Brain gonadotropin releasing hormone3 expression variation during oogenesis and sexual behavior and its effect on pituitary hormonal expression in the blue gourami.

To gain a better insight regarding the roles of gonadotropin releasing hormone3 (GnRH3) in the regulation of reproduction in the suborder Labyrinthici, GnRH3 expression was investigated during the reproduction cycle of the male and female blue gourami (Trichogaster trichopterus). The full-length blue gourami brain GnRH3 gene was sequenced (EMBL acc. no. EU107388) and was found to be expressed in both the brain and pituitary of the blue gourami. High mRNA levels were detected only in the brain of females with oocytes in the maturation stage. Correspondingly, significantly greater mRNA levels of GnRH3 were detected in mature males than in immature ones. In primary cultures of dispersed pituitary cells, GnRH3 significantly increased betaFSH and betaLH subunit mRNA levels in cells from both females and males, whereas GH gene transcription was affected differently by GnRH3 in females, as compared to males. Thus, we propose that GnRH3 can act differentially in the blue gourami females and males. In females, GnRH3 may be involved in the final maturation stage of the oocyte and induces betaFSH, betaLH and GH gene expression; in males, it is engaged in sexual behavior and spermatogenesis regulation via betaFSH and betaLH stimulation and dowregulation of GH transcription.

Early development of forebrain gonadotrophin-releasing hormone (GnRH) neurones and the role of GnRH as an autocrine migration factor.

Normal migration of the gonadotrophin-releasing hormone (GnRH) neurones during early development, from the olfactory region to the hypothalamus, is crucial for reproductive development in all vertebrates. The establishment of the GnRH system includes tangential migration of GnRH perikarya as well as extension of GnRH fibres to various areas of the central nervous system (CNS). The exact spatio-temporal nature of this process, as well as the factors governing it, are not fully understood. We studied the development of the GnRH system and the effects of GnRH knockdown using a newly developed GnRH3:EGFP transgenic zebrafish line. We found that enhanced green fluorescent protein is specifically and robustly expressed in GnRH3 neurones and fibres. GnRH3 fibres in zebrafish began to extend as early as 26 h post-fertilisation and by 4-5 days post-fertilisation had developed into an extensive network reaching the optic tract, telencephalon, hypothalamus, midbrain tegmentum and hindbrain. GnRH3 fibres also innervated the retina and projected into the trunk via the spinal cord. GnRH3 perikarya were observed migrating along their own fibres from the olfactory region to the preoptic area (POA) via the terminal nerve ganglion and the ventral telencephalon. GnRH3 cells were also observed in the trigeminal ganglion. The establishment of the GnRH3 fibre network was disrupted by morpholino-modified antisense oligonucleotides directed against GnRH3 causing abnormal fibre development and pathfinding, as well as anomalous GnRH3 perikarya localisation. These findings support the hypothesis that GnRH3 neurones migrate from the olfactory region to the POA and caudal hypothalamus. Novel data regarding the early development of the GnRH3 fibre network in the CNS and beyond are described. Moreover we show, in vivo, that GnRH3 is an important factor regulating GnRH3 fibre pathfinding and neurone localisation in an autocrine fashion.

Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

Zebrafish arylalkylamine-N-acetyltransferase genes - targets for regulation of the circadian clock.

Daily rhythms of melatonin production are controlled by changes in the activity of arylalkylamine-N-acetyltransferase (AANAT). Zebrafish possess two aanats, aanat1 and aanat2; the former is expressed only in the retina and the latter is expressed in both the retina and the pineal gland. Here, their differential expression and regulation were studied using transcript quantification and transient and stable in vivo and in vitro transfection assays. In the pineal gland, the aanat2 promoter exhibited circadian clock-controlled activity, as indicated by circadian rhythms of Enhanced green fluorescent protein (EGFP) mRNA in AANAT2:EGFP transgenic fish. In vivo transient expression analyses of the aanat2 promoter indicated that E-box and photoreceptor conserved elements (PCE) are required for expression in the pineal gland. In the retina, the expression of both genes was characterized by a robust circadian rhythm of their transcript levels. In constant darkness, the rhythmic expression of retinal aanat2 persisted while the aanat1 rhythm disappeared; indicating that the former is controlled by a circadian clock and the latter is also light driven. In the light-entrainable clock-containing PAC-2 zebrafish cell line, both stably transfected aanat1 and aanat2 promoters exhibited a clock-controlled circadian rhythm, characteristic for an E-box-driven expression. Transient co-transfection experiments in NIH-3T3 cells revealed that the two, E-box- and PCE-containing, promoters are driven by the synergistic action of BMAL/CLOCK and orthehodenticle homeobox 5. This study has revealed a shared mechanism for the regulation of two related genes, yet describes their differential phases and photic responses which may be driven by other gene-specific regulatory mechanisms and tissue-specific transcription factor profiles.

Functional development of the zebrafish pineal gland: light-induced expression of period2 is required for onset of the circadian clock.

In zebrafish, the pineal gland is a photoreceptive organ that contains an intrinsic circadian oscillator and exhibits rhythmic arylalkylamine-N-acetyltransferase (zfaanat2) mRNA expression. In the present study, we investigated the role of light and of a clock gene, zperiod2 (zper2), in the development of this rhythm. Analysis of zfaanat2 mRNA expression in the pineal gland of 3-day-old zebrafish embryos after exposure to different photoperiodic regimes indicated that light is required for proper development of the circadian clock-controlled rhythmic expression of zfaanat2, and that a 1-h light pulse is sufficient to initiate this rhythm. Analysis of zper2 mRNA expression in zebrafish embryos exposed to different photoperiodic regimes indicated that zper2 expression is transiently up-regulated by light but is not regulated by the circadian oscillator. To establish the association between light-induced zper2 expression and light-induced clock-controlled zfaanat2 rhythm, zPer2 knock-down experiments were performed. The zfaanat2 mRNA rhythm, induced by a 1-h light pulse, was abolished in zPer2 knock-down embryos. These experiments indicated that light-induced zper2 expression is crucial for establishment of the clock-controlled zfaanat2 rhythm in the zebrafish pineal gland.

Genetic, temporal and developmental differences between melatonin rhythm generating systems in the teleost fish pineal organ and retina.

Complete melatonin rhythm generating systems, including photodetector, circadian clock and melatonin synthesis machinery, are located within individual photoreceptor cells in two sites in Teleost fish: the pineal organ and retina. In both, light regulates daily variations in melatonin secretion by controlling the activity of arylalkylamine N-acetyltransferase (AANAT). However, in each species examined to date, marked differences exist between the two organs which may involve the genes encoding the photopigments, genes encoding AANAT, the times of day at which AANAT activity and melatonin production peak and the developmental schedule. We review the fish pineal and retinal melatonin rhythm generating systems and consider the evolutional pressures and other factors which led to these differences.

Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function.

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish larvae indicated that zfAANAT-2 mRNA abundance exhibits a dramatic 24-h rhythm in a 14-h light, 10-h dark cycle, with high levels at night. This rhythm persists in constant darkness, indicating that the zfAANAT-2 mRNA rhythm is driven by a circadian clock at this stage. The techniques described in this report were also used to determine that zfAANAT-2 expression is altered in two well characterized genetic mutants, mindbomb and floating head. The observations described here suggest that zfAANAT-2 mRNA may be a useful marker to study development of the pineal gland and of circadian clock mechanisms in zebrafish.

Levels of the native forms of GnRH in the pituitary of the gilthead seabream, Sparus aurata, at several characteristic stages of the gonadal cycle.

Brains of the gilthead seabream, Sparus aurata, contain three different forms of gonadotropin-releasing hormone (GnRH): seabream (sb) GnRH, chicken (c) GnRH-II, and salmon (s) GnRH. In the present study, we developed three specific enzyme-linked-immunosorbent assays (ELISA) for sbGnRH, cGnRH-II, and sGnRH and used them to measure the levels of each GnRH form in the pituitary of male and female seabream at different stages of gametogenesis. The sensitivity was 6 pg/well for the sbGnRH assay, 7 pg/well for the cGnRH-II assay, and 2 pg/well for the sGnRH assay. Levels of each of the three GnRH forms were measured in pituitaries from fish sampled at the beginning of gonadal recrudescence and during the spawning season. Of the three forms, only sbGnRH and cGnRH-II were detected in the pituitary, irrespective of reproductive state or sex. Recrudescent fish had similar levels of sbGnRH and cGnRH-II in the pituitary. In sexually mature fish, the levels of sbGnRH were higher than those in recrudescent fish while pituitary cGnRH-II content remained unchanged. Consequently, sbGnRH levels were 3- to 17-fold higher than cGnRH-II levels in mature fish. Positive correlations also existed between pituitary sbGnRH content and pituitary and plasma gonadotropin (GtH) II levels. Surprisingly, mature 1-year-old males had significantly higher levels of sbGnRH in the pituitary than mature 3-year-old males, while pituitary and plasma GtH II levels were similar between these two groups. Although the reason for this difference in sbGnRH levels is unclear, a possible role of sbGnRH in the processes of puberty or sex-inversion is implied. Based on the present results, it can be suggested that in the gilthead seabream, sbGnRH is the most relevant form of GnRH in the control of reproduction.

Multiple GnRHs present in a teleost species are encoded by separate genes: analysis of the sbGnRH and cGnRH-II genes from the striped bass, Morone saxatilis.

GnRH is a neuropeptide which plays an essential role in the control of reproductive fitness for all vertebrates. Increasing evidence suggests that multiple forms of GnRH may exist in most vertebrate brains. Southern blot analysis of the three GnRHs known to be present in perciform fish, the seabream (sb)GnRH, the salmon(s) GnRH and the chicken (c) GnRH-II, demonstrates that each is present as a single gene copy in the genome of the striped bass, Morone saxatilis. In order to investigate the physiological consequences of multiple GnRHs in a single vertebrate species, we have isolated and characterized two of the GnRH genes, those for sbGnRH and cGnRH-II. Computer analysis of 3.5 kb of sequence upstream of the sbGnRH gene reveals a number of consensus DNA binding sites which implicate steroids, such as estrogen and glucocorticoids, and the steroidogenic transcription factor, SF-1, as being involved in the regulation of sbGnRH gene expression. Sequence analysis of the cGnRH-II gene reveals evidence of multiple promoters. Expression studies using (1) solution hybridization-RNAse protection mapping with several RNA probes directed at various regions of the proGnRH gene, (2) primer extension assays using two specific oligonucleotide primers, and (3) reverse transcription PCR with several oligonucleotide primers on cGnRH-II transcripts demonstrate that the cGnRH-II gene initiates transcription at numerous sites using a TATA-less promoter within the brains of sexually mature striped bass. This study is the first to characterize and compare the promoter structures of two GnRH genes present in a single vertebrate species.

Gonadotropin-I and -II subunit gene expression of male striped bass (Morone saxatilis) after gonadotropin-releasing hormone analogue injection: quantitation using an optimized ribonuclease protection assay.

In fish, both gonadotropin (GtH)-I and -II are involved in the spermatogenic process, but the differential regulation of these hormones by GnRH is still poorly understood. To gain further insight into the GnRH regulation of GtH-I and -II gene expression in the male striped bass, we have developed and optimized a ribonuclease protection assay for the simultaneous measurement of all GtH subunit mRNAs in a single pituitary gland. The RNA extraction protocol enables the determination of GtH protein content in the same sample, thus enhancing the power of the method. Maturing striped bass males were injected intramuscularly with [D-Ala6,Pro9Net]-LHRH (GnRHa) and sampled at 6 and 24 h postinjection. The mRNA levels of the alpha subunit and GtH-IIbeta increased after 6 h (4- and 6-fold, respectively), while the GtH-Ibeta mRNA levels increased only 2-fold after 24 h. Interestingly, GnRHa stimulation caused a significant increase in beta-actin mRNA levels. GnRHa treatment also resulted in a 2-fold decrease in pituitary GtH-II content, associated with a dramatic increase of plasma GtH-II levels from undetectable levels (< 0.2 ng/ml) to 13+/-2 ng/ml after 6 h. These results demonstrate that both GtH-Ibeta and -Ilbeta are expressed during striped bass spermatogenesis and that the two genes are subjected to differential regulation by GnRHa.

Preovulatory changes in the levels of three gonadotropin-releasing hormone-encoding messenger ribonucleic acids (mRNAs), gonadotropin beta-subunit mRNAs, plasma gonadotropin, and steroids in the female gilthead seabream, Sparus aurata.

Gilthead seabream females undergo daily cycles of final oocyte maturation (FOM), ovulation, and spawning throughout their spawning season. FOM consists of lipid droplet and yolk granule coalescence, germinal vesicle (GV) migration, and GV breakdown. Plasma maturational gonadotropin (GtH-II) levels fluctuate throughout the day, reaching a peak at 8 h before spawning, when the GV is at the periphery of the oocyte. The preovulatory GtH-II surge is accompanied by an increase in the plasma levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one and estradiol, while testosterone and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one levels remain unchanged. Concurrent with the preovulatory GtH-II surge, there is an increase in pituitary GtH-II beta subunit mRNA levels followed by an increase in GtH-Ibeta mRNA levels. Gilthead seabream brain contains three different forms of GnRH: salmon (s)GnRH, seabream (sb)GnRH, and chicken (c)GnRH-II. All three GnRH-encoding mRNAs fluctuate throughout the day, reaching highest levels 8 h before spawning, concurrent with the preovulatory GtH-II surge. On the basis of these correlations and of the anatomical organization of the three GnRH systems, it is hypothesized that in the daily-spawning gilthead seabream females, preovulatory GtH-II secretion, and probably synthesis, are induced by a surge of sbGnRH secretion. The involvement of the other two GnRH forms, sGnRH and cGnRH-II, in the control of ovulation and spawning is presumed, on the basis of the elevation of their mRNA levels at the time of the preovulatory GtH-II secretion and spawning.

Three forms of gonadotropin-releasing hormone in a perciform fish (Sparus aurata): complementary deoxyribonucleic acid characterization and brain localization.

Three forms of GnRH-salmon (sGnRH), seabream (sb-GnRH), and chicken (cGnRH-II)-have been described in the gilthead seabream (Sparus aurata) brain, and the cDNA encoding the sbGnRH precursor was recently isolated. In the present study, the cDNAs encoding the sGnRH and cGnRH-II were isolated and characterized, and the neurons producing the three GnRHs were localized in the seabream brain. Fragments of sGnRH and cGnRH-II cDNAs were amplified by polymerase chain reaction and used as probes to isolate the full-length cDNAs from a brain cDNA library. The cDNA encoding the cGnRH-II precursor is 573 nucleotides (nt) long, and the cDNA encoding the sGnRH precursor is 1971 nt in length with an unusually long 5' untranslated region. Specific single-strand DNA probes for in situ detection of mRNA were designed according to nonconserved regions among the three GnRH c-DNAs. Localization of GnRH mRNA-producing cells in the brain revealed five distinct populations of cells: sGnRH-producing cells in the ventromedial olfactory bulbs and the terminal nerve, sbGnRH-producing cells in the preoptic area and the ventral thalamus, and cGnRH-II-producing cells in the midbrain tegmentum. The discrete sites of expression of the three forms of GnRH indicate that only sbGnRH is directly involved in the control of gonadotropin secretion.

Molecular cloning and characterization of a novel gonadotropin-releasing hormone from the gilthead seabream (Sparus aurata).

A novel form of gonadotropin-releasing hormone (GnRH) had been recently isolated and characterized from the gilthead seabream (Sparus aurata). This novel form, designated sbGnRH, was suggested to be the endogenous stimulator of gonadotropin release. This paper reports on the isolation and characterization of a 360-bp cDNA encoding the complete sbGnRH precursor. This precursor is composed of a 25 amino acid leader sequence, the biologically active sbGnRH, the cleavage site (Gly-Lys-Arg), and a 57 amino acid associated peptide (GAP). Comparison of the nucleotide and amino acid sequence of the sbGnRH precursor with precursors of other GnRH forms places the sbGnRH precursor evolutionarily closer to the mammalian GnRH and chicken GnRH-I, which have also been shown to be the relevant forms for gonadotropin release in mammals and birds, respectively. The characterization of the sbGnRH precursor cDNA lays the foundation for future studies aimed toward understanding the neuroendocrine control of reproduction in seabream and other fish species.

Long-term interactions between endogenous and exogenous opiates.

The in vitro biological effect of various opiates was studied in the guinea pig ileum bioassay. Besides their direct, immediate effect, certain opiates induced sensitivity changes which persisted after their removal and the complete recovery of the preparation. These specific, asymmetrical interactions may represent long-term modulation of the opiate receptor. The in vivo pharmacological implication of opiate interaction is discussed.

Characterization of humoral endorphin.

Utilizing a radioimmunoassay (RIA) with antibodies produced against leu-enkephalin, the presence of humoral endorphin in various body fluids and tissues has been shown. Pretreatment with trichloroacetic acid (TCA) was needed in all cases in order to detect the immunoreactivity. However, both treated and untreated samples of humoral endorphin were active in the opiate receptor assay. Gel filtration on Bio-Gel P-2 as well as on Sephadex G-10 columns of human cerebrospinal fluid (CSF), amniotic fluid (AF) and blood shows that they all contain a similar material with an apparent molecular weight of 1,000-1,400 daltons. Chromatography of rat brain homogenate exhibited two peaks of immunoreactivity, one of which is probably due to enkephalins and the other to humoral endorphin. The latter fraction was found to be very stable when incubated in CSF, while its degradation in blood was slightly faster. This opioid compound inhibited the electrically stimulated contractions of the guinea pig ileum; the specificity of this action was indicated by its reversal with low concentrations of naloxone. In pregnant women, humoral endorphin levels in maternal and cord blood remains stable during pregnancy, while there is a significantly higher concentration of humoral endorphin in the amniotic fluid at mid-trimester as compared to that in term pregnancies during labor.

Humoral endorphin: can in vitro experiments explain in vivo results?

Humoral endorphin, an endogenous substance isolated from brain, blood and cerebrospinal fluid, reveals non-conventional interactions with both opiate agonist (morphine) and antagonist (naloxone) in the guinea pig ileum bioassay. The opioid activity of humoral endorphin is potentiated by pretreatment of the preparation with morphine and vice versa. Naloxone, a specific opiate antagonist, interacts with humoral endorphin in a distinct manner which distinguishes it from opiates: while low concentrations of naloxone antagonize the effect of humoral endorphin, high concentrations of the antagonist are less effective and even potentiate its opiate activity. These interactions between opiate agents can be explained assuming conformational transformation of the opiate receptor. The in vitro interactions shed new light on paradoxical and conflicting results of in vivo experiments and indicate the physiological function of humoral endorphin.