Loss of function of Vasoactive-intestinal peptide alters sex ratio and reduces male reproductive fitness in zebrafish.

Vasoactive-intestinal peptide (Vip) is a pleiotropic peptide with a wide range of distribution and functions. Zebrafish possess two isoforms of Vip (a and b), in which Vipa is most homologous to the mammalian form. In female zebrafish, Vipa can stimulate LH secretion from the pituitary but is not essential for female reproduction, as vipa-/- females display normal reproduction. In contrast, we have found that vipa-/- males are severely sub-fertile and sex ratio of offspring is female-biased. By analyzing all aspects of male reproduction with WT males, we show that the testes of vipa-/- are underdeveloped and contain ∼70% less spermatids compared to WT counterparts. The sperm of vipa-/- males displayed reduced potency in terms of fertilization (by ∼80%) and motility span and duration (by ∼50%). In addition, vipa-/- male attraction to WT females was largely non-existent, indicating decreased sexual motivation. We show that vipa mRNA and protein is present in Leydig cells and in developing germ cells in the testis of WT, raising the possibility that endogenous Vipa contributes to testicular function. Absence of Vipa in vipa-/- males resulted in downregulation of three key genes in the androgen synthesis chain in the testis, 3ß-hsd, 17ß-hsd1 and cyp11c1 (11ß-hydrogenase), associated with a pronounced decrease in 11-ketotestosterone production and, in turn, compromised reproductive fitness. Altogether, this study establishes a crucial role for Vipa in the regulation of male reproduction in zebrafish, like in mammals, with the exception that Vipa is also expressed in zebrafish testis.

Chemogenetic Depletion of Hypophysiotropic GnRH Neurons Does Not Affect Fertility in Mature Female Zebrafish.

The hypophysiotropic gonadotropin-releasing hormone (GnRH) and its neurons are crucial for vertebrate reproduction, primarily in regulating luteinizing hormone (LH) secretion and ovulation. However, in zebrafish, which lack GnRH1, and instead possess GnRH3 as the hypophysiotropic form, GnRH3 gene knockout did not affect reproduction. However, early-stage ablation of all GnRH3 neurons causes infertility in females, implicating GnRH3 neurons, rather than GnRH3 peptides in female reproduction. To determine the role of GnRH3 neurons in the reproduction of adult females, a Tg(gnrh3:Gal4ff; UAS:nfsb-mCherry) line was generated to facilitate a chemogenetic conditional ablation of GnRH3 neurons. Following ablation, there was a reduction of preoptic area GnRH3 neurons by an average of 85.3%, which was associated with reduced pituitary projections and gnrh3 mRNA levels. However, plasma LH levels were unaffected, and the ablated females displayed normal reproductive capacity. There was no correlation between the number of remaining GnRH3 neurons and reproductive performance. Though it is possible that the few remaining GnRH3 neurons can still induce an LH surge, our findings are consistent with the idea that GnRH and its neurons are likely dispensable for LH surge in zebrafish. Altogether, our results resurrected questions regarding the functional homology of the hypophysiotropic GnRH1 and GnRH3 in controlling ovulation.

Vasoactive Intestinal Peptide Indirectly Elicits Pituitary LH Secretion Independent of GnRH in Female Zebrafish.

Vasoactive intestinal peptide (Vip) regulates luteinizing hormone (LH) release through the direct regulation of gonadotropin-releasing hormone (GnRH) neurons at the level of the brain in female rodents. However, little is known regarding the roles of Vip in teleost reproduction. Although GnRH is critical for fertility through the regulation of LH secretion in vertebrates, the exact role of the hypophysiotropic GnRH (GnRH3) in zebrafish is unclear since GnRH3 null fish are reproductively fertile. This phenomenon raises the possibility of a redundant regulatory pathway(s) for LH secretion in zebrafish. Here, we demonstrate that VipA (homologues of mammalian Vip) both inhibits and induces LH secretion in zebrafish. Despite the observation that VipA axons may reach the pituitary proximal pars distalis including LH cells, pituitary incubation with VipA in vitro, and intraperitoneal injection of VipA, did not induce LH secretion and lhß mRNA expression in sexually mature females, respectively. On the other hand, intracerebroventricular administration of VipA augmented plasma LH levels in both wild-type and gnrh3-/- females at 1 hour posttreatment, with no observed changes in pituitary GnRH2 and GnRH3 contents and gnrh3 mRNA levels in the brains. While VipA's manner of inhibition of LH secretion has yet to be explored, the stimulation seems to occur via a different pathway than GnRH3, dopamine, and 17ß-estradiol in regulating LH secretion. The results indicate that VipA induces LH release possibly by acting with or through a non-GnRH factor(s), providing proof for the existence of functional redundancy of LH release in sexually mature female zebrafish.

A half century of fish gonadotropin-releasing hormones: Breaking paradigms.

The field of fish gonadotropin-releasing hormones (GnRHs) is also celebrating its 50th anniversary this year. This review provides a chronological history of fish GnRH biology over the past five decades. It demonstrates how discoveries in fish regarding GnRH and GnRH receptor multiplicity, dynamic interactions between GnRH neurons, and additional neuroendocrine factors acting alongside GnRH, amongst others, have driven a paradigm shift in our understanding of GnRH systems and functions in vertebrates, including mammals. The role of technological innovations in enabling scientific discoveries is portrayed, as well as how fundamental research in fish GnRH led to translational outcomes in aquaculture. The interchange between fish and mammalian GnRH research is discussed, as is the value and utility of using fish models for advancing GnRH biology. Current challenges and future perspectives are presented, with the hope of expanding the dialogue and collaborations within the neuroendocrinology scientific community at large, capitalizing on diversifying model animals and the use of comparative strategies.

Gnrh2 maintains reproduction in fasting zebrafish through dynamic neuronal projection changes and regulation of gonadotropin synthesis, oogenesis, and reproductive behaviors.

Restricted food intake, either from lack of food sources or endogenous fasting, during reproductive periods is a widespread phenomenon across the animal kingdom. Considering previous studies show the canonical upstream regulator of reproduction in vertebrates, the hypothalamic Gonadotropin-releasing hormone (Gnrh), is inhibited in some fasting animals, we sought to understand the neuroendocrine control of reproduction in fasted states. Here, we explore the roles of the midbrain neuropeptide, Gnrh2, in inducing reproduction via its pituitary prevalence, gonadotropin synthesis, gametogenesis, and reproductive outputs in the zebrafish model undergoing different feeding regimes. We discovered a fasting-induced four-fold increase in length and abundance of Gnrh2 neuronal projections to the pituitary and in close proximity to gonadotropes, whereas the hypothalamic Gnrh3 neurons are reduced by six-fold in length. Subsequently, we analyzed the functional roles of Gnrh2 by comparing reproductive parameters of a Gnrh2-depleted model, gnrh2-/-, to wild-type zebrafish undergoing different feeding conditions. We found that Gnrh2 depletion in fasted states compromises spawning success, with associated decreases in gonadotropin production, oogenesis, fecundity, and male courting behavior. Gnrh2 neurons do not compensate in other circumstances by which Gnrh3 is depleted, such as in gnrh3-/- zebrafish, implying that Gnrh2 acts to induce reproduction specifically in fasted zebrafish.

Ectopic over expression of kiss1 may compensate for the loss of kiss2.

Kisspeptin (KISS) is a neuropeptide which plays a central role in the regulation of the hypothalamic-pituitary-gonadal axis, and is essential for sexual maturation and fertility in mammals. Unlike mammals, which possess only one KISS gene, two paralogous genes, kiss1 and kiss2, have been identified in zebrafish and other non-mammalian vertebrates. Previous studies suggest that Kiss2, but not Kiss1, is the reproduction relevant form amongst the two. To better understand the role of each of these isoforms in reproduction, a loss of function approach was applied. Two genetic manipulation techniques-clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN)-were used to generate kiss1 and kiss2 knockout (KO) zebrafish lines, respectively. Examination of these KO lines showed that reproductive capability was not impaired, confirming earlier observations. Further analysis revealed that KO of kiss2 caused a significant increase in expression levels of kiss1, kiss2r and tac3a, while KO of kiss1 had no effect on the expression of any of the examined genes. In situ hybridization analysis revealed that kiss1 mRNA is expressed only in the habenula in wild type brains, while in kiss2 KO fish, kiss1 mRNA-expressing cells were identified also in the ventral telencephalon, the ventral part of the entopeduncular nucleus, and the dorsal and ventral hypothalamus. Interestingly, these regions are known to express kiss2r, and the ventral hypothalamus normally expresses kiss2. These results suggest that a compensatory mechanism, involving ectopic kiss1 expression, takes place in the kiss2 KO fish, which may substitute for Kiss2 activity.

The gonadotropin-releasing hormones: Lessons from fish.

Fish have been of paramount importance to our understanding of vertebrate comparative neuroendocrinology and the mechanisms underlying the physiology and evolution of gonadotropin-releasing hormones (GnRH) and their genes. This review integrates past and recent knowledge on the Gnrh system in the fish model. Multiple Gnrh isoforms (two or three forms) are present in all teleosts, as well as multiple Gnrh receptors (up to five types), which differ in neuroanatomical localization, pattern of projections, ontogeny and functions. The role of the different Gnrh forms in reproduction seems to also differ in teleost models possessing two versus three Gnrh forms, Gnrh3 being the main hypophysiotropic hormone in the former and Gnrh1 in the latter. Functions of the non-hypothalamic Gnrh isoforms are still unclear, although under suboptimal physiological conditions (e.g. fasting), Gnrh2 may increase in the pituitary to ensure the integrity of reproduction under these conditions. Recent developments in transgenesis and mutagenesis in fish models have permitted the generation of fish lines expressing fluorophores in Gnrh neurons and to elucidate the dynamics of the elaborate innervations of the different neuronal populations, thus enabling a more accurate delineation of their reproductive roles and regulations. Moreover, in combination with neuronal electrophysiology, these lines have clarified the Gnrh mode of actions in modulating Lh and Fsh activities. While loss of function and genome editing studies had the premise to elucidate the exact roles of the multiple Gnrhs in reproduction and other processes, they have instead evoked an ongoing debate about these roles and opened new avenues of research that will no doubt lead to new discoveries regarding the not-yet-fully-understood Gnrh system.

Agouti-Related Protein 2 Is a New Player in the Teleost Stress Response System.

Agouti-related protein (AgRP) is a hypothalamic regulator of food consumption in mammals. However, AgRP has also been detected in circulation, but a possible endocrine role has not been examined. Zebrafish possess two agrp genes: hypothalamically expressed agrp1, considered functionally equivalent to the single mammalian agrp, and agrp2, which is expressed in pre-optic neurons and uncharacterized pineal gland cells and whose function is not well understood. By ablation of AgRP1-expressing neurons and knockout of the agrp1 gene, we show that AgRP1 stimulates food consumption in the zebrafish larvae. Single-cell sequencing of pineal agrp2-expressing cells revealed molecular resemblance to retinal-pigment epithelium cells, and anatomic analysis shows that these cells secrete peptides, possibly into the cerebrospinal fluid. Additionally, based on AgRP2 peptide localization and gene knockout analysis, we demonstrate that pre-optic AgRP2 is a neuroendocrine regulator of the stress axis that reduces cortisol secretion. We therefore suggest that the ancestral role of AgRP was functionally partitioned in zebrafish by the two AgRPs, with AgRP1 centrally regulating food consumption and AgRP2 acting as a neuroendocrine factor regulating the stress axis.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

Gonadotropin-releasing hormone (GNRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a noncell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

The gonadotropin-inhibitory hormone (Lpxrfa) system's regulation of reproduction in the brain-pituitary axis of the zebrafish (Danio rerio).

Gonadotropin-inhibitory hormone (GNIH) was discovered in quail with the ability to reduce gonadotropin expression/secretion in the pituitary. There have been few studies on GNIH orthologs in teleosts (LPXRFamide (Lpxrfa) peptides), which have provided inconsistent results. Therefore, the goal of this study was to determine the roles and modes of action by which Lpxrfa exerts its functions in the brain-pituitary axis of zebrafish (Danio rerio). We localized Lpxrfa soma to the ventral hypothalamus, with fibers extending throughout the brain and to the pituitary. In the preoptic area, Lpxrfa fibers interact with gonadotropin-releasing hormone 3 (Gnrh3) soma. In pituitary explants, zebrafish peptide Lpxrfa-3 downregulated luteinizing hormone beta subunit and common alpha subunit expression. In addition, Lpxrfa-3 reduced gnrh3 expression in brain slices, offering another pathway for Lpxrfa to exert its effects on reproduction. Receptor activation studies, in a heterologous cell-based system, revealed that all three zebrafish Lpxrfa peptides activate Lpxrf-R2 and Lpxrf-R3 via the PKA/cAMP pathway. Receptor activation studies demonstrated that, in addition to activating Lpxrf receptors, zebrafish Lpxrfa-2 and Lpxrfa-3 antagonize Kisspeptin-2 (Kiss2) activation of Kisspeptin receptor-1a (Kiss1ra). The fact that kiss1ra-expressing neurons in the preoptic area are innervated by Lpxrfa-ir fibers suggests an additional pathway for Lpxrfa action. Therefore, our results suggest that Lpxrfa may act as a reproductive inhibitory neuropeptide in the zebrafish that interacts with Gnrh3 neurons in the brain and with gonadotropes in the pituitary, while also potentially utilizing the Kiss2/Kiss1ra pathway.

Neurokinin B regulates reproduction via inhibition of kisspeptin in a teleost, the striped bass.

Kisspeptin and neurokinin B (NKB) are neuropeptides co-expressed in the mammalian hypothalamus and coordinately control GnRH signaling. We have found that Nkb and kisspeptin neurons are distinct in the teleost, striped bass (STB) and capitalized on this phenomenon to study the mode of action of Nkb and its related neuropeptide-F (Nkf), both of which are encoded by the tac3 gene. In vitro brain slices and in vivo administration studies revealed that Nkb/f consistently downregulated kiss2, whereas antagonist (AntD) administration restored this effect. Overall, a minor effect was noted on gnrh1 expression, whereas Gnrh1 content in the pituitaries was reduced after Nkb/f treatment and increased with AntD. Concomitantly, immunostaining demonstrated that hypothalamic Nkb neurons border and densely innervate the largest kiss2 neuronal population in the hypothalamus, which also coexpresses Nkb receptor. No expression of Nkb receptor or Nkb neuronal projections was detected near/in Gnrh1 soma in the preoptic area. At the level of the pituitary, however, the picture was more complex: both Nkb/f and AntD upregulated lhb and fshb expression and Lh secretion in vivo Together with the stimulatory effect of Nkb/f on Lh/Fsh secretion from pituitary cells, in vitro, this may indicate an additional independent action of Nkb/f within the pituitary, in which the hypothalamic pathway is more dominant. The current study demonstrates that Nkb/f utilizes multiple pathways to regulate reproduction in the STB and that in the brain, Nkb mainly acts as a negative modulator of kiss2 to regulate the release of Gnrh1.

Targeted Mutagenesis of the Hypophysiotropic Gnrh3 in Zebrafish (Danio rerio) Reveals No Effects on Reproductive Performance.

Gnrh is the major neuropeptide regulator of vertebrate reproduction, triggering a cascade of events in the pituitary-gonadal axis that result in reproductive competence. Previous research in mice and humans has demonstrated that Gnrh/GNRH null mutations result in hypogonadotropic hypogonadism and infertility. The goal of this study was to eliminate gnrh3 (the hypophysiotropic Gnrh form) function in zebrafish (Danio rerio) to determine how ontogeny and reproductive performance are affected, as well as factors downstream of Gnrh3 along the reproductive axis. Using the TALEN technology, we developed a gnrh3-/- zebrafish line that harbors a 62 bp deletion in the gnrh3 gene. Our gnrh3-/- zebrafish line represents the first targeted and heritable mutation of a Gnrh isoform in any organism. Using immunohistochemistry, we verified that gnrh3-/- fish do not possess Gnrh3 peptide in any regions of the brain. However, other than changes in mRNA levels of pituitary gonadotropin genes (fshb, lhb, and cga) during early development, which are corrected by adulthood, there were no changes in ontogeny and reproduction in gnrh3-/- fish. The gnrh3-/- zebrafish are fertile, displaying normal gametogenesis and reproductive performance in males and females. Together with our previous results that Gnrh3 cell ablation causes infertility, these results indicate that a compensatory mechanism is being activated, which is probably primed early on upon Gnrh3 neuron differentiation and possibly confined to Gnrh3 neurons. Potential compensation factors and sensitive windows of time for compensation during development and puberty should be explored.

Influence of tidal cycles on the endocrine control of reproductive activity in common snook (Centropomus undecimalis).

The aim of our study was to confirm the role of tidal pattern on the coordination of oocyte maturation and spawning in common snook Centropomus undecimalis. To do so, we studied oocyte maturation during the spawning season in relation to the tidal pattern in both males and females by means of histology and hormonal profiling along the pituitary-gonadal axis. Plasma LH levels, as well as transcript levels of gonadotropin genes (fshß and lhß) from the pituitaries of sexually mature male and female common snook were analyzed using a heterologous ELISA and quantitative RT-PCR, respectively. The fshß and lhß cDNAs were isolated and phylogenetic analysis of the deduced amino acid sequences revealed strong identity with other teleosts (75-90%). A strong link was found between tide and follicular development irrespective of the time of the day: female snook sampled on the rising tide were all found to have oocytes in the Secondary Growth Stage whereas females sampled at high tide or on the falling tide had oocytes in the later stages of maturation and ovulation. In addition, LH plasma and mRNA levels of fshß and lhß increased during the later stages of vitellogenesis peaking at ovulation in females. Plasma estradiol and testosterone significantly increased in late vitellogenesis (Secondary Growth Stage) and oocyte maturation (Eccentric Germinal Vesicle Step) respectively. Among male common snook sampled, no correlation was identified between tide and gonadal development. In addition, lhß mRNA expression in males peaked at the mid germinal epithelium stage as for testosterone and 11-KT in the blood while fshß expression and plasma LH levels peaked at late germinal epithelium stage. This study confirms the role played by tidal cycle on the entrainment of the later stages of oogenesis of common snook and provides a better understanding of the link between environmental and endocrine control of reproduction in this species.

Kisspeptin Antagonists Reveal Kisspeptin 1 and Kisspeptin 2 Differential Regulation of Reproduction in the Teleost, Morone saxatilis.

The importance of kisspeptin in regulating vertebrate reproduction has been well established, but the exact mechanism continues to unfold. Unlike mammals, many lower vertebrates possess a dual kisspeptin system, Kiss1 and Kiss2. To decipher the roles of the kisspeptins in fish, we identified two potential kisspeptin antagonists, pep 234 and pep 359, by screening analogs for their ability to inactivate striped bass Kiss1 and Kiss2 receptors expressed in COS7 cells. Pep 234 (a mammalian KISS1 antagonist) antagonizes Kiss1r signaling activated by Kiss1 and Kiss2, and pep 359 (a novel analog) antagonizes Kiss2 activation of both receptors. In vitro studies using brain slices demonstrated that only Kiss2 can upregulate the expression of the hypophysiotropic gnrh1, which was subsequently diminished by pep 234 and pep 359. In primary pituitary cell cultures, the two antagonists revealed a complex network of putative endogenous and exogenous regulation by kisspeptin. While both kisspeptins stimulate Fsh expression and secretion, Kiss2 predominately induces Lh secretion. Pep 234 and 359 treatment of spawning males hindered sperm production. This effect was accompanied with decreased brain gnrh1 and gnrh2 mRNA levels and peptide content in the pituitary, and increased levels of pituitary Lh, probably due to attenuation of Lh release. Strikingly, the mRNA levels of arginine-vasotocin, the neurons of which in the preoptic area coexpress kiss2r, were dramatically reduced by the antagonists. Our results demonstrate differential actions of Kiss1 and Kiss2 systems along the hypothalamic-pituitary axis and interactions with other neuropeptides, and further reinforce the importance of kisspeptin in the execution of spawning.

Seasonal variations in reproductive activity of the blue crab, Callinectes sapidus: Vitellogenin expression and levels of vitellogenin in the hemolymph during ovarian development.

In general, season affects the physiology and behavior of most animals. Warmer temperatures accelerate growth and reproduction of ectotherms, whereas these processes are slowed or halted in colder temperatures. Female blue crabs, Callinectes sapidus inhabiting the Chesapeake Bay, exhibit a seasonal migratory behavior that is closely tied with spawning and the release of larvae. To better understand reproductive activities of the migratory adult females, we examined two reproductive parameters of these crabs sampled monthly (April-December, 2006): the levels of vitellogenin (VtG) in the hemolymph and VtG expression in the hepatopancreas and ovary. The full-length cDNA of VtG (CasVtG-ova) has been isolated from the ovary. The putative CasVtG sequence found in the ovary is >99% identical to that of the hepatopancreas and is related most closely to the sequences reported in other crab species. In female C. sapidus, the hepatopancreas produces over 99% of the total VtG toward the ovarian development. Ovarian stages 2 and 3 in the sampled females are characterized by significant high levels of VtG in hemolymph and VtG expression in both the hepatopancreas and ovary. However, during the southbound migration in fall, females at ovarian stages 2 and 3 have decreased VtG levels, compared to those in spring and summer. The decreased vitellogenesis activity during the fall migration suggests seasonal adaptation to ensure successful spawning and the larval release.

The medio-basal hypothalamus as a dynamic and plastic reproduction-related kisspeptin-gnrh-pituitary center in fish.

Kisspeptin regulates reproductive events, including puberty and ovulation, primarily via GnRH neurons. Prolonged treatment of prepubertal striped bass females with kisspeptin (Kiss) 1 or Kiss2 peptides failed to enhance puberty but suggested a gnrh-independent pituitary control pathway. Kiss2 inhibited, but Kiss1 stimulated, FShß expression and gonadal development, although hypophysiotropic gnrh1 and gnrh receptor expression remained unchanged. In situ hybridization and immunohistochemistry on brains and pituitaries revealed a differential plasticity between the 2 kisspeptin neurons. The differences were most pronounced at the prespawning phase in 2 regions along the path of gnrh1 axons: the nucleus lateralis tuberis (NLT) and the neurohypophysis. Kiss1 neurons appeared in the NLT and innervated the neurohypophysis of prespawning males and females, reaching Lh gonadotropes in the proximal pars distalis. Males, at all reproductive stages, had Kiss2 innervations in the NLT and the neurohypophysis, forming large axonal bundles in the former and intermingling with gnrh1 axons. Unlike in males, only preovulatory females had massive NLT-neurohypophysis staining of kiss2. Kiss2 neurons showed a distinct appearance in the NLT pars ventralis-equivalent region only in spawning zebrafish, indicating that this phenomenon is widespread. These results underscore the NLT as important nuclei for kisspeptin action in 2 facets: 1) kisspeptin-gnrh interaction, both kisspeptins are involved in the regulation of gnrh release, in a stage- and sex-dependent manner, especially at the prespawning phase; and 2) gnrh-independent effect of Kiss peptides on the pituitary, which together with the plastic nature of their neuronal projections to the pituitary implies that a direct gonadotropic regulation is plausible.

Comprehensive analysis of GnRH2 neuronal projections in zebrafish.

The presence and conservation of GnRH2 across vertebrate species suggest important biological roles. However, the function of GnRH2 remains unclear. A good research model for GnRH2 functional studies is still lacking largely due to the absence of GnRH2 in the widely used mouse model. Hence, we used the zebrafish, for which powerful genetic tools are available, and developed a transgenic (Tg) line expressing enhanced green fluorescence protein (eGFP). The high sensitivity of eGFP, which can diffuse throughout the neuron, enables us to document the complete projectome of GnRH2 neurons at different developmental stages. Fine projection structures were observed without sacrificing the fish. Crossed with the GnRH3:tdTomato Tg line, the GnRH2:eGFP Tg line provides us with an opportunity to visualize the entire GnRH system simultaneously in one organism. This work will provide a framework to understand the function of the highly-conserved GnRH2 system.

A novel hormone is required for the development of reproductive phenotypes in adult female crabs.

The crustacean male-specific androgenic hormone is widely accepted as a key factor in sexual differentiation and in the development of secondary sex characteristics. However, the mechanism by which the plethora of different reproductive strategies are controlled and executed in crustaceans is not known. We discovered in the blue crab, Callinectes sapidus, a hitherto unknown neurohormone, named crustacean female sex hormone (CFSH), in distinct neurosecretory cells in the eyestalk ganglia. CFSH is highly expressed in females but weakly in males, and its crucial role in developing adult female phenotypes has now been established. CFSH cDNA encodes a 225-amino acid (aa) novel protein composed of a 23-aa predicted signal peptide, 33-aa precursor-related peptide and 167-aa mature protein that did not match any other sequence in GenBank. CFSH RNA interference knockdown by multiple administrations of double-stranded RNA at the prepubertal stage causes abnormal development of brooding and mating systems upon puberty. These systems include a pair of gonopores and an egg attachment system for brooding, comprised of an enlarged semicircular abdomen and ovigerous setae. The ovigerous setae in CFSH knocked-down females were fewer and 50% shorter and the gonopores were either significantly smaller than those of controls, misplaced, or absent. We also identified CFSH in the green crab, Carcinus maenas, a species that shares a similar reproductive strategy with C. sapidus. Together, our data provide the first evidence for the presence of a female hormone in crustaceans and its importance in positively controlling anatomic features associated with brooding and mating systems. From an evolutionary standpoint, the endocrine control supporting a female-specific reproductive strategy, as previously described for many vertebrate species, has now been demonstrated for the first time in crustaceans.

Stimulation of molt by RNA interference of the molt-inhibiting hormone in the crayfish Cherax quadricarinatus.

In crustaceans, molting is known to be under the control of neuropeptide hormones synthesized and secreted from the eyestalk ganglia. While the role of molt-inhibiting hormone (MIH) in regulating molting has been described in several species using classical methods, an in vivo specific MIH targeted manipulation has not been described yet. In the present study, an MIH cDNA was isolated and sequenced from the eyestalk ganglia of the Australian freshwater red claw crayfish Cherax quadricarinatus (Cq) by 5' and 3' RACE. We analyzed the putative Cq-MIH based on sequence homology, a three dimensional structure model and transcript's tissue specificity. We further examined the involvement of Cq-MIH in the control of molt in the crayfish through RNAi by in vivo injections of Cq-MIH double-stranded RNA, which resulted in, similarly to eyestalk ablation, acceleration of molt cycles. This acceleration was reflected by a significant reduction (up to 32%) in molt interval and an increased rate in molt mineralization index (MMI), which correlated with the induction of ecdysteroid hormones compared to control. Altogether, this study provides a proof of function for the involvement of the Cq-MIH gene in molt regulation in the crayfish.

Differential and gonad stage-dependent roles of kisspeptin1 and kisspeptin2 in reproduction in the modern teleosts, morone species.

Kisspeptin is an important regulator of reproduction in many vertebrates. The involvement of the two kisspeptins, Kiss1 and Kiss2, and their receptors, Gpr54-1 and Gpr54-2, in controlling reproduction was studied in the brains of the modern teleosts, striped and hybrid basses. In situ hybridization and laser capture microdissection followed by quantitative RT (QRT)-PCR detected coexpression of kiss1 and kiss2 in the hypothalamic nucleus of the lateral recess. Neurons expressing gpr54-1 and gpr54-2 were detected in several brain regions. In the preoptic area, gpr54-2 was colocalized in GnRH1 neurons while gpr54-1 was expressed in cells attached to GnRH1 fibers, indicating two different modes of GnRH1 regulation. The expression of all four genes was measured in the brains of males and females at different life stages using QRT-PCR. The levels of kiss1 and gpr54-1 mRNA, the latter being expressed in minute levels, were consistently lower than those of kiss2 and gpr54-2. While neither gene's expression increased at prepuberty, all were dramatically elevated in mature females. The levels of kiss2 mRNA increased also in mature males. Kiss1 peptide was less potent than Kiss2 in elevating plasma luteinizing hormone levels and in up-regulating gnrh1 and gpr54-2 expression in prepubertal hybrid bass in vivo. In contrast, during recrudescence, Kiss1 was more potent than Kiss2 in inducing luteinizing hormone release, and Kiss2 down-regulated gnrh1 and gpr54-2 expression. This is the first report in fish to demonstrate the alternating actions and the importance of both neuropeptides for reproduction. The organization of the kisspeptin system suggests a transitional evolutionary state between early to late evolving vertebrates.