Bernadette Vidal

Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty

Abstract 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.

Dopaminergic inhibition of reproduction in teleost fishes: ecophysiological and evolutionary implications.

Abstract: In many teleosts, dopamine (DA) exerts direct inhibitory control on gonadotropes, counteracting the stimulatory effect of gonadotropin‐releasing hormone (GnRH) on gonadotropin release. This dual control by GnRH and DA has been demonstrated in various adult teleosts and has major implications for aquaculture. Because of its unique life cycle, the European eel has provided a powerful model for demonstrating the key role of DA in the control of puberty. Data from tetrapods suggest that the inhibitory role of DA on reproduction is not restricted to the teleosts. Thus, DA inhibitory control could represent an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been differentially maintained throughout vertebrate evolution. The intensity of DA inhibition, its main site of action, and its involvement in the control of puberty, seasonal reproduction, ovulation, spermiation, or even sex change may differ among classes of vertebrates, as well as within smaller phylogenetic units such as teleosts or mammals. An inhibitory role for DA has been reported also in some invertebrates, indicating that neuronal DA pathways may have been recruited in various groups of metazoa to participate in the control of reproduction. In addition to the incontestable GnRH neurons, the recruitment of DA neurons for the neuroendocrine control of reproduction provides an additional brain pathway for the integration of various species‐specific, internal, and environmental cues. In teleosts, the plasticity of the DA neuroendocrine role may have contributed to their large diversity of biological cycles and to their successful adaptation to various environments.

Reconstruction of human maxillary defects with nacre powder: histological evidence for bone regeneration

The defective areas in the premolar-molar region of maxillary alveolar bone of eight patients were reconstructed using powdered nacre from the giant oyster Pinctada maxima. Histological, microradiographic and polarized light studies of drill biopsies taken 6 months postoperatively showed that nacre was tightly bound to newly-formed bone. The nacre was gradually and centripetally biodissolved and replaced with immature and then mature lamellar bone. These results are in agreement with our previous experimental in vitro data indicating that nacre has good osteogenic properties.

Distribution of the mRNA encoding the four dopamine D1 receptor subtypes in the brain of the european eel (Anguilla anguilla): Comparative approach to the function of D1 receptors in vertebrates

Four subtypes of D1 dopamine receptors are expressed in the brain of the European eel (Anguilla anguilla), an elopomorph teleost. To correlate this molecular multiplicity with specific localisation and functions, the distribution of the D1 receptor transcripts was analysed by in situ hybridisation. The four D1 receptor transcripts exhibit largely overlapping expression territories. In telencephalon, they are found in the olfactory bulb and the dorsal telencephalon (except its lateral part) but are most abundant in the subpallial areas. More caudally, the entopeduncular nucleus, preoptic nuclei, preglomerular nuclear complex, ventral thalamus, periventricular hypothalamus, optic tectum and cerebellum, all contain various amounts of D1 receptor transcripts. Finally, D1 receptor mRNAs are present in nuclei associated with the cranial nerves. The two D1A receptor subtypes are generally the most abundant and present a different distribution in several areas. The D1B mRNA, although present in fewer areas than D1A transcripts, is the most abundant in ventrolateral telencephalon and torus semicircularis. The D1C receptor transcript, which has not been found in mammals, is restricted to diencephalon and cerebellum. In view of the expression territories of D1 receptor transcripts and previous data, some areas of the everted telencephalon of teleost may be homologous to regions of the tetrapod brain. In particular, D1 expression territories of the ventral telencephalon are likely to be equivalent to striatal areas. These observations suggest an evolutionary scenario in which the D1A receptor subtype was highly conserved after the first gene duplication during the evolution of craniates, whereas D1B and D1C, and their associated specific characteristics, appeared later, probably in the gnathostome lineage. J. Comp. Neurol. 419:320–343, 2000.

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.

Immunocytochemical localization of mammalian GnRH (gonadotropin-releasing hormone) and chicken GnRH-II in the brain of the European silver eel (Anguilla anguilla L.)

Using specific antibodies for the two molecular forms of gonadotropin-releasing hormone (GnRH) present in the European eel, Anguilla anguilla, (mammalian GnRH, mGnRH, and chicken GnRH II, cGnRH-II), we employed immunocytochemistry to determine the distribution of these two peptides in the brain and in the pituitary. The results indicate that mGnRH and cGnRH-II are localized in different neurons: mGnRH-immunoreactive (ir) perikaria were observed in the olfactory bulbs, the junction between olfactory bulbs and telencephalon (nucleus olfactoretinalis), the telencephalon, the preoptic region and the mediobasal hypothalamus. These cell bodies are located along a continuum of ir-fibers that could be traced from the olfactory nerve to the pituitary. Mammalian GnRH-ir fibers were detected in many parts of the brain (olfactory bulbs, ventral telencephalon, hypothalamus, optic tectum, mesencephalon) and in the pituitary. Chicken GnRH-II-ir cell bodies were detected in the nucleus of the medial longitudinal fasciculus of the midbrain tegmentum, but only scattered fibers could be detected in different parts of the brain. The pituitary exhibited very few cGnRH-II-ir fibers, contrasting with an extensive mGnRH innervation. These results are in agreement with our previous data obtained in the same species using specific radioimmunoassays for mGnRH and cGnRH-II. They demonstrate a differential distribution of the two forms of GnRH in the brain of the eel, as in the brain of some other vertebrate species, and suggest differential physiological roles for the two GnRH forms in the eel. They also provide information concerning the evolution of the GnRH 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 FSHβ subunit was cloned, sequenced and together with earlier isolated eel LHβ and glycoprotein hormone α (GPα) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHβ and LHβ are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHβ and FSHβ subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHβ and GPα increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHβ 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 LHβ and GPα mRNA levels, without any significant effect on FSHβ. In contrast, T treatment induced a decrease in FSHβ 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.

Immunocytochemical detection in eel corpuscles of Stannius of a mammalian parathyroid-like hormone.

Stannius corpuscles of the eel synthesize and secrete a mammalian parathyroid-like hormone called parathyrin of CS (PCS). PCS has been localized in the cytoplasm of all cells in the corpuscles, detection being by indirect immunofluorescence with an antiserum anti-1-84 bovine hormone (PTH). The specificity of the reaction was demonstrated by inhibition of the fluorescent staining with 1-84 bovine PTH and the active fragment 1-34 of human PTH. Variations of the cellular localization of the PCS or a complete depletion of the hormonal content, in all cells, were observed in eels made hypercalcemic by Ca overloading. The secretory activity of the two types of CS cells may be regulated by the plasma Ca2+ concentration.

Long-Term Inhibitory Effects of Somatostatin and Insulin-Like Growth Factor 1 on Growth Hormone Release by Serum-Free Primary Culture of Pituitary Cells from European Eel (Anguilla anguilla)

To investigate the ability of hypothalamic and peripheral factors to directly regulate growth hormone (GH) release in a primitive teleost, the European eel (Anguilla anguilla L.), we used primary cultures of dispersed pituitary cells. When cultured for 12 days in a serum-free medium, pituitary cells continuously released large amounts of GH, which exceeded the initial cellular content. Somatotropin-release inhibiting hormone (SRIH-14) dose-dependently inhibited GH release (EC50 0.75 nM) up to a maximal inhibitory effect of 95%. No desensitization of somatotropes to SRIH was observed over the 12 days of culture. Use of receptor subtype-selective SRIH agonists suggests the existence on eel somatotropes of SRIH receptor(s) related to the mammalian sst2/sst3/sst5 class rather than to the sst1/sst4 class. Insulin-like growth factor 1 (IGF1) dose-dependently inhibited GH release (EC50 0.03 nM) up to a maximal inhibitory effect of 85%, without desensitization. IGF1 and IGF2 were equipotent in inhibiting GH release, whereas insulin was 1,000 times less active, suggesting the implication of a receptor related to the mammalian IGF type 1 receptor. These results indicate that eel somatotropes are active in vitro without any specific additional factors, and suggest the existence of a dominant inhibitory control of GH release in vivo. Two potential candidates for this chronic negative regulation are a neurohormone, SRIH and a circulating factor, IGF1. These data underline the early evolutionary origin of the molecular and functional SRIH-GH-IGF1 neuroendocrine axis in vertebrates.

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.