The caudal neurosecretory system: a still enigmatic second neuroendocrine complex in fish.

The caudal neurosecretory system (CNSS) is a neuroendocrine complex, whose existence is specific to fishes. In teleosts, it consists of neurosecretory cells (Dahlgren cells) whose fibers are associated with a neurohemal terminal tissue (urophysis). In other actinopterygians as well as in chondrichthyes, the system is devoid of urophysis, so that Dahlgren cells end in a diffuse neurohemal region. Structurally, it has many similarities with the hypothalamic-neurohypophysial system. However, it differs regarding its position at the caudal end of the spinal cord and the nature of the hormones it secretes, the most notable ones being urotensins. The CNSS was first described more than 60 years ago, but its embryological origin is still hypothetical, and its role is poorly understood. Observations and experimental data gave some evidences of a possible involvement in osmoregulation, stress and reproduction. But one may question the benefit for fish to possess this second neurosecretory system, while the central hypothalamic-pituitary complex already controls such functions. As an introduction of our review, a brief report on the discovery of the CNSS is given. A description of its organization follows, and our review then focuses on the neuroendocrinology of the CNSS with the different factors it produces and secretes. The current knowledge on the ontogenesis and developmental origin of the CNSS is also reported, as well as its evolution. A special focus is finally given on what is known on its potential physiological roles.

Exposure to environmentally relevant doses of plasticizers alters maternal behavior and related neuroendocrine processes in primiparous and multiparous female mice.

Phthalates are organic pollutants frequently detected in the environment. The effects of these substances on male reproduction have been extensively studied but their potential impact on female reproductive behaviors in particular at environmental doses still remains to be documented. In the present study, we examined the effects of chronic exposure to di (2-ethylhexyl) phthalate (DEHP) alone at 5 or 50 µg/kg/d, or in an environmental phthalate mixture on maternal behavior of lactating female mice after a first (primiparous) and a second gestation (multiparous). Exposure of DEHP alone or in a phthalate mixture reduced pup-directed behaviors, increased self-care and forced nursing behaviors and altered nest quality for both primiparous and multiparous dams. In pup-retrieval test, primiparous and multiparous dams exposed to DEHP alone or in a phthalate mixture retrieved their pups more rapidly, probably due to a higher emission of ultrasonic vocalizations by the pups. At lactational day 2 following the third and last gestational period, the neural circuitry of maternal behavior was analyzed. A lower number of oxytocin-immunoreactive neurons in the paraventricular and anterior commissural nuclei was found in dams exposed to DEHP alone or in a phthalate mixture, while no changes were observed in the number of arginine-vasopressin immunoreactive cells. In the medial preoptic area, exposure to DEHP alone or in a phthalate mixture reduced ERα-immunoreactive cell number. Dendritic spine density assessed for DEHP at 5 µg/kg/d was also reduced. Thus, exposure to DEHP alone or in a phthalate mixture altered maternal behavior probably through a neuroendocrine mode of action involving oxytocin and estrogen through ERα, key pathways necessary for neuroplasticity and behavioral processing.

Deletion of neural estrogen receptor alpha induces sex differential effects on reproductive behavior in mice.

Estrogen receptor (ER) α is involved in several estrogen-modulated neural and peripheral functions. To determine its role in the expression of female and male reproductive behavior, a mouse line lacking the ERα in the nervous system was generated. Mutant females did not exhibit sexual behavior despite normal olfactory preference, and had a reduced number of progesterone receptor-immunoreactive neurons in the ventromedial hypothalamus. Mutant males displayed a moderately impaired sexual behavior and unaffected fertility, despite evidences of altered organization of sexually dimorphic populations in the preoptic area. In comparison, males deleted for both neural ERα and androgen receptor (AR) displayed greater sexual deficiencies. Thus, these data highlight a predominant role for neural ERα in females and a complementary role with the AR in males in the regulation of sexual behavior, and provide a solid background for future analyses of neuronal versus glial implication of these signaling pathways in both sexes.

[The caudal neurosecretory system, the other "neurohypophysial" system in fish]. / Le système neurosécréteur caudal, l'autre système « neurohypophysaire ¼ des poissons.

The caudal neurosecretory system (CNSS) is a neuroendocrine complex whose existence is specific to fishes. Structurally, it has many similarities with the hypothalamic-neurohypophyseal complex of other vertebrates. However, it differs regarding its position at the caudal end of the spinal cord and the nature of the hormones it secretes, the most important being urotensins. The CNSS was first described more than 60 years ago, but its embryological origin is totally unknown and its role is still poorly understood. Paradoxically, it is almost no longer studied today. Recent developments in imaging and genome editing could make it possible to resume investigations on CNSS in order to solve the mysteries that still surround it.

Title: Le système neurosécréteur caudal, l'autre système « neurohypophysaire ¼ des poissons. Abstract: Le système neurosécréteur caudal (SNSC) est un complexe neuroendocrinien propre aux poissons. Sur le plan structural, il présente de nombreuses similitudes avec le complexe hypothalamo-neurohypophysaire d'autres vertébrés. Il s'en distingue toutefois par sa position, à l'extrémité caudale de la moelle épinière, et par la nature des hormones qu'il sécrète, les plus importantes étant les urotensines. Le SNSC a été décrit pour la première fois il y a plus de 60 ans, mais son origine embryologique est totalement inconnue et son rôle reste mal compris. Paradoxalement, il n'est presque plus étudié aujourd'hui. Les développements récents en imagerie et en génie génétique pourraient justifier la reprise d'investigations sur le SNSC afin de lever les mystères qui continuent de l'entourer.

Effects of neural estrogen receptor beta deletion on social and mood-related behaviors and underlying mechanisms in male mice.

Estradiol derived from neural aromatization of testosterone plays a key role in the organization and activation of neural structures underlying male behaviors. This study evaluated the contribution of the estrogen receptor (ER) ß in estradiol-induced modulation of social and mood-related behaviors by using mice lacking the ERß gene in the nervous system. Mutant males exhibited reduced social interaction with same-sex congeners and impaired aggressive behavior. They also displayed increased locomotor activity, and reduced or unaffected anxiety-state level in three paradigms. However, when mice were exposed to unescapable stress in the forced swim and tail suspension tests, they spent more time immobile and a reduced time in swimming and climbing. These behavioral alterations were associated with unaffected circadian and restraint stress-induced corticosterone levels, and unchanged number of tryptophan hydroxylase 2-immunoreactive neurons in the dorsal raphe. By contrast, reduced mRNA levels of oxytocin and arginine-vasopressin were observed in the bed nucleus of stria terminalis, whereas no changes were detected in the hypothalamic paraventricular nucleus. The neural ERß is thus involved to different extent levels in social and mood-related behaviors, with a particular action on oxytocin and arginine-vasopressin signaling pathways of the bed nucleus of stria terminalis, yet the involvement of other brain areas cannot be excluded.

Neural Mechanisms Underlying the Disruption of Male Courtship Behavior by Adult Exposure to Di(2-ethylhexyl) Phthalate in Mice.

BACKGROUND: Courtship behavior plays a critical role in attracting females and reproduction success. However, the effects of exposure to a ubiquitous contaminant di(2-ethylhexyl) phthalate (DEHP) on these behaviors and, in particular, on courtship vocalizations have not been examined. OBJECTIVE: The effects of adult exposure to DEHP on courtship and mating behaviors and gonadotropic axis and neural mechanisms involved in DEHP-induced effects were analyzed in male mice. METHODS: Adult C57BL/6J males were orally exposed to DEHP (0, 0.5, 5, and 50µg/kg/d) for 4 wk. Olfactory preference, ultrasonic vocalizations (USVs), partner preference and mating, as well as locomotor activity and motor coordination, were measured. The kisspeptin system and testosterone levels were analyzed. Proteomic and molecular studies were conducted on the hypothalamic preoptic nucleus, the key region involved in sexual motivation to vocalize and mate. RESULTS: DEHP at 50µg/kg/d reduced the emission of USVs, whereas lower doses changed the ratio of syllable categories. This was associated with diminished sexual interest of female partners toward males exposed to 5 or 50µg/kg/d and increased latency to mate, despite normal olfactory preference. The kisspeptin system and circulating testosterone levels were unaffected. In DEHP-exposed males, proteomic analysis of the preoptic nucleus identified differentially expressed proteins connected to the androgen receptor (AR). Indeed, exposure to 5 or 50µg/kg/d of DEHP induced selective AR downregulation in this nucleus and upstream chemosensory regions. The involvement of AR changes in the observed alterations was further supported by the reduced emission of courtship vocalizations in males with disrupted neural AR expression. CONCLUSIONS: These data demonstrate the critical role of neural AR in courtship vocalizations and raises the possibility that the vulnerability of this signaling pathway to exposure to endocrine disrupters may be detrimental for courtship communication and mating in several species. https://doi.org/10.1289/EHP1443.

Revisiting the neural role of estrogen receptor beta in male sexual behavior by conditional mutagenesis.

Estradiol derived from neural aromatization of gonadal testosterone plays a key role in the perinatal organization of the neural circuitry underlying male sexual behavior. The aim of this study was to investigate the contribution of neural estrogen receptor (ER) ß in estradiol-induced effects without interfering with its peripheral functions. For this purpose, male mice lacking ERß in the nervous system were generated. Analyses of males in two consecutive tests with a time interval of two weeks showed an effect of experience, but not of genotype, on the latencies to the first mount, intromission, pelvic thrusting and ejaculation. Similarly, there was an effect of experience, but not of genotype, on the number of thrusts and mating length. Neural ERß deletion had no effect on the ability of males to adopt a lordosis posture in response to male mounts, after castration and priming with estradiol and progesterone. Indeed, only low percentages of both genotypes exhibited a low lordosis quotient. It also did not affect their olfactory preference. Quantification of tyrosine hydroxylase- and kisspeptin-immunoreactive neurons in the preoptic area showed unaffected sexual dimorphism of both populations in mutants. By contrast, the number of androgen receptor- and ERα-immunoreactive cells was significantly increased in the bed nucleus of stria terminalis of mutant males. These data show that neural ERß does not play a crucial role in the organization and activation of the neural circuitry underlying male sexual behavior. These discrepancies with the phenotype of global ERß knockout models are discussed.

Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta.

Ovarian oestradiol is essential for pubertal maturation and adult physiology of the female reproductive axis. It acts at central and peripheral sites through two main oestrogen receptors (ER) α and ß. Here we investigate the role of ERß on central effects of oestradiol, by generating a mouse line specifically lacking the ERß gene in neuronal and glial cells. Central ERß deletion delays the age at vaginal opening and first oestrous and reduces uterine weight without affecting body growth. Analysis of factors necessary for pubertal progression shows reduced levels of Kiss1 transcripts at postnatal (P) day 25 in the preoptic area, but not in the mediobasal hypothalamus (MBH) of mutant females. In agreement with these data, the number of kisspeptin-immunoreactive neurons was decreased by 57-72% in the three subdivisions of the rostral periventricular area of the third ventricle (RP3V), whereas the density of kisspeptin-immunoreactive fibres was unchanged in the arcuate nucleus of mutant mice. These alterations do not involve changes in ERα mRNAs in the preoptic area and protein levels in the RP3V. The number and distribution of GnRH-immunoreactive cells were unaffected, but gonadotropin-releasing hormone (GnRH) transcript levels were higher in the P25 preoptic area of mutants. At adulthood, mutant females have normal oestrous cyclicity, kisspeptin system and exhibit unaltered sexual behaviour. They display, however, reduced ovary weight and increased anxiety-related behaviour during the follicular phase. This argues for the specific involvement of central ERß in the regulation of pubertal onset in female reproduction, possibly through prepubertal induction of kisspeptin expression in the RP3V.

Reactive oxygen species are physiological mediators of the noradrenergic signaling pathway in the mouse supraoptic nucleus.

Free radicals are essential for the vasopressin (AVP) response to plasmatic hyperosmolarity. Noradrenergic afferents are the major projections on the supraoptic nucleus (SON) of the hypothalamus and stimulate the expression of AVP via a nitric oxide (NO) pathway. In this study, we investigated the mechanisms linking free radicals and noradrenaline (NA)-induced regulation of AVP. Analysis of Tg8 transgenic mice, invalidated for the monoamine oxidase-A gene and with consequently high levels of brain monoamines and AVP in the SON, showed that free radicals are more abundant in their SON than in that of wild-type mice (WT). Antioxidant superoxide dismutase 1 and 2 and catalase enzyme activities were also higher in these mice than in WT. This may explain the observed absence of cytotoxicity that would otherwise be associated with such high level of free radicals. Treatment of Tg8 mice with α-MPT, a blocking agent for NA synthesis, decreased both the production of free radicals and the AVP levels in the SON. Furthermore, incubation of ex vivo slices including the SON with NA increased the production of free radicals and AVP levels in wild-type mice. When NA was associated with α-lipoic acid, an antioxidant blocking the production of free radicals, AVP remained at its control level, indicating that free radicals are required for the effect of NA on the expression of AVP. In slices incubated with SNP, a producer of NO, free radicals and AVP levels increased. When NA was associated with L-NAME (a NO synthase blocker), the levels of free radicals and AVP were the same as in controls. Thus, the noradrenaline-NO pathway, which stimulates the expression of vasopressin, involves free radicals. This study provides further evidence of the physiological importance of free radicals, which should no longer be considered solely as cytotoxic factors.

Neuroendocrine and behavioral effects of maternal exposure to oral bisphenol A in female mice.

Bisphenol A (BPA) is a widespread estrogenic compound. We investigated the effects of maternal exposure to BPA at reference doses on sexual behavior and neuroendocrine functions of female offspring in C57BL/6J mice. The dams were orally exposed to vehicle alone or vehicle-containing BPA at doses equivalent to the no observed adverse effect level (5 mg/kg body weight per day) and tolerable daily intake (TDI, 0.05 mg/kg body weight per day) level from gestational day 15 until weaning. Developmental exposure to BPA increased the lordosis quotient in naive females exposed to BPA at the TDI dose only. BPA exposure had no effect on olfactory preference, ability to express masculine behaviors or number of calbindin-positive cells, a sexually dimorphic population of the preoptic area. BPA at both doses selectively increased kisspeptin cell number in the preoptic periventricular nucleus of the rostral periventricular area of the third ventricle in adult females. It did not affect the number of GNRH-positive cells or percentage of kisspeptin appositions on GNRH neurons in the preoptic area. These changes were associated with higher levels of estradiol (E2) at the TDI dose while levels of LH, estrus cyclicity, ovarian and uterine weights, and fertility remained unaffected. Delay in the time of vaginal opening was observed during the postnatal period at TDI dose, without any alteration in body growth. This shows that developmental exposure to BPA at reference doses did not masculinize and defeminize the neural circuitry underlying sexual behavior in female mice. The TDI dose specifically exacerbated responses normally induced by ovarian E2, through estrogen receptor α, during the postnatal/prepubertal period.

Reactive oxygen species are required for the hypothalamic osmoregulatory response.

Free radicals, or reactive oxygen species (ROS), are highly reactive byproducts of oxygen degradation. They are well known for their cellular toxicity, but few studies have analyzed their potential role in homeostatic processes. We investigated ROS production and function during the arginine vasopressin (AVP) hypothalamic response to hyperosmolarity. Six-week-old male C3H/HeJ mice were subjected to salt loading for 2 or 8 d. The osmotic axis was progressively activated and reached a new steady-state status at 8 d as demonstrated by monitoring of plasmatic osmolality and c-Fos and AVP expression in the supraoptic nucleus (SON). Free radicals, visualized by dihydroethidine staining and measured by 2'-7'dichlorofluorescein diacetate assays, were detected after 2 d of salt loading. The activity and expression of superoxide dismutase 2 and catalase were concomitantly up-regulated in the SON, suggesting that free radicals are detoxified by endogenous antioxidant systems, thereby avoiding their deleterious effects. The early phase of the osmoregulatory response has been investigated using an acute hyperosmotic model; free radicals were produced 45 min after an ip injection of 1.5 m NaCl. This was followed by an increase in c-Fos and AVP expression and an increase in superoxide dismutase 2 and catalase activities. α-Lipoic acid, a ROS scavenger, administrated during the 3 d before the hypertonic ip injection, abolished the increase of AVP. These findings establish that hyperosmolarity causes ROS production in the SON, which is essential for AVP increase. This demonstrates the importance of free radicals as physiological signaling molecules in the regulation of body-fluid balance.

Direct evidence for the co-expression of URP and GnRH in a sub-population of rat hypothalamic neurones: anatomical and functional correlation.

Urotensin-II-related peptide (URP) is an eight amino-acid neuropeptide recently isolated from rat brain and considered as the endogenous ligand for the GPR14 receptor. Using single and double immunohistochemical labelling, in situ hybridization and ultrastructural immunocytochemistry, we explored the cellular and subcellular localization of URP in the male rat brain. URP peptide was detected in numerous varicose fibres of the median eminence (ME) and organum vasculosum laminae terminalis (OVLT) as well as in neuronal cell bodies of the medial septal nucleus and diagonal band of Broca where corresponding mRNA were also detected. Combining in situ hybridization with immunohistochemistry, we showed that cell bodies of the rat anterior hypothalamus contained both URP mRNA and GnRH peptide. In addition, double ultrastructural immunodetection of URP and GnRH peptides clearly revealed, in the median eminence, the co-localization of both peptides in the same neuronal processes in the vicinity of fenestrated portal vessels. This remarkable cellular and subcellular distribution led us to test the effect of URP on the GnRH-induced gonadotrophins release in the anterior pituitary, and to discuss its putative role at the level of the median eminence.

Occurrence of two distinct urotensin II-related peptides in zebrafish provides new insight into the evolutionary history of the urotensin II gene family.

The urotensin II (UII) family is currently known to consist of two paralogous peptides, namely UII and UII-related peptide (URP). In contrast to UII, which has been identified in all vertebrate classes so far, URP has only been characterized in tetrapods. We report here the occurrence of two distinct URP genes in teleosts, which we have named URP1 and URP2. Synteny analysis revealed that teleost URP1 and URP2 genes and tetrapod URP genes represent three distinct paralog genes that, together with the UII gene, probably arose from the two rounds of tetraploidization, which took place early in vertebrate evolution. The absence of URP in fish indicates that the corresponding gene has been lost in the teleost lineage, whereas it is likely that both the URP1 and URP2 genes have been lost in the tetrapod lineage. Quantitative RT-PCR analysis revealed that the URP2 gene is mainly expressed in the spinal cord and the brain in adult zebrafish. In situ hybridization experiments showed that in zebrafish embryos, URP2 mRNA-containing cells are located in the floor plate of the neural tube. In adult, URP2-expressing cells occur in close contact with the ventral side of the ependymal canal along the whole spinal cord, whereas in the brain, they are located below the fourth ventricle. These URP-expressing cells may correspond to cerebrospinal fluid-contacting neurons. In conclusion, our study reveals the occurrence of four distinct UII paralogous systems in vertebrates that may exert distinct functions, both in tetrapods and teleosts.

Comparative distribution of the mRNAs encoding urotensin I and urotensin II in zebrafish.

The neural neurosecretory system of fishes produces two biologically active neuropeptides, i.e. the corticotropin-releasing hormone paralog urotensin I (UI) and the somatostatin-related peptide urotensin II (UII). In zebrafish, we have recently characterized two UII variants termed UIIalpha and UIIbeta. In the present study, we have investigated the distribution of UI, UIIalpha and UIIbeta mRNAs in different organs by quantitative RT-PCR analysis and the cellular localization of the three mRNAs in the spinal cord by in situ hybridization (ISH) histochemistry. The data show that the UI gene is mainly expressed in the caudal portion of the spinal cord and, to a lesser extent, in the brain, while the UIIalpha and the UIIbeta genes are exclusively expressed throughout the spinal cord. Single-ISH labeling revealed that UI, UIIalpha and UIIbeta mRNAs occur in large cells, called Dahlgren cells, located in the ventral part of the caudal spinal cord. Double-ISH staining showed that UI, UIIalpha and UIIbeta mRNAs occur mainly in distinct cells, even though a few cells were found to co-express the UI and UII genes. The differential expression of UI, UIIalpha and UIIbeta genes may contribute to the adaptation of Dahlgren cell activity during development and/or in various physiological conditions.

Comparative genomics provides evidence for close evolutionary relationships between the urotensin II and somatostatin gene families.

Although urotensin II (UII) and somatostatin 1 (SS1) exhibit some structural similarities, their precursors do not show any appreciable sequence identity and, thus, it is widely accepted that the UII and SS1 genes do not derive from a common ancestral gene. The recent characterization of novel isoforms of these two peptides, namely urotensin II-related peptide (URP) and somatostatin 2 (SS2)/cortistatin (CST), provides new opportunity to revisit the phylogenetic relationships of UII and SS1 using a comparative genomics approach. In the present study, by radiation hybrid mapping and in silico sequence analysis, we have determined the chromosomal localization of the genes encoding UII- and somatostatin-related peptides in several vertebrate species, including human, chicken, and zebrafish. In most of the species investigated, the UII and URP genes are closely linked to the SS2/CST and SS1 genes, respectively. We also found that the UII-SS2/CST locus and the URP/SS1 locus are paralogous. Taken together, these data indicate that the UII and URP genes, on the one hand, and the SS1 and SS2/CST genes, on the other hand, arose through a segmental duplication of two ancestral genes that were already physically linked to each other. Our results also suggest that these two genes arose themselves through a tandem duplication of a single ancestral gene. It thus appears that the genes encoding UII- and somatostatin-related peptides belong to the same superfamily.