A reproductive role for the nonapeptides vasotocin and isotocin in male zebrafish (Danio rerio).

Two distinct nonapeptide systems, vasotocin- and oxytocin-related peptides, evolved in vertebrates. Their role in male zebrafish reproduction has not been formally investigated. We hypothesized that the teleost nonapeptides vasotocin and isotocin stimulate male zebrafish reproductive physiology and success by affecting central neuronal and/or peripheral endocrine pathways. Pharmacological inhibition experiments revealed that both vasotocin and isotocin contribute significantly to male reproductive success, which in the case of vasotocin correlated significantly with indices of male courtship behavior. Interestingly, co-administration of vasotocin and isotocin antagonists completely abolished male reproductive success without affecting male courtship behavior and endocrine indices, possibly linked to a synergistic action of nonapeptides on male pheromone release. To further probe the nonapeptides' role in male zebrafish reproduction, we subsequently tested whether male zebrafish nonapeptide systems were acutely activated by the female releaser pheromone PGF2α, a strong chemoattractant and important reproductive cue in males which stimulates courtship behavior. Male zebrafish attracted to PGF2α in a choice assay exhibited acute increases in neuronal activation marker p-ERK immunoreactivity in the ventral glomerulus of the olfactory bulb and the preoptic area, however no co-localization with isotocin was observed. Conversely, PGF2α time-dependently stimulated whole brain isotocin mRNA abundance, suggesting secondary longer-term effects of PGF2α exposure on the central isotocinergic system. While the current lack of vasotocin-specific antibodies for zebrafish does not allow to probe acute activation of vasotocinergic neurons, whole brain vasotocin mRNA was not significantly affected by PGF2α exposure. Together, our results identify a role for nonapeptides in male zebrafish reproductive physiology and success.

Female-biased sex difference in vasotocin-immunoreactive neural structures in the developing quail brain.

The bed nucleus of the stria terminalis pars medialis (BSTM), medial preoptic nucleus (POM), and lateral septal region (LS) exhibit more vasotocin-immunoreactive (VT-ir) neural structures in male than in female adult quail. VT-ir cells and fibers in these regions are sensitive to gonadal steroids only in males. The insensitivity of adult female VT-ir neural structures to sex steroids is attributed to estradiol exposure during a critical period in embryonic life. Although the VT-ir system has been intensively examined in adult quail, information is limited in embryos and juveniles. Therefore, we herein investigated the development of VT-immunoreactive neural structures from embryonic day (E) 9 to adulthood with a particular focus on the BSTM, POM and LS of both sexes. VT-ir neural structures were more evident in female than in male embryos from E9 (BSTM and POM) and E11 (LS). This sex difference disappeared between E15 and post-hatch day 1 in the BSTM and POM, and during the first week of life in the LS. Male-biased sex differences in VT-ir structures appeared at puberty. Female-biased sexual dimorphism in the density of the VT-ir structures of BSTM was reflected by the stronger expression of VT mRNA in females than in males. However, the density of VT mRNA somata was comparable in the two sexes. The exposure of male embryos to estradiol resulted in the feminization of VT-ir neural structures in the BSTM, but not in the POM or LS at E11. Collectively, these results suggest that sex differences in VT-ir neural structures changes drastically throughout quail life. In embryos, endogenous estradiol may stimulate the expression of VT in females, resulting in a robust sex difference in VT-ir cells and fibers in favor of this sex.

The hypothalamic photoreceptors regulating seasonal reproduction in birds: a prime role for VA opsin.

Extraretinal photoreceptors located within the medio-basal hypothalamus regulate the photoperiodic control of seasonal reproduction in birds. An action spectrum for this response describes an opsin photopigment with a λmax of ∼ 492 nm. Beyond this however, the specific identity of the photopigment remains unresolved. Several candidates have emerged including rod-opsin; melanopsin (OPN4); neuropsin (OPN5); and vertebrate ancient (VA) opsin. These contenders are evaluated against key criteria used routinely in photobiology to link orphan photopigments to specific biological responses. To date, only VA opsin can easily satisfy all criteria and we propose that this photopigment represents the prime candidate for encoding daylength and driving seasonal breeding in birds. We also show that VA opsin is co-expressed with both gonadotropin-releasing hormone (GnRH) and arginine-vasotocin (AVT) neurons. These new data suggest that GnRH and AVT neurosecretory pathways are endogenously photosensitive and that our current understanding of how these systems are regulated will require substantial revision.

Vasotocin mRNA expression is sensitive to testosterone and oestradiol in the bed nucleus of the stria terminalis in female Japanese quail.

Vasotocin-producing parvocellular neurones in the medial part of the bed nucleus of the stria terminalis (BSTM) of many species of birds and mammals show sexual dimorphism and great plasticity in response to hormonal and environmental stimuli. In the BSTM of Japanese quail, vasotocin-immunoreactive neurones are visible and sensitive to testosterone exclusively in males. In males, gonadectomy decreases and testosterone restores vasotocin-immunoreactive cells and fibres by acting on vasotocin mRNA transcription. The insensitivity of female vasotocin-immunoreactive neurones to the activating effects of testosterone is the result of organisational effects of early exposure to oestradiol. Female quail also show vasotocin mRNA-expressing neurones in the BSTM, although it is not known whether the insensitivity of the vasotocinergic neurones to testosterone originates at the level of vasotocin gene transcription in this sex. Therefore, initially, the present study analysed the effects of acute treatment with testosterone on vasotocin mRNA expression in the BSTM of gonadectomised male and female quail using in situ hybridisation. Gonadectomy decreased (and a single injection of testosterone increased) the number of vasotocin mRNA-expressing neurones and intensity of the vasotocin mRNA hybridisation signal similarly in both sexes. Notably, testosterone increased vasotocin mRNA expression in ovariectomised females over that shown by intact quail. However, this treatment had no effect on vasotocin immunoreactivity. A second experiment analysed the effects of testosterone metabolites, oestradiol and 5α-dihydrotestosterone, on vasotocin mRNA expression in female quail. Oestradiol (but not 5α-dihydrotestosterone) fully mimicked the effects of testosterone on the number of vasotocin mRNA-expressing neurones and the intensity of the vasotocin mRNA hybridisation signal. Taken together, these results show, for the first time, that gonadal steroids strongly activate vasotocin mRNA expression in the BSTM of female quail.

Pheromone exposure influences preoptic arginine vasotocin gene expression and inhibits social approach behavior in response to rivals but not potential mates.

The nonapeptides arginine vasotocin (AVT) and vasopressin mediate a variety of social behaviors in vertebrates. However, the effects of these peptides on behavior can vary considerably both between and within species. AVT, in particular, stimulates aggressive and courtship responses typical of dominant males in several species, although it can also inhibit social interactions in some cases. Such differential effects may depend upon AVT influences within brain circuits that differ among species or between males that adopt alternative reproductive phenotypes and/or upon the differential activation of those circuits in different social contexts. However, to date, very little is known about how social stimuli that promote alternative behavioral responses influence AVT circuits within the brain. To address this issue, we exposed adult male goldfish to androstenedione (AD), a pheromonal signal that is released by both males and females during the breeding season, and measured social approach responses of males towards same- and other-sex individuals before and after AD exposure. In a second experiment, we measured AD-induced AVT gene expression using in situ hybridization. We found that brief exposure to AD induces social avoidance in response to rival males, but does not affect the level of sociality exhibited in response to sexually receptive females. Exposure to AD also increases AVT gene expression in the preoptic area of male goldfish, particularly in the parvocellular population of the preoptic nucleus. Together, these data suggest that AD is part of a social signaling system that induces social withdrawal specifically during male-male interactions by activating AVT neurons.

Arginine-vasotocin expression and participation in reproduction and social behavior in males of the cichlid fish Cichlasoma dimerus.

In non-mammalian vertebrates, the nonapeptide arginine-vasotocin (AVT) is involved in the regulation of social behavior related to reproduction and aggression. The cichlid fish Cichlasoma dimerus is a monogamous species with complex social hierarchies. Males are found in one of two basic alternative phenotypes: Non-territorial and territorial males. In this work we characterize the vasotocinergic system in males of C. dimerus in relation to social status with particular emphasis on the various putative sites of action of AVT across the hypothalamic-pituitary-gonad (HPG) axis, and its effects on reproductive and social behavior. The location and distribution of vasotocinergic neurons in the brain was studied, highlighting a morphometric analysis of AVT producing neurons in males of different social status. The effect of AVT on pituitary gonadotropin secretion was analyzed by single pituitary culture while expression of AVT in peripheral organs was studied by RT-PCR using specific primers. Finally, the role of AVT on testicular androgen release was assessed by in vitro incubation of testis. Results showed a positive effect of AVT on gonadotropin secretion, where ß-LH showcased a triphasic response under increasing AVT concentration, while ß-FSH's response was dose-dependent and directly proportional. AVT showed a positive and concentration-dependent effect over testicular androgens synthesis and secretion in vitro. Vasotocin expression was observed in testicular somatic tissue located in the interstitial compartment. Thus, the AVT system in C. dimerus appears to be of high complexity, with multiple sites of action in the hypothalamus-pituitary-gonadal axis.

Sex differences in the expression of vasotocin/isotocin, gonadotropin-releasing hormone, and tyrosine and tryptophan hydroxylase family genes in the medaka brain.

In teleost fish, sex differences in several behavioral and physiological traits have been assumed to reflect underlying sex differences in the central expression of neurotransmitter/neuromodulator-related molecules, including vasotocin (VT)/isotocin (IT), gonadotropin-releasing hormone (GnRH), and tyrosine and tryptophan hydroxylases (TH and TPH). However, the sex-dependent expression patterns of these molecules have not been fully characterized in the teleost brain. In the present study, we therefore systematically evaluated sex differences in their expression in the medaka (Oryzias latipes) brain. The most prominent sex difference was observed in vt expression in the nucleus posterior tuberis (NPT) and the posterior part of the nucleus ventral tuberis (NVT) in the hypothalamus, where the expression was completely male-specific. Male-biased expression of gnrh1, tph1, and tph2 was also evident in the supracommissural and posterior nuclei of the ventral telencephalic area (Vs/Vp), medial nucleus of the dorsal telencephalic area (Dm), and thalamic dorsal posterior nucleus (DP), respectively. In contrast, the overall expression levels of it and gnrh3 were higher in the female brain than in the male brain. Equally importantly, no conspicuous sex differences were observed in the expression of gnrh2, th1, and th2, despite several previous reports of their sex-biased expression in the brains of other teleost species. Taken together, these data have uncovered previously unidentified sex differences in the expression of VT/IT, GnRH, and TPH in the teleost brain, which may possibly be relevant to sexual dimorphism in some behavioral and/or physiological traits, and have simultaneously highlighted potential species differences in the roles of these molecules.

Brain levels of arginine-vasotocin and isotocin in dominant and subordinate males of a cichlid fish.

The nonapeptides arginine-vasotocin (AVT) and isotocin (IT), which are the teleost homologues of arginine-vasopressin and oxytocin in mammals, have well established peripheral effects on osmoregulation and stress response, and central effects on social behavior. However, all studies that have looked so far into the relationship between these nonapeptides and social behavior have used indirect measures of AVT/IT activity (i.e. immunohistochemistry of AVT/IT immunoreactive neurons, or AVT/IT or their receptors mRNA expression with in situ hybridization or qPCR) and therefore direct measures of peptide levels in relation to social behavior are still lacking. Here we use a recently developed high-performance liquid chromatography analysis with fluorescence detection (HPLC-FL) method to quantify the levels of both AVT and IT in macro-dissected brain areas [i.e. olfactory bulbs, telencephalon, diencephalon, optic tectum, cerebellum, and hindbrain (= rhombencephalon minus cerebellum)] and pituitary of dominant and subordinate male cichlid fish (Oreochromis mossambicus). The pituitary shows higher levels of both peptides than any of the brain macroareas, and the olfactory bulbs have the highest AVT among all brain areas. Except for IT in the telencephalon there is a lack of correlations between central levels and pituitary peptide levels, suggesting an independent control of hypophysial and CNS nonapeptide secretion. There were also no correlations between AVT and IT levels either for each brain region or for the pituitary gland, suggesting a decoupled activity of the AVT and IT systems at the CNS level. Subordinate AVT pituitary levels are significantly higher than those of dominants, and dominant hindbrain IT levels are significantly higher than those of subordinates, suggesting a potential involvement of AVT in social stress in subordinate fish and of IT in the regulation of dominant behavior at the level of the hindbrain. Since in this species dominant males use urine to communicate social status and since AVT is known to have an antidiuretic effect, we have also investigated the effect of social status on urine storage. As predicted, dominant males stored significantly more urine than subordinates. Given these results we suggest that AVT/IT play a key role in orchestrating social phenotypes, acting both as central neuromodulators that promote behavioral plasticity and as peripheral hormones that promote integrated physiological changes.

Diurnal rhythms in hypothalamic/pituitary AVT synthesis and secretion in rainbow trout: evidence for a circadian regulation.

Arginine vasotocin (AVT) and isotocin (IT) are two neurohypophysial peptide hormones for which a role in adaptation to environmental changes has been suggested in fish. In teleosts, there are only a few available studies about circadian changes of AVT and IT levels, and a role of those peptides in the circadian system has been mainly suggested on the basis of the role of the homologous hormone AVP in mammals. Herein, we evaluated the diurnal rhythms in plasma AVT, pituitary AVT and IT content and the hypothalamic pro-vasotocin (pro-VT) expression in rainbow trout kept under a natural photoperiod, as well as their persistence in constant darkness as a tool for defining circadian dependence. Trout kept under a natural light cycle showed clear diurnal rhythms in both circulating and pituitary AVT levels with peak values around the last hours of the light phase. Hypothalamic pro-VT mRNA was also rhythmically expressed with similar peak characteristics. These rhythms persisted in fish kept under constant darkness for nearly two consecutive days, although peaks were progressively attenuated and phase-advanced. An IT rhythm was also found in pituitary of the trout maintained under a natural photoperiod, but not in those kept under continuous darkness. These results suggest that rhythms of hypothalamic AVT synthesis might be regulated by endogenous circadian mechanisms, and these rhythms contribute to maintain a similar fluctuation in pituitary AVT secretion into the blood. A potential role for AVT in the circadian and seasonal time-keeping system of teleost fish, either as a component of the neural machinery that participates in the adaptation to cyclic environmental changes, or as a circadian/seasonal output signal, is also discussed.

TonEBP regulates hyperosmolality-induced arginine vasotocin gene expression in the chick (Gallus domesticus).

Arginine vasotocin (AVT) is expressed mainly in the paraventircular and supraoptic nuclei of the hypothalamus in chicken. This peptide is known to act as an antidiuretic hormone and its gene expression is stimulated by hyperosmolality. However, the transcription factors that regulate the AVT gene expression induced by hyperosmolality are still unknown. In this study, we examined the role of hyper-tonicity enhancer binding protein (TonEBP) in the transcriptional regulation of AVT gene in chicken. TonEBP mRNA expression levels increased at 1h after salt-loading treatment in the hypothalamus. This increase preceded that in AVT and c-fos mRNA expression. Intracerebroventricular injections of TonEBP antisense oligonucleotides, before the salt-loading treatment, prevented the increase in AVT gene expression. These results, all together, suggest that the transcription factor TonEBP may be involved in the regulation of AVT genes expression in response to a hyperosmotic environment in chicken.

Osmoregulatory responses of expression of vasotocin, isotocin, prolactin and growth hormone genes following hypoosmotic challenge in a stenohaline marine teleost, tiger puffer (Takifugu rubripes).

To examine possible roles of vasotocin (VT), isotocin (IT), prolactin (PRL) and growth hormone (GH) in osmoregulation of a stenohaline marine teleost, tiger puffer (Takifugu rubripes), changes in expression levels of these genes following hypoosmotic challenge, were examined in two experiments. Fish were transferred from 100% seawater (SW) to 33% SW, 10% SW and fresh water (FW), and left for 3days in experiment I. In experiment II, fish were transferred to FW, and left for 1day. Changes in plasma osmolality, concentrations of Na(+) and Cl(-), and Na(+)/K(+)-ATPase activity in the gills and kidney were examined. Changes in the absolute amounts of VT, IT, PRL and GH mRNAs were determined by real-time PCR. In experiment I, almost all fish survived over 3days of acclimation. The plasma parameters decreased on day 1, and remained at similar levels until day 3. The renal Na(+)/K(+)-ATPase activity significantly increased in 10% SW and FW on day 1. The amounts of VT and IT mRNAs tended to decrease in the hypoosmotic conditions on day 1. The amounts of PRL mRNA significantly increased in the hypoosmotic conditions, whereas those of GH mRNA decreased in FW. In experiment II, the amount of VT mRNA significantly decreased in FW concomitantly with the changes in PRL and GH mRNAs. The present results suggest that the hyperosmotic responses may be regulated by neuroendocrine factors such as VT, PRL and GH in tiger puffer, as in case of euryhaline teleosts. Particularly, the present study first shows that the expression of VT gene may be down-regulated following hypoosmotic challenge in the stenohaline marine fish.

Expression of arginine vasotocin in distinct preoptic regions is associated with dominant and subordinate behaviour in an African cichlid fish.

Neuropeptides have widespread modulatory effects on behaviour and physiology and are associated with phenotypic transitions in a variety of animals. Arginine vasotocin (AVT) is implicated in mediating alternative male phenotypes in teleost fish, but the direction of the association differs among species, with either higher or lower AVT related to more territorial behaviour in different fishes. To clarify the complex relationship between AVT and alternative phenotype, we evaluated AVT expression in an African cichlid in which social status is associated with divergent behaviour and physiology. We compared AVT mRNA expression between territorial and non-territorial (NT) males in both whole brains and microdissected anterior preoptic areas using transcription profiling, and in individual preoptic nuclei using in situ hybridization. These complementary methods revealed that in the posterior preoptic area (gigantocellular nucleus), territorial males exhibit higher levels of AVT expression than NT males. Conversely, in the anterior preoptic area (parvocellular nucleus), AVT expression is lower in territorial males than NT males. We further correlated AVT expression with behavioural and physiological characteristics of social status to gain insight into the divergent functions of individual AVT nuclei. Overall, our findings highlight a complex association between AVT and social behaviour.

Characterization of CRF, AVT, and ACTH cDNA and pituitary-adrenal axis function in Japanese quail divergently selected for tonic immobility.

Higher corticosterone (CORT) responses to acute stress have previously been reported in quail selected for short (STI) duration of tonic immobility (TI) than for long TI (LTI), although behavioral studies indicated that LTI quail were more fearful. To investigate adrenal and pituitary function in these quail lines and their possible involvement in the differences in hypothalamic-pituitary-adrenal (HPA) axis reactivity, we measured CORT responses to adrenocorticotropin (1-24 ACTH), corticotropin-releasing factor (CRF), and arginine vasotocin (AVT) after characterizing the nucleotide acid sequences of these peptides in quail. Although maximum adrenal responses, assessed by ACTH challenge, were higher in STI quail, adrenal sensitivity was comparable for the two genotypes. It is therefore unlikely that differences in HPA axis reactivity involved the adrenal level. AVT and ACTH induced comparable CORT responses in both genotypes, whereas those induced by CRF were much lower. AVT is thus more potent than CRF in quail, but the respective maximum pituitary capacity of both genotypes to secrete ACTH was similar, and it is doubtful that the AVT pathway is involved in the difference in HPA axis reactivity between genotypes. On the other hand, the higher CORT responses induced by CRF in STI quail suggest that CRF might be involved in the differences in HPA axis reactivity between LTI and STI genotypes.

Novel expression and functional role of ghrelin in chicken ovary.

Ghrelin has recently emerged as pleiotropic regulator of a wide array of endocrine and non-endocrine functions. The former likely includes the control of gonadal function, as expression of ghrelin and its putative receptor, the GH secretagogue receptor type 1a (GHS-R1a), has been described in mammalian gonads, and direct effects of ghrelin in the control of testicular secretion and cell proliferation have been reported. Yet, the expression and/or functional role of ghrelin in gonads from non-mammalian species remain to be analyzed. The present study aimed to evaluate the expression of ghrelin and GHS-R genes in the chicken ovary, and to assess the potential involvement of ghrelin in the direct control of chick ovarian function. To this end, RT-PCR assays for ghrelin and GHS-R1a mRNAs were performed in ovarian tissue, and cultures of chicken ovarian cells were conducted in the presence of increasing doses (1, 10 or 100 ng/ml) of the ghrelin analog, ghrelin 1-18. Our results demonstrate that both ghrelin and GHS-R1a mRNAs are expressed in chick ovarian tissue. Moreover, challenge of ovarian granulosa cells with ghrelin 1-18 was able to induce markers of proliferation (i.e. expression of both PCNA and cyclin), and to modulate markers of apoptosis (i.e. decreased expression of caspase-3, bax, bcl-2 and TUNEL-positive cells). Moreover, ghrelin 1-18 increased the expression of PCNA, cyclin, bax and p53 in cultures of ovarian follicular fragments, where it also stimulated the release of progesterone, estradiol, arginine-vasotocin (AVT) and IGF-I, but not of testosterone. In conclusion, our study provides novel evidence for the gonadal expression of the genes encoding ghrelin and its cognate receptor in a non-mammalian species, i.e. the chicken ovary, and unravels the potential involvement of this newly discovered molecule in the control of key gonadal functions in the chick, such as proliferation, apoptosis, and hormone release.

The medial preoptic nucleus receives vasotocinergic inputs in male quail: a tract-tracing and immunocytochemical study.

The sexually dimorphic testosterone-sensitive medial preoptic nucleus (POM) of quail can be identified by the presence of a dense network of vasotocinergic fibers. This innervation is sexually differentiated (present in males only) and testosterone sensitive. The origin of these fibers has never been formally identified although their steroid sensitivity suggests that they originate in parvocellular vasotocinergic neurons that are found in quail only in the medial part of the bed nucleus striae terminalis (BSTm) and in smaller numbers within the POM itself. We report here that following injections of a retrograde tracer into the POM of male quail, large populations of retrogradely labeled cells can be identified in the BSTm. The POM also receives afferent projections from magnocellular vasotocinergic nuclei, the supraoptic and paraventricular nuclei. Double labeling for vasotocin immunoreactivity of the retrogradely labeled sections failed however to clearly identify magnocellular vasotocin-immunoreactive cells that were retrogradely labeled from POM. In contrast a substantial population of vasotocin-immunoreactive neurons in the BSTm contained tracer retrogradely transported from the POM. These data therefore demonstrate that a significant part of the vasotocinergic innervation of the quail POM originates in the medial part of the BST. An intrinsic innervation could however also contribute to this network. This interaction between BSTm and POM could play a key role in the control of male-typical sexual behavior and in its sex dimorphism in quail.

Development of sexually dimorphic vasotocinergic system in the bed nucleus of stria terminalis in chickens.

The bed nucleus of stria terminalis (BnST) of the domestic fowl contains two groups of parvicellular vasotocinergic neurons that are sexually dimorphic. In adult cockerels, arginine vasotocin (AVT) synthesis is well expressed in the dorsolateral and ventromedial portions of the BnST, whereas in corresponding brain areas of hens, AVT synthesis is completely lacking. In the present study, in situ hybridization and immunocytochemical methods were used to compare the ontogeny of sexually dimorphic AVT gene expression in the BnST of male and female chickens from day 12 of embryonic development (E12) until the onset of sexual maturation. By E12, both parvicellular groups of AVT-immunoreactive (AVT-ir) perikarya in the developing BnST can be distinguished in some males, whereas in females their presence is questionable. A quantitative analysis, beginning at E14, showed that the parvicellular dorsolateral portion of the BnST of male embryos had more AVT perikarya compared with females. In contrast, no evident sex difference in distribution pattern and number of AVT mRNA containing neurons in this BnST portion was observable by in situ hybridization at E15. At E18, as well as on the first and second days posthatch (D1 and D2), no differences in the number of AVT synthesizing cells and intensity of immunoreactive staining in male versus female chickens were found. Between D2 and D7, the number of AVT-ir cells in the BnST declined rapidly in both sexes until it disappeared completely in females before D35. In males, another increase in sexually dimorphic AVT-ir cells and innervation of the lateral septum was associated with the onset of puberty and fully matched a pattern observed in adult fowls. These results demonstrate that the sexually dimorphic part of the AVT system undergoes sexual differentiation during early stages of ontogeny.

Arginine vasotocin mRNA revealed by in situ hybridization in bovine pineal gland cells.

Arginine vasopressin (AVP) is the main antidiuretic hormone in mammals and arginine vasotocin (AVT) in submammalian vertebrates. The possibility that the genetic material encoding AVT is maintained in mammals is controversial. In this study, we investigated by radioactive in situ hybridization the possible presence of the mRNA encoding AVP and AVT, and using immunocytochemistry the presence of structures immunoreactive for AVP and AVT in the bovine pineal gland. In situ hybridization was performed by use of 35S-labelled oligoprobes. Immunocytochemistry was performed using specific polyclonal rabbit antibodies and the avidin-biotin-complex method. In situ hybridization revealed positive signals for both AVP mRNA and AVT mRNA in a few cells scattered throughout the pineal body. Immunocytochemistry revealed thin AVP-immunoreactive fibres in the pineal stalk and the pineal gland. It also revealed staining of several AVT-immunoreactive nerve fibres in both the pineal stalk and the gland. In addition, polyhedral, neuron-like cell bodies from which two to three processes emerged were also AVT-immunoreactive. Thus, our investigation shows the presence of AVP/AVT-immunoreactive cellular structures in the bovine pineal gland. Our data further show the presence of mRNAs encoding both AVT and AVP. We therefore suggest that AVT mRNA is translated into an AVT-like peptide in the bovine pineal.

Effects of testosterone on sexually dimorphic parvocellular neurons expressing vasotocin mRNA in the male quail brain.

In situ hybridization with a P33-labelled cDNA probe was used to analyze the effects of castration and replacement therapy by testosterone on the number of neurons expressing vasotocin mRNA in the male quail brain. Castration completely eliminated neurons expressing vasotocin mRNA in the previously described parvocellular vasotocin cell groups, located in the medial preoptic nucleus and in the anterior and posterior part of the medial subdivision of the bed nucleus of the stria terminalis. These effects were completely reversed by a 3-week treatment with exogenous testosterone. No marked change in vasotocin expression could be detected in the magnocellular cell groups located in the paraventricular and supraoptic nuclei. These data indicate that the testosterone-induced changes in the vasotocinergic innervation of the quail medial preoptic region and bed nucleus of the stria terminalis result from controlling mechanisms at the pretranslational, presumably transcriptional level. These control mechanisms are therefore very similar to those described for the rat brain despite the existence of major differences in the neuroanatomical organization of this peptidergic system in the two species.

Sites of origin and patterns of migration of vasotocin/mesotocin neurons in developing brain of the chick.

Vasotocin/mesotocin (VT/MT) producing neurons are known to migrate extensively during development of the hypothalamus. Birthdating studies as well as immunohistochemical studies suggested the possibility that VT/MT producing neurons originate from specific sites of the neural tube. Furthermore, a relationship between the site of origin and the eventual fate of VT/MT cells has been suggested. This study proposes to identify the sites of origin of VT/MT cells and to establish whether magnocellular and parvocellular VT/MT, and neuromodulatory and neurosecretory VT/MT arise from common or different areas of the developing neural tube. To do so, the embryological distribution of VT/MT producing neurons of the chick was studied with immunohistochemistry. Analysis of the youngest brains in which VT/MT cells could be detected (embryonic day 7.25, E7.25) suggested the presence of two separate sites of origin. The first site was located in the hypothalamic anlage, next to the third ventricle, and the second in the mesencephalon, next to the fourth ventricle. Three-dimensional reconstructions of the location of VT/MT cells throughout development substantiated the hypothesis that diencephalic VT/MT cells originate from the first site while mesencephalic ones originate from the second site. Mesencephalic VT/MT producing cells were confined to the nucleus of Edinger-Westphal and were only detectable during a brief period in development (E7.25-E10). Diencephalic VT/MT producing neurons were noted to form two main paths from their sites of origin to the rostral diencephalon. Quantitative analysis confirmed this caudal to rostral displacement. Magnocellular and parvocellular VT/MT+ cells were intermingled at the diencephalic site of origin as well as in the migratory paths. Neuromodulatory and neurosecretory VT/MT cells of the diencephalon appeared to be derived from a common diencephalic site of origin. These studies support the hypothesis that while specific groups of progenitors may be important in allowing their offspring to produce VT/MT, they do not appear to influence the morphological attributes (magnocellular vs. parvocellular), nuclear locations, or functional characteristics of these cells.

Divergence of gene expression in neurohypophysial hormone precursors among salmonids.

Salmonid fish have pairs of genes for various hypothalamic and pituitary hormones including neurohypophysial hormones, vasotocin, and isotocin, probably because they are tetraploid. The problem here is whether two genes for the same hormone are expressed equally or differently. We therefore examined expression patterns of vasotocin and isotocin genes in four salmonid species using Northern blot analysis with chum salmon cDNAs as hybridization probes. The presence of two vasotocin and also two isotocin genes was confirmed by Southern blot analysis in rainbow trout and sockeye salmon which were not examined previously. Prior to Northern blot analysis, isotocin-I cDNA of sockeye salmon was determined and compared to those of chum salmon and masu salmon, since molecular probes are so specific that cross-species hybridization often leads misinterpretation in a quantitative study. The nucleotide sequence of sockeye salmon isotocin-I cDNA showed sufficiently high homology (> 96.0%) to those of chum salmon and masu salmon for cross-species hybridization among salmonids. Northern blot analysis showed that both isotocin-I and isotocin-II genes were well expressed in all species examined. Expression of isotocin-I gene tended to be relatively higher than that of isotocin-II gene in all species. However, expression pattern of vasotocin-I and vasotocin-II genes did not coincide among species. Expression of vasotocin-II genes was very weak or scarce in masu salmon and rainbow trout, while that in sockeye salmon was stronger than vasotocin-I gene expression. The present result may reflect complicated molecular evolution of salmonid vasotocin genes probably both in regulatory and coding regions.