological effects of native GnRHs included the induction of synthesis and secretion of sex steroids from ciona gonads and the secretion of luteinizing hormone from rat pituitary. These results suggest that the primary structure and functional roles of mGnRH and cGnRH-I have been highly conserved throughout evolution of chordates.

Ontogenetic profile of FSH and LH in Rana esculenta.

Circulating levels and pituitary content of FSH and LH were determined by specific radioimmunoassays in Rana esculenta starting a few days after hatching until the completion of metamorphosis. Both gonadotropins were found in the pituitary as well as in the blood plasma at all stages of development examined here. The plasma concentrations of FSH and LH were more or less uniform during pre- and prometamorphosis, but increased significantly at the onset of metamorphic climax. The plasma levels of FSH and LH remained high at the completion of metamorphosis. The pituitary content of FSH and LH was low in early premetamorphosis. It increased slightly through prometamorphosis and metamorphic climax, following which a highly significant increase occurred. Whereas plasma concentrations of FSH and LH were essentially similar within a single stage of development, the pituitary FSH content was severalfold higher than pituitary LH. The significance of these results is discussed in relation to the functional maturation of the brain-pituitary-gonadal axis in the frog.

Comparative analysis of GnRH neuronal systems in the amphibian brain.

We have investigated the GnRH-ir neuronal systems in the brain of the oviparous urodele, Triturus vulgaris, ovoviviparous urodele, Salamandra salamandra, and viviparous caecilian, Typhlonectes compressicauda, and have reexamined Xenopus laevis, Ambystoma mexicanum, and Rana esculenta. Results showed that mGnRH neuronal system was diffused along the medioventral telencephalon and diencephalon with the numerical preponderance of GnRH cell bodies in the rostral mediobasal telencephalon in T. vulgaris and S. salamandra and in medial septal area and preoptic area respectively in Typhlonectes compressicauda and X. laevis. The cGnRH-II-ir perikarya were restricted to the midbrain tegmentum in X. laevis and T. compressicauda. In T. vulgaris, two distinct groups of cGnRH-II neurons were distinguished, one in the midbrain tegmentum and another in the paraventricular organ. The former was composed of comparatively bigger perikarya than the latter. In X. laevis brain, besides those in the rostralmost dorsomedial and ventromedial telencephalon and septopreoptic area, mGnRH neurons were also found in the habenulae and habenular commissure as well the infundibular hypothalamus. In A. mexicanum, reexamined, the preoptic area-located mGnRH neurons were distributed in the ependymal lining of the preoptic recess. In this neotenic urodele, furthermore, cGnRH-II neurons were also present in the rhombencephalon, as well as in the infundibular hypothalamus. It is thus clear that while GnRH-ir cell bodies are distributed in the fore-, mid- and hindbrain, their precise neuroanatomical localization varies somewhat within and among groups. Altogether, it is evident that mGnRH neuronal system is confined mainly to the forebrain, whereas cGnRH-II system is commonly found in the mid- and hindbrain. Additional morphological investigations are required to eventually define the functional neuroanatomy of GnRH in the amphibian brain.

Distribution of somatostatin-like immunoreactivity in the brain of the frog, Rana esculenta, during development.

The anatomical distribution of somatostatin-like immunoreactivity in the central nervous system of the frog, Rana esculenta, during development and in juvenile specimens was investigated by indirect immunofluorescence. Soon after hatching, at stages II-III, somatostatin-like immunoreactive structures were found in the preoptic-median eminence complex. In stage VI tadpoles, new groups of immunopositive perikarya and nerve fibers appeared in the diencephalon, within the ventral infundibular nucleus and in the ventral area of the thalamus, as well as in the medial pallium. In stages XII-XIV of development, immunopositive perikarya were also present in the dorsal infundibular nucleus of the hypothalamus and ventrolateral area of the thalamus. A small group of somatostatin-like immunoreactive neurons appeared in the posteroventral nucleus of the rhombencephalon. However, these neurons were not seen in later stages of development. Tadpoles in stages XVIII, XXI-XXII and in juveniles were characterized by a wider distribution of immunoreactive cell bodies and fibers in the pallium. New groups of immunoreactive neurons were found in the dorsal and lateral pallium. The presence of positive perikarya in the lateral pallium is a transient expression found only in these stages. The organization of the somatostatinergic system was most complex during the metamorphic climax, with the appearance of positive cell bodies in the posterocentralis area of the thalamus, and in juvenile animals with the presence of perikarya in the ventral part of the medial pallium and within the central grey rhombencephali. In contrast to the adult frog, somatostatin neurons were not observed in the mesencephalon of tadpoles and juveniles.

Distribution of gonadotropin-releasing hormone immunoreactivity in the brain of Ichthyophis beddomei (Amphibia: Gymnophiona).

From a comparative viewpoint, we have investigated the presence and neuroanatomical distribution of gonadotropin-releasing hormone (GnRH)-immunoreactive material in the brain of a gymnophione amphibian, Ichthyophis beddomei. Immunocytochemical analysis of the adult brain and terminal nerves in both sexes shows the presence of neurons and fibers containing mammalian GnRH (mGnRH)- and chicken GnRH-II (cGnRH-II)-like peptides. With respect to GnRH-immunoreactive material, there are two distinct neuronal systems in the brain: one containing mGnRH, which is located in the forebrain and terminal nerve, and the other containing cGnRH-II, which is restricted to the midbrain tegmentum. Basically, this distribution pattern parallels that of many species of anurans and a urodele. Whereas the presence of cGnRH-II-immunoreactive fibers in the dorsal pallium of L. beddomei is a feature in common with a urodele amphibian, the total absence of cGnRH-II-like material in the median eminence is unique to this species. It is suggested here that the distribution profile of GnRH-like material within the brain and terminal nerve of I. beddomei represents a primitive pattern.

Sex and reproductive status related brain content of mammalian and chicken-II GnRHs in Rana esculenta.

Mammalian and chicken-II forms of gonadotropin-releasing hormone (mGnRH and cGnRH-II, respectively) have been measured simultaneously in the brain, pituitary, and peripheral terminal nerves (nasal area tissue) of adult males and females of a representative amphibian, Rana esculenta, during the annual reproductive cycle. Whereas in the male, brain concentrations of both GnRH forms showed significant reproductive status-related fluctuations, in the female brain only cGnRH-II content showed significant changes. The highest GnRH levels were recorded just prior to breeding in both sexes. In the pituitary both GnRHs were present in all seasons. In the peripheral terminal nerves, instead, only mGnRH was detected in all seasons confirming our previous immunohistochemical data. In both sexes furthermore, the brain and pituitary mGnRH levels were consistently much higher than those of cGnRH-II and there were no sex-related differences in the brain and pituitary content of GnRHs. Seasonal changes in brain GnRH levels may correlate with plasma sex steroid levels reinforcing the postulate that sex steroids affect GnRH neuronal systems.

Immunohistochemical demonstration of FSH and LH in the pituitary of the developing frog, Rana esculenta.

The ontogenetic pattern of immunohistochemically detectable FSH beta and LH beta cells was investigated in the pars distalis of the pituitary of the frog, Rana esculenta. The appearance, distribution, and percentage of these cells were examined in tadpoles from soon after hatching to the end of metamorphosis and in juveniles. We used monoclonal antibodies against bullfrog FSH beta and LH beta for single staining, and either mouse anti-bullfrog LH beta + guinea pig anti-rat FSH beta or rabbit polyclonal anti-bullfrog LH beta + mouse monoclonal anti-bullfrog FSH beta for double staining. The first appearance of gonadotropes, immunopositive for FSH beta, was revealed in stage 26 tadpoles. In successive stages of development the percentage of FSH beta-positive cells increased progressively and significantly. The mean percentage of these in the pars distalis cells increased from 0.7% in stage 26 to nearly 10% during the metamorphic climax (stages 31-33). In juveniles, the mean percentage of FSH beta-positive cells increased more than twofold compared to the climax value. The appearance of LH beta-positive cells was first recorded during the climax, and the mean percentage of LH beta-positive cells in juveniles reached levels as high as 30% or more, exceeding the number of FSH beta-positive cells. In climax, all LH beta-positive cells stained with anti-FSH beta as well. In juveniles, however, up to 80% of gonadotropes demonstrated colocalization of FSH beta and LH beta staining. We argue that both gonadotropins may be synthesized in all gonadotropes, and a small number of cells immunoreactive to either of the two gonadotropins may simply indicate that at that particular moment the cell contained detectable amounts of only one form of gonadotropin. These observations are discussed in relation to the possible involvement of hypothalamic influence in the differentiation of gonadotropes of the pituitary.

Reproduction in the Mexican leaf frog, Pachymedusa dacnicolor. VI. Presence and distribution of multiple GnRH forms in the brain.

The presence and distribution of gonadotropin-releasing hormone (GnRH) has been investigated in the Mexican leaf frog, Pachymedusa dacnicolor, brain during development and in the adult. The ontogenetic pattern of GnRH neurons illustrates their extracranial as well as intracranial sites. Immunohistochemical analysis indicates that GnRH-immunoreactive neurons appear during the metamorphic climax. They are located in the mesencephalon and subsequently other GnRH neurons appear in the peripheral terminal nerve and anterior preoptic area of the brain. Use of specific antisera and homologous combined with heterologous preabsorption tests indicate that mammalian and chicken GnRH-II-like peptide-containing neurons are differentially located within the brain, the former in the anterior preoptic area and peripheral terminal nerve and the latter in the mesencephalon. HPLC and RIA data suggest the presence of three forms of immunoreactive GnRH in the P. dacnicolor brain. A mammalian GnRH-like molecule and a chicken GnRH-II-like molecule are present. A third form, suspected to be [hydroxyproline9]mGnRH elutes before the mammalian GnRH.

Immunoreactive mammalian and chicken-II GnRHs in Rana esculenta brain during development.

Two forms of gonadotropin-releasing hormone (mammalian, mGnRH and chicken-II, cGnRH-II) were measured by radioimmunoassay in the nasal area (containing peripheral terminal nerve), brain and pituitary of Rana esculenta during larval development, metamorphosis, and until prior to becoming reproductively active. Small amounts of both forms of GnRH were first detected in the brain extract of early tadpoles (stage 26-27, when hindlimbs begin to develop). Later, there was a gradual, but constant, stage-dependent increase in the brain content of GnRHs, with the most remarkable increase recorded at postclimax and in young frogs. In tadpoles, postclimax froglets, and young frogs, the brain concentration of mGnRH was higher than that of cGnRH-II, with a ratio of approximately 2:1 in favor of mGnRH. In juveniles, however, the brain extract contained more cGnRH-II than mGnRH. No GnRH immunoreactivity was detected in the nasal area until stage 31. In successive stages of development, however, only mGnRH was present in the nasal area, and this confirmed our previous immunohistochemical analysis which showed that the peripheral terminal nerve contains only mGnRH-immunoreactive neurons and fibers. Although both GnRH forms were detected in the anterior (telencephalon, diencephalon) and posterior (mesencephalon, rhombencephalon) brain halves from juveniles, mGnRH content predominated in the anterior half, whereas in the posterior half cGnRH-II was present in greater amounts. Pituitaries from male and female postclimax froglets and young frogs contained both forms of GnRH in a ratio of approximately 10:1 in favor of mGnRH. This finding may shed light on the question of which GnRH(s) regulate gonadotropin release from the pituitary. The developmental changes in GnRH-immunoreactive content of the brain and pituitary have been discussed in the light of functional maturation of the brain-pituitary-gonad axis.

Development and distribution of gonadotropin-releasing hormone neuronal systems in the frog (Rana esculenta) brain: immunohistochemical analysis.

The ontogenesis of the gonadotropin-releasing hormone (GnRH) neuronal systems was studied in the brain of the frog, Rana esculenta. Attention was also focussed on the differential distribution of molecular forms of GnRH during development. The first GnRH-immunoreactive neurons appear in the mesencephalon of posterior limb-stage tadpoles. These neurons are shown to contain only chicken [His5,Trp7,Tyr8]GnRH (cGnRH-II). Later in development, mammalian [Tyr5,Leu7,Arg8] GnRH (mGnRH)-like peptide-containing neurons appear simultaneously in the terminal nerve as well as in the anterior preoptic area of the telencephalon. Subsequently, only after metamorphosis, mGnRH-containing neurons appear in the medial septal area of the posterior telencephalon. It is here shown that neurons containing the two forms of GnRH are distributed in distinct brain areas during development and in the adult: mGnRH-immunoreactive neurons in the terminal nerve, olfactory bulb, mediobasal telencephalon, medial septal area, anterior preoptic area, ventrolateral thalamus and infundibulum, whereas cGnRH-II neurons are located in the mesencephalon. We hypothesize that the terminal nerve/forebrain-located GnRH neurons express immunohistochemically late in development and originate extracranially migrating centrally, along the terminal nerve, during development, whereas those located in the mesencephalon express earlier and may have an intracranial site of origin.

Neuroanatomical organization of GnRH neuronal systems in the lizard (Podarcis s. sicula) brain during development.

The ontogenesis of the GnRH neuronal systems was studied in the brain of the lizard, Podarcis s. sicula, by immunohistochemistry. The first GnRH neurons were seen in the mesencephalon on the 45th day of incubation. One week later GnRH-ir neurons appeared in the infundibulum as well. These neurons never appeared to be contiguous with midbrain GnRH neurons. Thus, the adult pattern of distribution of GnRH neurons was reached before hatching, which occurred on the 66th day of incubation at a temperature of 28 +/- 2 degrees C. Although mesencephalic and infundibular GnRH neurons and their fiber projections appeared to be distributed in anatomically distinct brain areas, both systems showed a positive reaction to chicken-I GnRH (cGnRH-I), chicken-II GnRH (cGnRH-II) and salmon GnRH (sGnRH). From the time of hatching, GnRH-ir fibers in the mesencephalon appeared to be reaching the optic tectum, tegmentum, cerebellum and rostral dorsal rhombencephalon, whereas GnRH fibers in the infundibulum were projecting to the caudal basal telencephalon, median eminence and rostral basal rhombencephalon. In 60-day-old juvenile lizards, the central area of telencephalon contained neurons reacting only with anti-cGnRH-I and anti-sGnRH. Such neurons were absent in the adult. Neither GnRH cells nor fibers were observed in the nasal area, terminal nerve and olfactory bulbs at any stage of development and in the adult. We hypothesize that the two GnRH neuronal systems have separate embryonic origins.

Influence of gonadectomy and steroid hormone replacement therapy on the gonadotropin-releasing hormone neuronal system in the anterior preoptic area of the frog (Rana esculenta) brain.

Effects of gonadectomy and sex steroid replacement therapy were studied on the anterior preoptic area-located GnRH-immunoreactive neurons in males and females of the frog, Rana esculenta. Removal of gonads caused a drastic reduction in the immunostaining as well as in the number of GnRH neurons in the anterior preoptic area of the brain in both sexes. Replacement therapy with estradiol-17 beta, testosterone, and 5 alpha-dihydrotestosterone, given alone or in combination, enhanced the somal accumulation of immunoreactive material in GnRH neurons and also their number. These effects, however, varied to some extent from steroid to steroid, a combination of testosterone and 5 alpha-dihydrotestosterone being most effective in gonadectomized males, whereas that of estradiol-17 beta and testosterone was most effective in gonadectomized females. The present data are the first to demonstrate that androgen and estrogen can influence the immunoreactive GnRH neurons in the anterior preoptic area of the amphibian brain.

Immunohistochemical localization of GnRH in the crested newt (Triturus carnifex) brain and terminal nerve: a developmental study.

Localization of GnRH-immunoreactive neuronal system was studied by immunohistochemistry in the nasal-brain area of the crested newt, Triturus carnifex. Besides adults, developmental stages were those from hatchlings up to complete metamorphosis. Neurons containing immunoreactive GnRH were first detected in the nasal area of larvae with yet undifferentiated gonads. Subsequently, in prometamorphic stages, GnRH-immunoreactive cell bodies and fibers were detected in the proximal part of the terminal nerve as well as along the ventromedial surface of the olfactory bulbs. In older larvae with sexually differentiated gonads and up to the metamorphic climax GnRH-neurons were detected, as a rostral to caudal continuum, along the ventromedial surface of the olfactory bulbs and midtelencephalon. This is exactly the route followed by the terminal nerve. In the adult brain, besides the presence of occasional GnRH-neurons and fibers in the terminal nerve proximal to olfactory bulbs, olfactory bulbs and the mid-basal telencephalon, another aggregate of immunoreactive neurons was present in the anterior preoptic area, and a greater number of fibers in the habenular area as well as in the infundibular floor, median eminence and pars nervosa. These data suggest the nasal area to forebrain migration (along the course of the terminal nerve) of GnRH-neurons during development in the crested newt.

Immunohistochemical demonstration of the presence and localization of diverse molecular forms of gonadotropin-releasing hormone in the lizard (Podarcis s. sicula) brain.

The immunohistochemical presence and the distribution pattern of four different molecular forms of gonadotropin-releasing hormone (GnRH) were investigated in the brain of both sexes of the lizard, Podarcis s. sicula. Animals used in this study were collected in November and April, representing two different periods of the reproductive cycle. The antisera used were those raised against synthetic mammalian GnRH, chicken GnRH-I and II, and salmon GnRH. Strong immunoreaction was obtained for salmon, chicken-I, and chicken-II GnRHs, whereas a very weak reaction was seen for the mammalian form of GnRH. The distribution of immunoreactive-GnRH perikarya and fibers did not vary with the sex, the reproductive condition of the animals, or the antiserum used. Also, the intensity of immunoreaction with any one antiserum was quite similar in both periods of the year and in all brains examined. The immunoreactive perikarya was seen as two distinct groups, one in the mesencephalon and the other in the infundibulum. Immunoreactive fiber endings were seen in the telencephalon, the optic tectum, the anterior preoptic area, the median eminence, the central grey matter, the rhombencephalon, and the cerebellum. No immunoreactive perikarya were seen in the telencephalon or the anterior preoptic area.

[Echographic profile of senile disciform macular degeneration and problems of differential diagnosis]. / Profilo ecografico della degenerazione disciforme senile della macula e problemi di diagnosi differenziale.

Authors describe the echographic characteristics A and B-scan of the disciform macular degeneration. They also examine the problems of an echographic differential diagnosis between the upper described pathological pattern and the melanoma of small dimensions growing from the posterior pole. With the B-scan, both pathological patterns show a zone of echoes which is of solid aspect, not so much raised from the retina. The only characteristic element is the choroideal excavation that we always have in the choroideal melanoma and never in the disciform macular degeneration. Even the A-scan is useful mostly if we reduce the amplification level we have a separation of the internal echoes better in the disciform macular degeneration than in the melanoma. Moreover in some cases when the pathological area is small, we cannot have an echographic differential diagnosis in order to recognize the lesion.