Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids.

Two full-length cDNA encoding putative dopamine D2-like receptors were cloned from the brain of female European eel. The deduced protein sequences, termed D2A- and D2B-R, exhibit closer phylogenetic relationships to vertebrate D2 receptors compared with D3 and D4 or D1 receptors. The two protein sequences share 100% identity within the transmembrane domains containing the highly conserved amino acids involved in dopamine binding. Accordingly, an apparent single population of sites on eel brain membranes bound [(3)H]spiperone, a D2-R-specific antagonist, with a K(d) of 0.2 +/- 0.04 nM. However, D2A- and D2B-R significantly differ within the amino terminus and the third intracellular loop. As analyzed by quantitative PCR and in situ hybridization, both receptor transcripts were found, with different relative abundance, in the majority of brain areas and in the pituitary, whereas in the retina, olfactory epithelium, spinal cord, and adipose tissue, only D2A-R gene was expressed. Because sex steroid hormones recently have been shown to regulate eel brain dopamine systems, we analyzed the effect of steroids on the amount of D2-R transcripts by quantitative PCR and in situ hybridization. In eels treated with testosterone, the gene expression of the D2B-R, but not D2A-R, was increased in a region-dependent manner. The effect of testosterone on D2B-R transcript levels was mimicked by dihydrotestosterone, a nonaromatizable androgen, whereas estradiol had no stimulatory action, evidencing an androgen receptor-dependent mechanism. Although functionality of the two receptors awaits determination of D2-R proteins, we hypothesize that differences in the tissue expression pattern and hormonal regulation of eel D2A- and D2B-R gene expression could represent selective forces that have contributed to the conservation of the duplicated D2-R.

Androgen-dependent stimulation of brain dopaminergic systems in the female European eel (Anguilla anguilla).

Dopamine (DA), a neurotransmitter present in all vertebrates, is involved in processes such as motor function, learning and behavior, sensory activities, and neuroendocrine control of pituitary hormone release. In the female eel, we analyzed how gonadal steroids regulate brain expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of DA. TH mRNA levels were assayed by quantitative real-time RT-PCR. TH-positive nuclei were also localized by in situ hybridization (ISH) and immunohistochemistry, and the location of TH nuclei that project to the pituitary was determined using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindicarbocyanine perchlorate retrograde tracing. Chronic in vivo treatment with testosterone increased TH mRNA specifically in the periglomerular area of the olfactory bulbs and in the nucleus preopticus anteroventralis (NPOav). NPOav was labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindicarbocyanine perchlorate, showing that this nucleus is hypophysiotropic in the eel. The nonaromatizable 5alpha-dihydrotestosterone gave identical results in both areas, whereas 17beta-estradiol had no stimulatory effect, showing that the observed stimulatory effects of testosterone were androgen dependent. In teleosts, DA neurons originating from the NPOav directly inhibit gonadotropic function, and our results indicate an androgen-dependent, positive feedback on this neuroendocrine control in the eel. In mammals, DA interneurons in the olfactory bulbs are involved in the enhancement of olfactory sensitivity and discrimination. Our results in the European eel suggest an androgen-dependent stimulation of olfactory processing, a sensory function believed to be important in eel navigation during its reproductive migration toward the oceanic spawning grounds. To our knowledge, this is the first evidence from any vertebrate of an androgen-dependent effect on DAergic activity in the olfactory bulbs, providing a new basis for understanding the regulation by gonadal steroids of central DAergic systems in vertebrates.

Differential regulation of luteinizing hormone and follicle-stimulating hormone expression during ovarian development and under sexual steroid feedback in the European eel.

Pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are, in teleosts as in mammals, under the control of hypothalamic factors and steroid feedbacks. In teleosts, feedback regulations largely vary depending on species and physiological stage. In the present study the regulation of FSH and LH expression was investigated in the European eel, a fish of biological and phylogenetical interest as a representative of an early group of teleosts. The eel FSHbeta subunit was cloned, sequenced and together with earlier isolated eel LHbeta and glycoprotein hormone alpha (GPalpha) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHbeta and LHbeta are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHbeta and FSHbeta subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHbeta and GPalpha increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHbeta mRNA levels decreased. To understand the role of sex steroids in these opposite variations, immature eels were treated with estradiol (E2)and testosterone (T), both steroids being produced in eel ovaries during gonadal development. E2 treatment induced increases in both LHbeta and GPalpha mRNA levels, without any significant effect on FSHbeta. In contrast, T treatment induced a decrease in FSHbeta mRNA levels, without any significant effect on the other subunits. These data demonstrate that steroids exert a differential feedback on eel gonadotropin expression, with an E2-specific positive feedback on LH and a T-specific negative feedback on FSH, leading to an opposite regulation of LH and FSH during ovarian development.

Endocrine evidence that silvering, a secondary metamorphosis in the eel, is a pubertal rather than a metamorphic event.

Silvering (transition from yellow to silver eel) has been traditionally considered as a metamorphosis in view of the numerous morphological, physiological and behavioral changes preparing the eel for the oceanic migration. However, some changes, such as increases in gonad weight and steroidogenesis, suggest that silvering could also be considered as a pubertal event. In order to assess which endocrine axis may be involved in the induction of silvering, we compared the profiles of pituitary and peripheral hormones during the transition from yellow to silver female eels. A strong activation of the gonadotropic axis was shown during silvering. Follicle-stimulating hormone (FSH) mRNA levels increased during the early stages of silvering, followed by a later increase in luteinizing hormone (protein and mRNA) levels. In addition, plasma levels of sexual steroids (estradiol, E2; testosterone, T, and 11-ketotestosterone) and of vitellogenin significantly increased. In contrast, thyrotropin mRNA levels did not change and no or weak variations in plasma thyroid hormones were observed, indicating no or moderate change of the thyrotropic axis during silvering. Similarly, the somatotropic axis was not activated, as shown by pituitary growth hormone expression (protein and mRNA) and plasma levels. In addition, we studied the effects of chronic treatments of female yellow eels with thyroid hormone (thyroxine, T4) and sex steroids (T and E2) on biometrical parameters characteristics of silvering. T induced an increase in eye size and a reduction of digestive tract, whereas T4 and E2 had no effect. These hormonal profiles and experimental data lead to the conclusion that eel silvering should be considered as an onset of puberty rather than a 'genuine' metamorphosis.

Dopamine inhibits luteinizing hormone synthesis and release in the juvenile European eel: a neuroendocrine lock for the onset of puberty.

In various adult teleost fishes, LH ovulatory peak is under a dual neurohormonal control that is stimulatory by GnRH and inhibitory by dopamine (DA). We investigated whether DA could also be involved in the inhibitory control of LH at earlier steps of gametogenesis by studying the model of the European eel, Anguilla anguilla, which remains at a prepubertal stage until the oceanic reproductive migration. According to a protocol previously developed in the striped bass, eels received sustained treatments with GnRH agonist (GnRHa), DA-receptor antagonist (pimozide), and testosterone (T) either alone or in combination. Only the triple treatment with T, GnRHa, and pimozide could trigger dramatic increases in LH synthesis and release as well as in plasma vitellogenin levels and a stimulation of ovarian vitellogenesis. Thus, in the prepubertal eel, removal of DA inhibition is required for triggering GnRH-stimulated LH synthesis and release as well as ovarian development. To locate the anatomical support for DA inhibition, the distribution of tyrosine hydroxylase (TH) in the brain and pituitary was studied by immunocytochemistry. Numerous TH-immunoreactive cell bodies were observed in the preoptic anteroventral nucleus, with a dense tract of immunoreactive fibers reaching the pituitary proximal pars distalis, where the gonadotrophs are located. This pathway corresponds to that mediating the inhibition of LH and ovulation in adult teleosts. To our knowledge, this is the first demonstration of a pivotal role for DA in the control of LH and puberty in a juvenile teleost. These data support the view that DA inhibition on LH secretion is an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been partially maintained throughout vertebrate evolution.

[An antagonist to GnRH in the control of reproduction in teleost fish: dopaminergic inhibition. Ancestral origin and differential conservation within vertebrates?]. / Un contre-pouvoir au contrôle de la reproduction par la GnRH chez les Poissons Téléostéens : l'inhibition dopaminergique. Rôle ancestral et conservation différentielle chez les Vertébrés?

In mammals, the neurohormonal control of the pituitary gonadotropes is provided by the gonadoliberin GnRH. Several studies on teleost fish indicate that a single positive control by GnRH is not a general rule among vertebrates. Peter and colleagues presented the first evidence of an inhibitory neurohormonal factor, "GRIF" (gonadotropin-release inhibiting factor). They induced a preovulatory LH surge by injuring particular brain areas in the goldfish. Subsequent in vivo and in vitro studies identified dopamine as GRIF, and neuroanatomical investigations have demonstrated that dopaminergic neurones in the anterior preoptic area projecting to the pituitary represent the anatomical substrate for GRIF activity. An inhibitory role of dopamine on the control of LH and ovulation/spermiation has been evidenced in many adult teleosts, including its implications for aquaculture. However, dopamine does not play an inhibitory role in all adult teleosts. As regards the early stages of gametogenesis and especially the control of puberty, a role for dopamine has been suggested or rejected depending on species. The European eel has a unique life cycle with a long prepubertal stage, which has made it a useful model to demonstrate the key-role of dopamine in the control of puberty. Data from tetrapods suggest that the role of dopamine as a GRIF is not restricted to the teleosts, but that it may have an ancient evolutionary origin, and has been differentially conserved throughout vertebrate evolution.