phoenixin(smim20), a gene coding for a novel reproductive ligand,is expressed in the brain of adult zebrafish

Gonadotropin-releasing hormone (GnRH) is a highly conserved neuroendocrine decapeptide that is essential for the onset of puberty and the maintenance of the reproductive state. In addition to its role as hypothalamic releasing hormone, GnRH has multiple functions including modulator of neural activity within the nervous system and of resulting behaviors. These multiple functions are reflected by the existence of multiple isoforms. Despite its importance as a critical hypothalamic releasing hormone, the gnrh1 gene has been lost in zebrafish, and its reproductive function is not compensated for by other GnRH isoforms (GnRH2 and GnRH3), suggesting that, surprisingly, zebrafish do not use any of the GnRH peptides to control reproduction and fertility. Previously we proposed that Phoenixin/SMIM20, a novel peptide identified in mammals and the ligand for the orphan GPR173, is a potential candidate to control the initiation of sexual development and fertility in the zebrafish. Here we confirm the sequence of the zebrafish phoenixin/smim20 gene and by RT-PCR show that it is expressed early in development through adulthood. Subsequently we show that phoenixin/smim20 is expressed in the adult brain including the regions of the hypothalamus important in the control of fertility and reproduction.     

Design and synthesis of a gonadotropin-releasing hormone (GnRH) analogue, [Tyr(OMe)5,d-Glu6,Aze9]GnRH: Receptor binding, gonadotropin release and ovulation studies

Summary Gonadotropin-releasing hormone (GnRH) stimulates the release and synthesis of gonadotropin hormones (GtH) and is the key regulator of reproduction. The present study was carried out to design a potent GnRH analogue containing Tyr(OMe) at position 5 and ad-amino acid at position 6. This was based on a previous study in which [Tyr(OMe)⁵]GnRH was shown to have reduced potency compared to GnRH. A novel GnRH peptide containing Tyr(OMe)⁵ andd-Glu⁶ in combination with other substitutions at positions 9 and 10 was synthesized in the present study and tested for binding to the rat pituitary as well as potency in terms of gonadotropin (GtH) release in the goldfish pituitary and ovulation in sea bass. The results demonstrate that the replacement of the glycine residue at position 6 with ad-Glu in combination with the substitution of proline at position 9 with azetidine (Aze) increased the binding and biological activity of [Tyr(OMe)⁵]GnRH. The observed increased potency is likely to be related to the improved resistance to degradation. The present findings may lead to the development of a more potent GnRH agonist for inducing ovulation in fish.     

Synthesis and in vitro evaluation of small-molecule [18F] labeled gonadotropin-releasing hormone (GnRH) receptor antagonists as potential PET imaging agents for GnRH receptor expression

Two novel small molecule gonadotropin-releasing hormone (GnRH) receptor antagonists (12 and 13) of the furamide-class were synthesized and evaluated in vitro for their receptor binding affinities for the rat GnRH receptor. Radiolabeling with no carrier added fluorine-18 of the appropriate precursors was investigated in a one-step reaction. Log P (Octanol/PBS pH 7.4) and serum stability of the compounds were investigated. The antagonists showed low nM affinity for the rat GnRH receptor. ¹⁸F-radiolabled compounds were obtained in high radiochemical purity (>95%) and specific activity (>75 GBq/μmol). These findings suggest this class of compounds holds promise as potential probes for PET targeting of GnRH-receptor expression.     

Distribution of salmon gonadotrophin releasing-hormone in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Summary The distribution of salmon gonadotrophin-releasing hormone (sGnRH) was studied in the brain and pituitary of two-year-old immature sea bass (Dicentrarchus labrax) by means of an enzymoimmunoassay (EIA) for sGnRH and immunocytochemistry. The EIA for sGnRH is a competitive assay using a tracer made of sGnRH coupled to acetylcholinesterase from an electric eel. The separation of free and bound tracer is achieved by coating the plates with mouse anti-rabbit IgG monoclonal antibodies. Displacement curves generated by sGnRH and extracts from pituitary and different brain regions showed a good parallelism allowing the assay to be used for sGnRH measurements in this species. Although all parts of the brain contained measurable levels of sGnRH, the highest concentrations were found in the pituitary, the olfactory bulbs and the telencephalon. These data were confirmed by immunocytochemistry. Cell bodies were found in the olfactory bulbs, ventral telencephalon, preoptic region and mediobasal hypothalamus. Immunoreactive fibers could be observed in all parts of the brain including the optic tectum, the cerebellum (corpus and valvula), the vagal lobe, the medulla oblongata and the rostral spinal cord. In most cases, these fibers do not form well defined bundles; however, there was clearly a continuum of immunoreactive fibers, extending from the olfactory bulbs to the pituitary, and along which all the cell bodies described above were located. In the ventral telencephalon and the preoptic region, clear pictures of varicose positive fibers contacting immunoreactive perikarya could be observed. These data indicate that sGnRH is most likely an endogenous peptide in the brain of the sea bass, although the presence of other forms of GnRH cannot be excluded at this point. This study also demonstrates that the general organization of the GnRH systems in the sea bass is highly similar to what has been described in most freshwater teleost species, and provides basis for further studies on the neuroendocrine control of gonadotrophin release in this commercially important species.     

Cloning, expression, and purification of a highly immunogenic recombinant gonadotropin-releasing hormone (GnRH) chimeric peptide

To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3-hinge-MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3-hinge-MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE-Sephacel chromatography. The purified GnRH3-hinge-MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3-hinge-MVP could be considered as a candidate anti-GnRH vaccine.     

In vitro and in vivo effects of GABA, muscimol, and bicuculline on lamprey GnRH concentration in the brain of the sea lamprey (Petromyzon marinus)

gamma-Aminobutyric acid (GABA) is a neurotransmitter with a demonstrated neuroregulatory role in reproduction in most representative species of vertebrate classes via the hypothalamus. The role of GABA on the hypothalamus-pituitary axis in lampreys has not been fully elucidated. Recent immunocytochemical and in situ hybridization studies suggest that there may be a neuroregulatory role of GABA on the gonadotropin-releasing hormone (GnRH) system in lampreys. To assess possible GABA-GnRH interactions, the effects of GABA and its analogs on lamprey GnRH in vitro and in vivo were studied in adult female sea lampreys (Petromyzon marinus). In vitro perfusion of GABA and its analogs at increasing concentrations (0.1-100 microM) was performed over a 3-h time course. There was a substantial increase of GnRH-I and GnRH-III following treatment of muscimol at 100 microM. In in vivo studies, GABA or muscimol injected at 200 microg/kg significantly increased lamprey GnRH concentration in the brain 0.5 h after treatment compared to controls in female sea lampreys. No significant change in lamprey GnRH-I or GnRH-III was observed following treatment with bicuculline. These data provide novel physiological data supporting the hypothesis that GABA may influence GnRH in the brain of sea lamprey.     

Immunocytochemical characterization of adenohypophyseal cells in the greater weever fish (Trachinus draco)

The adenohypophysis of the greater weever fish (Trachinus draco) was studied using histochemical and immunocytochemical methods. The adenohypophysis comprised the rostral pars distalis (RPD), the proximal pars distalis (PPD), and the pars intermedia (PI). Neurohypophysis showed a patent hypophyseal stalk which was divided into several branches intermingled with the adenohypophysis. Salmon prolactin (PRL)-immunoreactive (ir) cells, arranged in follicles, resided in the RPD and the most rostral part of the ventral PPD. Human adrenocorticotropin (ACTH)-ir cells were located in the RPD between PRL-ir cells and the neurohypophyseal processes. Salmon and seabream somatotropin (GH)-ir cells were located in both the dorsal and the ventral PPD. Some GH-ir cells were seen in surrounding and in contact with neurohypophyseal branches, whereas other isolated or clustered GH-ir cells were embedded in adenohypophyseal cells of the PPD. In addition, isolated or clustered GH-ir cells were also detected in the tissue of the PPD covering the most rostral part of PI. Only one class of salmon and carp gonadotropin (GTH)-ir cells was detected. Isolated or clustered GTH-ir cells resided in both the dorsal and the ventral PPD and were seen surrounding the PI and in the tissue of the PPD covering the most rostral part of PI. In addition, a few scattered GTH-ir cells were observed in the ventral RPD. Scattered groups of thyrotropin (TSH)-ir cells were present in the anteroventral PPD. Salmon and seabream somatolactin (SL)-ir and bovine melanotropin (MSH)-ir cells were intermingled surrounding the neurohypophyseal tissue. SL-ir cells were negative to periodic acid-Schiff technique. MSH-ir cells showed a very weak immunoreactivity to anti-human ACTH((1-24)) serum. In addition to the PI location, few isolated or clustered SL- and MSH-ir cells were observed in the dorsal PPD.     

Reversible,long-term inhibition of ovulation with a gonadotropin-releasing hormone antagonist in the marmoset monkey (Callithrix jacchus)

The effects of weekly injections of a gonadotropin-releasing hormone (GnRH) antagonist (GnRHa) ([N-acetyl-DβNal1-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8D-Ala10] NH2 GnRH) on pituitary and ovarian function were examined in the marmoset monkey, Callithrix jacchus. In experiment 1, five cyclic females were given weekly injections of vehicle (50% propylene glycol in saline) for 6 weeks followed by GnRHa for 20 weeks, animals receiving either 200 μg GnRHa/injection (n = 2) or 67 μg GnRHa/injection (n = 3) for 10 weeks, after which the treatment was reversed. Bioactive luteinizing hormone (LH) and progesterone (Po) were measured in blood samples (0.2–0.4 ml) collected twice weekly until at least 8 weeks after the last GnRHa injection. GnRHa treatment, timed to begin in the midluteal phase, caused a rapid decline in LH and Po and luteal regression after a single injection (both doses). Po levels were consistently low (<10 ng/ml), and ovulation was inhibited throughout 200 μg treatment in all animals. Short periods of elevated Po (>10 ng/ml) were, however, occasionally seen during 67 μg treatment, indicating incomplete ovarian suppression. Mean LH levels were significantly lower during GnRHa treatment compared with the period of vehicle injection (all animals 200 μg; three animals 67 μg), and there were significant differences in LH levels between GnRHa treatments (200 μg vs. 67 μg) in four animals. Four animals resumed normal ovarian cycles after the end of GnRHa treatment (15/16 days, three animals; 59 days, one animal); the fifth animal died of unknown causes 32 days after the last GnRHa injection. In a second experiment, pituitary responsiveness to exogenous GnRH was tested 1 day after a single injection of vehicle or antagonist (200 or 67 μg). Measurement of bioactive LH indicated that pituitary response to 200 ng native GnRH was significantly suppressed in animals receiving the antagonist, the degree of suppression being dose related. A third experiment examined the effect of four weekly injections of 200 μg GnRHa on follicular size and granulosa cell responsiveness to human follicle-stimulating hormone (hFSH) in vitro. Follicular development beyond 1 mm was inhibited by GnRHa treatment (preovulatory follicles normally 2-4 mm) although granulosa cell responsiveness to FSH during 48 hr of culture was not impaired. These results suggest that the GnRHa-induced suppression of follicular development and ovulation was mediated primarily by an inhibition of pituitary gonadotropin secretion and not by a direct action at the level of the ovary.     

Ultrastructure of the extracutaneous pigment cells in the plaice (Pleuronectes platessa,L., Teleostei)

Summary The extracutaneous pigment cell system of the plaice (Pleuronectes platessa L.) was examined by light and electron microscopy in selected regions, including two cutaneous regions for comparison. The extracutaneous pigmentation consists of guanocytes and melanocytes with differing distributions within the body. The eyeless side lacks melanocytes. The pigment cells are differentiated as very flat elements with long processes. They display an affinity for loose connective tissue at boundary layers such as the peritoneal epithelium, organ capsules or blood vessels, to which they are parallelly arranged at a very constant distance. In some locations guanocytes are intimately associated with melanocytes forming reduced chromatophore units. Extracutaneous pigment cells are poor in mitochondria, endoplasmic reticulum, microfilaments, caveolae intracellulares, ribosomes and glycogen granules, all of which are more abundant in cutaneous pigment cells and pigment cells of the eye. In extracutaneous guanocytes the crystals are loosely arranged parallel to the cell surface, in cutaneous guanocytes perpendicular. Cells with rod-like vesicular cisternae are described as guanoblasts. No single pigment cell was found exhibiting different types of pigment granules. The varying colors of extracutaneous pigmentation arise from varying combinations of guanocytes and melanocytes in addition to the color of the tissue itself.In partial fulfillment for the degree of Doctor of Medicine under the direction of Prof. Dr. Dr. H.-R. Duncker (Giessen)     

Growth and sexual strategies in the fishThalassoma duperrey (Labridae), a protogynous hermaphrodite

Synopsis Adults of the fishThalassoma duperrey, a protogynous hermaphrodite, were collected and growth observed in captivity to study the relationship between growth and reproduction among primary males, females, and secondary males. Sexual maturity is reached at about 60 mm standard length, probably less than 1 year after fertilization. Gonosomatic index in both males and females peaks at about 120 mm, nearly 2 years later. Shortly thereafter females typically change sex, and both primary and secondary males undergo color change. At the same time gonosomatic index falls abruptly and remains low in large fish. The above changes appear to reflect differences in reproductive effort over a lifetime and are interpreted as the optimum strategy given the social and mating system of this fish. Unless they cannot acquire enough food to develop large gonads, small individuals put a much greater proportion of energy into growth than reproduction apparently to minimize the period of low fitness. Intermediate-sized males and females generally invest heavily in gametes, though some retain small gonads. Large individuals (both primary and secondary males) greatly reduce their investment in gametes, probably trading the energy required to maintain reproductive territories for it. This kind of gonad ontogeny involving gonad regression, as inT. duperrey and other labrid fishes, is unique among vertebrates.     

Sex difference in the neurotensin-immunoreactive cell populations of the preoptic area in quail (Coturnix japonica)

The distribution of neurotensin-immunoreactive cells and fibers was analyzed by immunocytochemistry in the forebrain of male and female Japanese quail (Coturnix japonica) by using an antibody directed against the C-terminal part of the molecule. Immunoreactive perikarya were located almost exclusively in the medial preoptic area with small populations also being present in the nucleus paraventricularis and in the tuberal region. Immunoreactive fibers were observed not only throughout the preoptic area-hypothalamus, but also in the septal region, nucleus intercollicularis, substantia grisea centralis and the classical catecholaminergic areas of the mesencephalon, such as the area ventralis of Tsai and the nucleus tegmenti pedunculo-pontinus, pars compacta. The preoptic neurotensin-immunoreactive cells were exclusively located within the boundaries of the sexually dimorphic medial preoptic nucleus. They were significantly more numerous in females than in males. In females, the number of neurotensin cells varied during the ovulatory cycle: fewer cells were observed in birds that were about to lay an egg (they had a calcified egg in the oviduct) than in those that had already laid or were not going to lay on that day. These data indicate major variations in the expression of neurotensin in response to neurochemical or neuroendocrine changes associated with ovulation.     

Endocrine correlates of alternative phenotypes in the white-throated sparrow (Zonotrichia albicollis)

Many vertebrate species exhibit alternative phenotypes (or morphs), in which one sex displays phenotypic variation equal to or greater than the variation between the sexes. Males in such species typically display differences in reproductive strategies and morphology. Steroid hormones such as testosterone are known modulators of reproductive behavior and morphology and therefore are obvious candidates for the mediation of phenotypic differences between morphs. We conducted a year-round study in the white-throated sparrow (Zonotrichia albicollis) that exhibits alternative phenotypes in plumage coloration and behavior in both sexes: during the breeding season, white-striped males and females are more aggressive and have higher song rates than tan-striped individuals. At the beginning of the breeding season, free-living white-striped males had higher plasma testosterone concentrations than tan-striped males. However, this finding might have been due to different social experiences because captive male morphs sampled at similar times of year did not differ in testosterone concentrations. Captive white-striped males had larger testis and cloacal protuberance sizes than tan-striped males, which might be related to the divergent mating strategies of the morphs. Male morphs showed similar increases in luteinizing hormone following injections of gonadotropin-releasing hormone, but white-striped males showed larger increases in testosterone, indicating differences between morphs in gonadal testosterone production. Females had low concentrations of testosterone, and morphs did not differ. Plasma dehydroepiandrosterone (DHEA) concentrations were elevated in both sexes and morphs during the breeding and non-breeding seasons. These data do not support the hypothesis that testosterone activates behavioral differences between alternative phenotypes in the white-throated sparrow. Alternative testable hypotheses include hormonal effects during early development and direct genetic effects.     

Immunocytochemical identification of crustacean hyperglycemic hormone-producing cells in the brain of a freshwater fairy shrimp,Streptocephalus dichotomus Baird (Crustacea: Anostraca)

Localization of crustacean hyperglycemic hormone (CHH) activity in the brain ganglia of Streptocephalus dichotomus was demonstrated by immunocytochemical method. For this, two different rabbit antisera, one raised against a crayfish Orconectus limosus, and the other with a crab Carcinus maenas, were used. Positive immunoreactivity was recorded with the antibody raised against Orconectus limosus CHH. However, the antibody raised against the CHH of the crab, Carcinus maenas failed to show any cross-reactivity. The biological specificity of these peptides was further confirmed by immunoblotting and immunodiffusion studies performed with the partially purified CHH. The results obtained with the immunodot-blotting and immunodiffusion studies with CHH, further confirmed the immunocytochemical studies. Bioassay experiments performed with a freshwater prawn, Macrobrachium rosenbergii indicate the interspecific biological activity of this neuropeptide. The present study indicates that besides the sinus gland, there are other sites of CHH synthesis and release in the brain ganglia of S. dichotomus.     

Testicular interstitial cells, and steroidogenic detection in the protogynous fish, Synbranchus marmoratus (Teleostei, Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a freshwater protogynic diandric species. Primary males develop directly as males while secondary males arise from the sex reversal of females. Fishes from Argentine and Brazil inland waters were collected, examined and compared for this study. In order to characterize the interstitial testicular compartment, light and electron microscopy techniques and an enzyme histochemical examination for steroidogenic cells detection were used. The interstitial compartment of S. marmoratus is composed of Leydig and myoid cells, collagen fibers, blood cells, macrophages,and amyelinic nerves. At the ultrastructural level, no differences were observed in the interstitial tissue, either between specimens from the different sampling sites or between primary and secondary males. Leydig cells are present in all testes examined throughout the year. A cytoplasmatic reaction of 3beta-HSD was detected only in Leydig cells during sex reversal and in both type of males, mainly during the regressed and early maturation classes (autumn and winter). Leydig cells possess the typical fine structural characteristics associated with steroidogenesis. Furthermore, in both type of males, during sex reversal and after the spawning period, the number of granulocytes and macrophages present in the testes increased, suggesting that they could be involved in phagocytosis and resorption of damaged cells.     

Ultrastructure of endocrine cells in the stomach of two teleost fish Perca fluviatilis L. and Ameiurus nebulosus L.

Summary In the gastric mucosa of two teleost species, the perch (Perca fluviatilis) and the catfish (Ameiurus nebulosus) three endocrine cell types were found, located predominantly between the mucoid cells of the gastric mucosa. A fourth cell type is present in the gastric glands of catfish. Each cell type was defined by its characteristic secretory granules. Type-I cells were predominant in both fish. These cells contained round or oval granules with a pleomorphic core. The average diameter of granules was 400 nm for the perch and 270 nm for the catfish. Type-II cells of both species displayed small, highly osmiophilic granules about 100 nm in diameter. The secretory granules of type-III cells (260 nm in the perch and 190 nm in the catfish) were round or slightly oval in shape and were filled with a finely particulate electron-dense material. Type-IV cells of the catfish were found in the gastric glands only. Their cytoplasm was filled with homogeneous, moderately electron-dense granules averaging 340 nm in diameter. The physiological significance of these different morphological types of gastric endocrine cells requires further investigation.     

Plasma membrane structure in spermatogenic cells of the swordtail (teleostei,Xiphophorus helleri)

The plasma membrane of spermatogenic cells of the teleost Xiphophorus helleri was examined ultrastructurally and cytochemically in order to characterize the temporal development of the membrane specializations characteristic of the mature spermatozoon. Mature sperm display a mosaic distribution of Concanavalin A and Ricinus comrnunis I binding sites; the anterior region of the head displays an intense binding that is not seen in other surface regions. This asymmetric binding is evident in early spermatids and the area of lectin binding appears associated with the plasma membrane overlying the nucleus. Transmission electron microscopy reveals that the plasma membrane over the anterior region of the head is characterized by an ordered glycocalyx and a tight adherence to the underlying nucleus. Additional membrane differentiations were revealed both in the midpiece region where a “submitochondrial net” is attached to the plasma membrane and at the base of the axoneme where the plasma membrane possesses a “collar-like” arrangement of circumferential rings. The possible functions of these differentiations, as well as their correlation to differentiations seen in sperm of other animal groups, are discussed.     

Induction of PER1 mRNA expression in immortalized gonadotropes by gonadotropin-releasing hormone (GnRH): Involvement of protein kinase C and MAP kinase signaling

The initiation and maintenance of reproductive function in mammals is critically dependent on the pulsatile secretion of gonadotropin-releasing hormone (GnRH). This peptide drives the pulsatile release of FSH and LH from the pituitary pars distalis via signaling pathways that are activated by the type I GnRH receptor (GnRH-R). Recently, a microarray analysis study reported that a number of genes, including mPer1, are induced by GnRH in immortalized gonadotrope cells. In view of these data, we have begun to analyze in detail the signaling pathways mediating the action of GnRH on mPer1 expression in these cells. Using quantitative real-time polymprose cho read (PCR), we could confirm that exposure of immortalized gonadotropes (LβT2 cells) to the GnRH analog, buserelin, markedly induces mPer1 (but not mPer2) expression. Consistent with GnRH receptor signaling via the protein kinase (PK)-C pathway, exposure of the cells to phorbol 12,13-dibutyrate rapidly elevates both mPer1 and LHβ subunit mRNA levels, while pharmacological inhibition of PKC prevents the mPer1 and LHβ response to buserelin. As GnRH is known to regulate gonadotropin synthesis via activation of p42/44 mitogen-activated protein kinase (MAPK) signaling pathways, we then examined the involvement of this pathway in regulating mPer1 expression in gonadotropes. Our data reveal that GnRH-induced mPer1 expression is blocked following acute exposure to a MAPK kinase inhibitor. Although the involvement of this signaling mechanism in the regulation of mPer1 is known in neurons, e.g., in the suprachiasmatic nuclei, the induction of mPer1 in gonadotropes represents a novel mechanism of GnRH signaling, whose functional significance is still under investigation.