Sinhcaf-dependent histone deacetylation is essential for primordial germ cell specification.

Primordial germ cells (PGCs) are the progenitor cells that give rise to sperm and eggs. Sinhcaf is a recently identified subunit of the Sin3 histone deacetylase complex (SIN3A-HDAC). Here, we provide evidence that Sinhcaf-dependent histone deacetylation is essential for germ plasm aggregation and primordial germ cell specification. Specifically, maternal-zygotic sinhcaf zebrafish mutants exhibit germ plasm aggregation defects, decreased PGC abundance and male-biased sex ratio, which can be rescued by re-expressing sinhcaf. Overexpression of sinhcaf results in excess PGCs and a female-biased sex ratio. Sinhcaf binds to the promoter region of kif26ab. Loss of sinhcaf epigenetically switches off kif26ab expression by increasing histone 3 acetylation in the promoter region. Injection of kif26ab mRNA could partially rescue the germ plasm aggregation defects in sinhcaf mutant embryos. Taken together, we demonstrate a role of Sinhcaf in germ plasm aggregation and PGC specialization that is mediated by regulating the histone acetylation status of the kif26ab promoter to activate its transcription. Our findings provide novel insights into the function and regulatory mechanisms of Sinhcaf-mediated histone deacetylation in PGC specification.

Rearing density and food variety impact growth, development, and survival of larvae in the declining amphibian, Pseudacris maculata.

Boreal chorus frogs (Pseudacris maculata Agassiz 1850) are a widespread amphibian in North America, but several populations are in decline. Specifically, we are developing captive breeding and reintroduction methods for the Great Lakes/St. Lawrence-Canadian Shield population. Here we present the effects of tadpole density, food variety, and addition of supplemental minerals to rearing water on the growth, development, and survival during the larval and metamorph/juvenile stages. We conducted two experiments using a factorial design. We found that low tadpole density (1 vs. 2 tadpoles/L) and high food variety (five food items vs. three food items) significantly increased tadpole body length and Gosner stage after 2 weeks (p < .001), increased survival to metamorphosis (p < .001), decreased time to metamorphosis (p < .001), and increased weight after metamorphosis (p < .001). On average, tadpoles in the high density/low food treatment, compared to the low density/high food, were 25% smaller after 2 weeks, had 3.9× lower survival to metamorphosis, took 1.25× longer to reach metamorphosis, and weighed 1.5× less after metamorphosis. In contrast, neither density (0.5 vs. 1 tadpole/L) nor mineral supplemented water affected growth and development, but tadpole survival was higher at 1 tadpole/L. Our findings demonstrate the feasibility of rearing boreal chorus frogs in captivity and provide guidelines for rearing this and similar species in a laboratory environment.

Fighting cichlids: An integrated multimodal analysis to understand female and male aggression in Cichlasoma dimerus.

Aggression has been historically linked to males and androgen levels and, even if females from different species also display aggressive behavior, female aggression is still widely understudied. The aim of the present work is to disentangle how sex differences in social plasticity can be explained by sex steroid hormone levels, gonadal state and/or morphometric characteristics. In this context, we performed intrasexual dyadic encounters to identify social plasticity after acquiring a winner or loser status in males and females of Cichlasoma dimerus. This integral analysis suggests that the reproductive and hormonal variables analyzed explain the behavioral variation among winner and loser males and females, and that there are significant differences between sexes and contest outcome when individual morphometric variables are excluded from the analysis. Interestingly, there are no sex differences in aggressive and submissive behaviors, and clustering into winners and losers is mainly explained by specific behavioral displays, such as bites, chases, approaches, passive copings, and escapes. Correlation heatmaps show a positive correlation between estradiol with aggression and a negative correlation with submission, suggesting estrogens may have a dual role regulating agonistic behavior. Finally, these results suggest that size difference can help to understand aggression in females but not in males, and that assessment of the opponent's body size is important to understand aggression also before the initiation of the contest in both sexes. Overall, this study constitutes an integral approach adding insights into the importance of reproductive and hormonal variables to understand social plasticity in males and females.

Targeted mutation of secretogranin-2 disrupts sexual behavior and reproduction in zebrafish.

The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. We previously reported that secretoneurin-a, a neuropeptide derived from the processing of secretogranin-2a (Scg2a), stimulates luteinizing hormone release, suggesting a role in reproduction. Here we provide evidence that mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. Large-scale spawning within-line crossings (n = 82 to 101) were conducted. Wild-type (WT) males paired with WT females successfully spawned in 62% of the breeding trials. Spawning success was reduced to 37% (P = 0.006), 44% (P = 0.0169), and 6% (P < 0.0001) for scg2a-/- , scg2b-/- , and scg2a-/-;scg2b-/- mutants, respectively. Comprehensive video analysis indicates that scg2a-/-;scg2b-/- mutation reduces all male courtship behaviors. Spawning success was 47% in saline-injected WT controls compared to 11% in saline-injected scg2a-/-;scg2b-/- double mutants. For these mutants, spawning success increased 3-fold following a single intraperitoneal (i.p.) injection of synthetic secretoneurin-a (P = 0.0403) and increased 3.5-fold with injection of human chorionic gonadotropin (hCG). Embryonic survival at 24 h remained on average lower in scg2a-/-;scg2b-/- fish compared to WT injected with secretoneurin-a (P < 0.001). Significant reductions in the expression of gonadotropin-releasing hormone 3 in the hypothalamus, and luteinizing hormone beta and glycoprotein alpha subunits in the pituitary provide evidence for disrupted hypothalamo-pituitary function in scg2a and scg2b mutant fish. Our results indicate that secretogranin-2 is required for optimal reproductive function and support the hypothesis that secretoneurin is a reproductive hormone.

A cross-species comparative approach to assessing multi- and transgenerational effects of endocrine disrupting chemicals.

A wide range of chemicals have been identified as endocrine disrupting chemicals (EDCs) in vertebrate species. Most studies of EDCs have focused on exposure of both male and female adults to these chemicals; however, there is clear evidence that EDCs have dramatic effects when mature or developing gametes are exposed, and consequently are associated with in multigenerational and transgenerational effects. Several publications have reviewed such actions of EDCs in subgroups of species, e.g., fish or rodents. In this review, we take a holistic approach synthesizing knowledge of the effects of EDCs across vertebrate species, including fish, anurans, birds, and mammals, and discuss the potential mechanism(s) mediating such multi- and transgenerational effects. We also propose a series of recommendations aimed at moving the field forward in a structured and coherent manner.

Conserved functions of hypothalamic kisspeptin in vertebrates.

Hypothalamic kisspeptin encoded by KISS1/Kiss1 gene emerged as a regulator of the reproductive axis in mammals following the discovery of the kisspeptin receptor (Kissr) and its role in reproduction. Kisspeptin-Kissr systems have been investigated in various vertebrates, and a conserved sequence of kisspeptin-Kissr has been identified in most vertebrate species except in the avian linage. In addition, multiple paralogs of kisspeptin sequences have been identified in the non-mammalian vertebrates. The allegedly conserved role of kisspeptin-Kissr in reproduction became debatable when kiss/kissr genes-deficient zebrafish and medaka showed no apparent effect on the onset of puberty, sexual development, maturation and reproductive capacity. Therefore, it is questionable whether the role of kisspeptin in reproduction is conserved among vertebrate species. Here we discuss from a comparative and evolutional aspect the diverse functions of kisspeptin and its receptor in vertebrates. Primarily this review focuses on the role of hypothalamic kisspeptin in reproductive and non-reproductive functions that are conserved in vertebrate species.

Life history traits and reproductive ecology of North American chorus frogs of the genus Pseudacris (Hylidae).

Amphibian biodiversity is declining globally, with over 40% of species being considered threatened to become extinct. Crucial to the success of conservation initiatives are a comprehensive understanding of life history and reproductive ecology of target species. Here we provide an overview of the Pseudacris genus, including breeding behaviour, reproduction, development, survival and longevity. We present an updated distribution map of the 18 species found throughout North America. We also summarize the conservation status at the national and subnational (state, provincial, and territorial) levels, in Canada, USA, and Mexico, to evaluate the relationship between life history traits and extinction risk. Results show a high degree of consistency in the life history traits of Pseudacris species considering their relative diversity and wide distribution in North America. However, data are lacking for several species, particularly in the Fat Frog and West Coast clades, causing some uncertainties and discrepancies in the literature. We also found that the most threatened populations of chorus frog were located in the east coast of the USA, potentially as a result of increased levels of anthropogenic disturbance. We suggest that the similarities in life history traits among chorus frog species provides an opportunity for collaboration and united efforts for the conservation of the genus.

Transgenerational hypocortisolism and behavioral disruption are induced by the antidepressant fluoxetine in male zebrafish Danio rerio.

The global prevalence of depression is high during childbearing. Due to the associated risks to the mother and baby, the selective serotonin reuptake inhibitor fluoxetine (FLX) is often the first line of treatment. Given that FLX readily crosses the placenta, a fetus may be susceptible to the disruptive effects of FLX during this highly plastic stage of development. Here, we demonstrate that a 6-day FLX exposure to a fetus-relevant concentration at a critical developmental stage suppresses cortisol levels in the adult zebrafish (F0). This effect persists for three consecutive generations in the unexposed descendants (F1 to F3) without diminution and is more pronounced in males. We also show that the in vivo cortisol response of the interrenal (fish "adrenal") to an i.p. injection of adrenocorticotropic hormone was also reduced in the males from the F0 and F3 FLX lineages. Transcriptomic profiling of the whole kidney containing the interrenal cells revealed that early FLX exposure significantly modified numerous pathways closely associated with cortisol synthesis in the male adults from the F0 and F3 generations. We also show that the low cortisol levels are linked to significantly reduced exploratory behaviors in adult males from the F0 to F2 FLX lineages. This may be a cause for concern given the high prescription rates of FLX to pregnant women and the potential long-term negative impacts on humans exposed to these therapeutic drugs.

A Review of the Effects of the Biopesticides Bacillus thuringiensis Serotypes israelensis (Bti) and kurstaki (Btk) in Amphibians.

Insecticides are important in agriculture, to reduce human disease, and to decrease the nuisance of biting insects. Despite this, many have the potential for environmental impacts and toxicity in nontarget organisms. We reviewed data on the effects of insecticides based on toxins from Bacillus thuringiensis var. israelensis (Bti) and Bacillus thuringiensis var. kurstaki (Btk) on amphibians. The few peer-reviewed publications that are available for Bti provide variable conclusions, ranging from few observable effects to evidence of acute toxicity at high concentrations. We briefly highlight the current controversies and identify key areas for future investigation.

Multimodal hypothalamo-hypophysial communication in the vertebrates.

The vertebrate pituitary is arguably one of the most complex endocrine glands from the evolutionary, anatomical and functional perspectives. The pituitary plays a master role in endocrine physiology for the control of growth, metabolism, reproduction, water balance, and the stress response, among many other key processes. The synthesis and secretion of pituitary hormones are under the control of neurohormones produced by the hypothalamus. Under this conceptual framework, the communication between the hypophysiotropic brain and the pituitary gland is at the foundation of our understanding of endocrinology. The anatomy of the connections between the hypothalamus and the pituitary gland has been described in different vertebrate classes, revealing diverse modes of communication together with varying degrees of complexity. In this context, the evolution and variation in the neuronal, neurohemal, endocrine and paracrine modes will be reviewed in light of recent discoveries, and a re-evaluation of earlier observations. There appears to be three main hypothalamo-pituitary communication systems: 1. Diffusion, best exemplified by the agnathans; 2. Direct innervation of the adenohypophysis, which is most developed in teleost fish, and 3. The median eminence/portal blood vessel system, most conspicuously developed in tetrapods, showing also considerable variation between classes. Upon this basic classification, there exists various combinations possible, giving rise to taxon and species-specific, multimodal control over major physiological processes. Intrapituitary paracrine regulation and communication between folliculostellate cells and endocrine cells are additional processes of major importance. Thus, a more complex evolutionary picture of hypothalamo-hypophysial communication is emerging. There is currently little direct evidence to suggest which neuroendocrine genes may control the evolution of one communication system versus another. However, studies at the developmental and intergenerational timescales implicate several genes in the angiogenesis and axonal guidance pathways that may be important.

Kisspeptin and GnRH interactions in the reproductive brain of teleosts.

It is well known that gonadotropin-releasing hormone (Gnrh) has a key role in reproduction by regulating the synthesis and release of gonadotropins from the anterior pituitary gland of all vertebrates. About 25 years ago, another neuropeptide, kisspeptin (Kiss1) was discovered as a metastasis suppressor of melanoma cell lines and then found to be essential for mammalian reproduction as a stimulator of hypothalamic Gnrh and regulator of puberty onset. Soon after, a kisspeptin receptor (kissr) was found in the teleost brain. Nowadays, it is known that in most teleosts the kisspeptin system is composed of two ligands, kiss1 and kiss2, and two receptors, kiss2r and kiss3r. Even though both kisspeptin peptides, Kiss1 and Kiss2, have been demonstrated to stimulate gonadotropin synthesis and secretion in different fish species, their actions appear not to be mediated by Gnrh neurons as in mammalian models. In zebrafish and medaka, at least, hypophysiotropic Gnrh neurons do not express Kiss receptors. Furthermore, kisspeptinergic nerve terminals reach luteinizing hormone cells in some fish species, suggesting a direct pituitary action. Recent studies in zebrafish and medaka with targeted mutations of kiss and/or kissr genes reproduce relatively normally. In zebrafish, single gnrh mutants and additionally those having the triple gnrh3 plus 2 kiss mutations can reproduce reasonably well. In these fish, other neuropeptides known to affect gonadotropin secretion were up regulated, suggesting that they may be involved in compensatory responses to maintain reproductive processes. In this context, the present review explores and presents different possibilities of interactions between Kiss, Gnrh and other neuropeptides known to affect reproduction in teleost fish. Our intention is to stimulate a broad discussion on the relative roles of kisspeptin and Gnrh in the control of teleost reproduction.

Secretoneurin is a secretogranin-2 derived hormonal peptide in vertebrate neuroendocrine systems.

Secretogranin-2 (SCG2) is a large precursor protein that is processed into several potentially bioactive peptides, with the 30-43 amino acid central domain called secretoneurin (SN) being clearly evolutionary conserved in vertebrates. Secretoneurin exerts a diverse array of biological functions including regulating nervous, endocrine, and immune systems in part due to its wide tissue distribution. Expressed in some neuroendocrine neurons and pituitary cells, SN is a stimulator of the synthesis and release of luteinizing hormone from both goldfish pituitary cells and the mouse LßT2 cell line. Neuroendocrine, paracrine and autocrine signaling pathways for the stimulation of luteinizing hormone release indicate hormone-like activities to regulate reproduction. Mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. A single injection of the SNa peptide enhanced reproductive outcomes in scg2a/scg2b double mutant zebrafish. Evidence in goldfish suggests a new role for SN to stimulate food intake by actions on other feeding-related neuropeptides. Expression and regulation of the Scg2a precursor mRNA in goldfish gut also supports a role in feeding. In rodent models, SN has trophic-like properties promoting both neuroprotection and neuronal plasticity and has chemoattractant properties that regulate neuroinflammation. Data obtained from several cellular models suggest that SN binds to and activates a G-protein coupled receptor (GPCR), but a bona fide SN receptor protein needs to be identified. Other signaling pathways for SN have been reported which provides alternatives to the GPCR hypothesis. These include AMP-activated protein kinase (AMPK), extracellular signal-regulated kinases (ERK), mitogen-activated protein kinase (MAPK)and calcium/calmodulin-dependent protein kinase II in cardiomyocytes, phosphatidylinositol 3-kinase (PI3K) and Akt/Protein Kinase B (AKT, and MAPK in endothelial cells and Janus kinase 2/signal transducer and activator of transcription protein (JAK2-STAT) signaling in neurons. Some studies in cardiac cells provide evidence for cellular internalization of SN by an unknown mechanism. Many of the biological functions of SN remain to be fully characterized, which could lead to new and exciting applications.

Cortisol disruption and transgenerational alteration in the expression of stress-related genes in zebrafish larvae following fluoxetine exposure.

Fluoxetine (FLX), the active ingredient in well-known therapeutic drugs such as Prozac, is highly prescribed worldwide to treat affective disorders even among pregnant women and adolescents. Given that FLX readily crosses the placenta, a fetus from a treated pregnant woman is potentially at risk from unintended effects of the chemical. Moreover, FLX reaches aquatic ecosystems at biologically active levels through sewage release, so fish may also be inadvertently affected. We previously demonstrated that FLX exposure to environmentally- (Low FLX Lineage; LFL) and human- (High FLX Lineage; HFL) relevant concentrations during the first 6 days of life in zebrafish (ZF; Danio rerio) reduced cortisol levels in the adults (F0), an effect that persisted across 3 consecutive unexposed generations (F1 to F3). Here, we show that the transcriptional profile of selected genes in the steroidogenesis pathway in the F0 whole-larvae varied in magnitude and direction in both FLX lineages, despite the same attenuated cortisol phenotype induced by both concentrations. We also observed an up-regulation in the transcript levels of some steroidogenic-related genes and a down-regulation of a gene involved in the inactivation of cortisol in the F3 HFL larvae. These findings on the transcript levels of the selected genes in the larvae from F0 and F3 suggest that specific coping mechanism(s) are activated in descendants to attempt to counteract the disruptive effects of FLX. Our data are cause for concern, given the increasing prescription rates of FLX and other antidepressants, and the potential long-term negative impacts on humans and aquatic organisms.

Naphthenic Acid Mixtures and Acid-Extractable Organics from Oil Sands Process-Affected Water Impair Embryonic Development of Silurana (Xenopus) tropicalis.

Naphthenic acids (NAs) are carboxylic acids naturally occurring in crude oils and bitumen and are suspected to be the primary toxic substances in wastewaters associated with oil refineries and mining of oil sands. Oil sands process-affected water (OSPW) generated by the extraction of bitumen from oil sands are a major source of NAs and are currently stored in tailings ponds. We report on the acute lethality and teratogenic effects of aquatic exposure of Silurana (Xenopus) tropicalis embryos to commercial NA extracts and from the acid extractable organics (AEOs) fraction of a Canadian OSPW. Using electrospray ionization-high resolution mass spectrometry, we determined that the O2 species proportion were 98.8, 98.9 and 58.6% for commercial mixtures Sigma 1 (S1M) and Sigma 2 (S2M), and AEOs, respectively. The 96h LC50 estimates were 10.4, 11.7, and 52.3 mg/L for S1M, S2M, and the AEOs, respectively. The 96h EC50 estimates based on frequencies of developmental abnormalities were 2.1, 2.6, and 14.2 mg/L for S1M, S2M, and the AEOs, respectively. The main effects observed were reduced body size, edema, and cranial, heart, gut and ocular abnormalities. Increasing concentrations of the mixtures resulted in increased severity and frequency of abnormalities ( p < 0.05). The rank-order potency was S1M > S2M > AEO based on LC50 and EC50 estimates. These data provide insight into the effects NAs in amphibian embryos and can contribute to the development of environmental guidelines for the management of OSPW.

Secretoneurin-A inhibits aromatase B (cyp19a1b) expression in female goldfish (Carassius auratus) radial glial cells.

In the teleost brain, radial glial cells (RGCs) are the main macroglia and are stem-like progenitors that express key steroidogenic enzymes, including the estrogen-synthesizing enzyme, aromatase B (cyp19a1b). As a result, RGCs are integral to neurogenesis and neurosteroidogenesis, however little is known about the regulatory factors and signaling mechanisms that control these functions. A potential new role of the secretogranin II-derived neuropeptide secretoneurin A (SNa) in the control of goldfish (Carassius auratus) RGC function is the subject of this study. Immunohistochemistry revealed a close neuroanatomical relationship between RGCs and soma of SNa-immunoreactive magnocellular and parvocellular neurons in the preoptic nucleus of female goldfish. Five hours following intracerebroventricular injection of 0.2ng/g SNa cyp19a1b mRNA levels were decreased by 86% (P<0.05) in the hypothalamus and by 88% (P<0.05) in the telencephalon. In vitro, 24 h incubation with 500nM SNa decreased cyp19a1b mRNA by 51% (P<0.05) in cultured RGCs. These data provide evidence that SNa can regulate aromatase expression in goldfish RGCs. By regulating neuroestrogen production in RGCs SNa may therefore be implicated in the control of major estrogen-dependent functions of the preoptic region such as reproductive behavior and osmoregulation.

Differential success in obtaining gametes between male and female Australian temperate frogs by hormonal induction: A review.

Most Australian frogs fall into two deeply split lineages, conveniently referred to as ground frogs (Myobatrachidae and Limnodynastidae) and tree frogs (Pelodryadidae). Species of both lineages are endangered because of the global chytrid pandemic, and there is increasing interest and research on the endocrine manipulation of reproduction to support the use of assisted reproductive technologies in conservation. Hormonal induction of gamete release in males and females is one such manipulation of the reproductive process. This paper reviews progress in temperate ground and tree frogs towards developing simple and efficient hormonal protocols for induction of spermiation and ovulation, and presents some new data, that together build towards an understanding of advances and obstacles towards progress in this area. We report that protocols for the non-invasive induction of sperm release, relying on single doses of gonadotropin-releasing hormone (GnRH) or human chorionic gonadotropin are very effective in both ground and tree frog species investigated to date. However, we find that, while protocols based on GnRH, and GnRH and dopamine antagonists, are moderately efficient in inducing ovulation in ground frogs, the same cannot be said for the use of such protocols in tree frogs. Although induced ovulation in the pelodryadid tree frogs has not been successfully implemented, and is difficult to explain in terms of the underlying endocrinology, we propose future avenues of investigation to address this problem, particularly the need for a source of purified or recombinant follicle-stimulating hormone and luteinising hormone for species from this group.

Neuroendocrine disruption of organizational and activational hormone programming in poikilothermic vertebrates.

In vertebrates, sexual differentiation of the reproductive system and brain is tightly orchestrated by organizational and activational effects of endogenous hormones. In mammals and birds, the organizational period is typified by a surge of sex hormones during differentiation of specific neural circuits; whereas activational effects are dependent upon later increases in these same hormones at sexual maturation. Depending on the reproductive organ or brain region, initial programming events may be modulated by androgens or require conversion of androgens to estrogens. The prevailing notion based upon findings in mammalian models is that male brain is sculpted to undergo masculinization and defeminization. In absence of these responses, the female brain develops. While timing of organizational and activational events vary across taxa, there are shared features. Further, exposure of different animal models to environmental chemicals such as xenoestrogens such as bisphenol A-BPA and ethinylestradiol-EE2, gestagens, and thyroid hormone disruptors, broadly classified as neuroendocrine disrupting chemicals (NED), during these critical periods may result in similar alterations in brain structure, function, and consequently, behaviors. Organizational effects of neuroendocrine systems in mammals and birds appear to be permanent, whereas teleost fish neuroendocrine systems exhibit plasticity. While there are fewer NED studies in amphibians and reptiles, data suggest that NED disrupt normal organizational-activational effects of endogenous hormones, although it remains to be determined if these disturbances are reversible. The aim of this review is to examine how various environmental chemicals may interrupt normal organizational and activational events in poikilothermic vertebrates. By altering such processes, these chemicals may affect reproductive health of an animal and result in compromised populations and ecosystem-level effects.

Time- and dose-related effects of a gonadotropin-releasing hormone agonist and dopamine antagonist on reproduction in the Northern leopard frog (Lithobates pipiens).

Gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone release to control ovulation and spermiation in vertebrates. Dopamine (DA) has a clear inhibitory role in the control of reproduction in numerous teleosts, and emerging evidence suggests that similar mechanisms may exist in amphibians. The interactions between GnRH and DA on spawning success and pituitary gene expression in the Northern leopard frog (Lithobates pipiens) were therefore investigated. Frogs were injected during the natural breeding season with a GnRH agonist [GnRH-A; (Des-Gly10, D-Ala6, Pro-NHEt9)-LHRH; 0.1µg/g and 0.4µg/g] alone and in combination with the dopamine receptor D2 antagonist metoclopramide (MET; 5µg/g and 10µg/g). Injected animals were allowed to breed in outdoor mesocosms. Time to amplexus and oviposition were assessed, and egg mass release, incidences of amplexus, egg mass weight, total egg numbers and fertilization rates were measured. To examine gene expression, female pituitaries were sampled at 12, 24 and 36h following injection of GnRH-A (0.4µg/g) alone and in combination with MET (10µg/g). The mRNA levels of the genes lhb, fshb, gpha, drd2 and gnrhr1 were measured using quantitative real-time PCR. Data were analyzed by a two-way ANOVA. Both GnRH-A doses increased amplexus, oviposition and fertilization alone. Co-injection of MET with GnRH-A did not further enhance spawning success. Injection of GnRH-A alone time-dependently increased expression of lhb, fshb, gpha and gnrhr1. The major effect of MET alone was to decrease expression of drd2. Importantly, the stimulatory effects of GnRH-A on lhb, gpha and gnrhr1 were potentiated by the co-injection of MET at 36h. At this time, expression of fshb was increased only in animals injected with both GnRH-A and MET. Spawning success was primarily driven by the actions of GnRH-A. The hypothesized inhibitory action of DA was supported by pituitary gene expression analysis. The results from this study provide a fundamental framework for future time- and dose-response investigations to improve current spawning methods in amphibians.

The nonapeptide isotocin in goldfish: Evidence for serotonergic regulation and functional roles in the control of food intake and pituitary hormone release.

Nonapeptides are a highly conserved family of peptides synthesized in the neuroendocrine brain and acting on central and peripheral receptors to regulate physiological functions in vertebrates. While the evolution of the two gene families of oxytocin-like and vasopressin-like nonapeptides and their receptors, as well as the neuroanatomy of their independent neuronal circuits have been well-characterized across vertebrate species, comparative studies on the physiological roles across vertebrates are lagging behind. In the current study, we focused on the comparative neuroendocrine functions and regulation of isotocin, the teleost homologue of mammalian oxytocin. Specifically, we address the hypothesis that isotocin exerts opposing effects on food intake and reproduction, which are well-established effects of its homologue oxytocin in mammalian species. Using goldfish, a well-characterized model of neuroendocrine regulation of both food intake and reproduction, we here showed that isotocin acts as an anorexigenic factor while exerting stimulatory effects on pituitary luteinizing hormone and growth hormone release. Given the dual inhibitory and stimulatory roles of serotonin on food intake and pituitary release of reproductive hormone in goldfish, we also investigated the potential crosstalk between both systems using immunohistochemistry and pharmacological approaches. Results provide neuroanatomical and pharmacological evidence for serotonergic regulation of magnocellular isotocinergic neurons in the preoptic area and pituitary. Together, these findings firstly provide the basis to investigate neuroendocrine cross-talk between serotonergic and nonapeptidergic systems in the regulation of both food intake and reproduction in goldfish, and secondly point to a conserved function of oxytocin-like peptides in the differential neuroendocrine control of both physiological processes in vertebrates.