Publications

Two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), are important players in the hypothalamic-pituitary-gonadal axis of vertebrates. In the present work, we describe the construction of recombinant (r) common carp (Cyprinus carpio; c) FSH (rcFSH) and LH (rcLH) using the Pichia pastoris system, the generation of specific antibodies against their respective β subunits, and their use in the development and validation of specific ELISAs. We produced carp rLH and rFSH as single-chain polypeptides, wherein the GTH subunit α was joined with either cLHβ or cFSHβ mature protein-coding sequences to form a fusion gene that encodes a yoked polypeptide, in which the GTH β-subunit forms the N-terminal part and the α-subunit forms the C-terminal part. Competitive ELISAs were developed, using primary antibodies against rcLHβ or rcFSHβ, respectively, and rcLHβα or rcFSHβα for the standard curves. The standard curves for cLH paralleled those of pituitary extracts of the homologous fish and also those of other cyprinids species like the black carp (Mylopharyngodon piceus), goldfish (Carassius auratus), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyngodon idella). We used the specific antibodies raised against cFSH and cLH to study the specific localization of the different GTH cells in the pituitary of carp and its taxonomic relative species - the zebrafish. Both FSH and LH cells are localized in the center of the proximal pars distalis enveloping both sides of the neurohypophysis. LH cells form a continuous population throughout the PPD, while FSH cells are more loosely distributed throughout the same area and form small aggregations. Marked annual changes were encountered in gonadosomatic index (GSI), follicle diameter, mRNA levels and protein levels of FSH and LH. From September to November, all fish had low GSI, and the ovary contained previtellogenic follicles. From December, the GSI level increased and remained high until March, the follicular diameter reached its maximum in January, where the ovary contained large fully grown follicles. Thereafter, spawning occurred through March and April and ended in May, and GSI level and follicle diameter increased again; and the ovary contained mid-vitellogenic follicles. LH pituitary content and mRNA levels were low at pre- and early vitellogenesis, increasing gradually during this process to reach a peak of LH mRNA levels in mid vitellogenic ovary and a peak of LH content in fully grown ovarian follicles. However, no significant change occurred in FSH pituitary content and mRNA levels in vitellogenic fish and in fish during final maturation stages. A dramatic difference was found in the total content of each gonadotropin in the pituitary, with higher LH than FSH. Moreover, follicle diameter was positively and significantly correlated with LH pituitary content and its transcript levels - but not with the pituitary content or mRNA levels of FSH. Taken together, these results indicate that in carp, LH alone is sufficient to regulate both vitellogenesis and final oocyte maturation while FSH may have another, yet undefined role.

A novel heterodimeric glycoprotein hormone (GpH) comprised of alpha (GpA2) and beta (GpB5) subunits was discovered in 2002 and called thyrostimulin for its ability to activate the TSH receptor in mammals, but its central function in vertebrates has not been firmly established. We report here the cloning and expression of lamprey (l)GpB5, and its ability to heterodimerize with lGpA2 to form a functional l-thyrostimulin. The full-length cDNA of lGpB5 encodes 174 amino acids with ten conserved cysteine residues and one glycosylation site that is conserved with other vertebrate GpB5 sequences. Phylogenetic and synteny analyses support that lGpB5 belongs to the vertebrate GpB5 clade. Heterodimerization of lGpB5 and lGpA2 was shown by nickel pull-down of histidine-tagged recombinant subunits. RNA transcripts of lGpB5 were detected in the pituitary of lampreys during both parasitic and adult life stages. Intraperitoneal injection with lGnRH-III (100 μg/kg) increased pituitary lGpA2, lGpB5, and lGpHβ mRNA expression in sexually mature, adult female lampreys. A recombinant l-thyrostimulin produced by expression of a fusion gene in Pichia pastoris activated lamprey GpH receptors I and II as measured by cAMP enzymeimmunoassay. In contrast to jawed vertebrates that have pituitary LH, FSH, and TSH, our data support that lampreys only have two functional pituitary GpHs, lGpH and l-thyrostimulin, which consist of lGpA2 and unique beta subunits. It is hypothesized that lGpH and l-thyrostimulin differentially regulate reproductive and thyroid activities in some unknown way(s) in lampreys.

OBJECTIVE: To assess the postpubertal outcome of ventral penile curvature repaired in infancy in terms of recurrence and aesthetics. MATERIALS AND METHODS: Postpubertal patients treated for hypospadias and ventral penile curvature in infancy at a tertiary medical center were invited to undergo assessment of the quality of the repair. Findings were compared between patients with a straight penis after skin release and patients who required dorsal plication. RESULTS: The cohort included 27 patients of mean age 16.5 years who were reported with straight penis after surgery. Postpubertal curvature was found in 6 of 14 patients (43%) successfully treated by skin release and 10 of 13 patients (77%) who underwent dorsal plication (P = .087). Significant curvature (≥30 degrees) was found in 1 of 14 patients in the skin-release group and 4 of 13 in the dorsal plication group (P = .16). Rates of redo urethroplasty were 2 of 14 (14%) and 5 of 10 (50%), respectively. Patient satisfaction with the appearance of the penis did not differ significantly. CONCLUSION: Ventral penile curvature repaired in infancy often recurs after puberty. The need for dorsal plication has a trend-level association with recurrence of penile curvature in puberty. It might also be related to the degree of postpubertal penile curvature and the need for redo urethroplasty. Procedure type does not affect patient satisfaction with the postpubertal appearance of the penis.

Currently, spawning is induced in carp species by carp pituitary extract (CPE) and a combination of synthetic agonist of GnRH combined with a dopamine antagonist. The main goal of this study was the production of recombinant gonadotropins (GtHs) on a large scale to serve as an alternative to currently used agents. We produced carp (c) recombinant (r) Lh as a single chain in the methylotrophic yeast Pichia pastoris Lha subunit was joined with Lhb subunit with a flexible linker of three glycine-serine repeats and six Histidines to form a mature protein, the β-subunit formed the N-terminal part and the α-subunit formed the C-terminal part. The ability of the rcLh to elicit biological response was tested by in vivo stimulation of estradiol (E2) and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and by its in vivo potency to induce ovulation and spawning induction. rcLh tested in this work significantly enhanced both E2 and DHP secretion in a dose-dependent manner similar to the results obtained with CPE. E2 levels showed a moderate rise following the priming injection and a subsequent decrease during the rest of the trial. DHP levels were only increased after the resolving injection, approximately 5 h before spawning. At the highest dose of rcLh (350 µg/kg BW), the recombinant protein was more efficient than CPE in terms of both spawning success and fertilization rate. It is shown here that rcLh can elicit the secretion of DHP in vivo and actually trigger spawning. These novel findings introduce the potential of utilizing recombinant gonadotropins in aquaculture.

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.

Biologically active recombinant yellowtail kingfish follicle stimulating hormone (rytkFsh) was produced in yeast Pichia pastoris and its biological activity was demonstrated by both in-vitro and in-vivo bioassays. Incubation of ovarian and testicular fragments with the recombinant hormone stimulated E and 11-KT secretion, respectively. In-vivo trial in immature female YTK resulted in a significant increase of plasma E levels and development of oocytes. In males at the early stages of puberty, advancement of spermatogenesis was observed, however plasma 11-KT levels were reduced when administered with rytkFsh.

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 gene. brain slices and administration studies revealed that Nkb/f consistently downregulated , whereas antagonist (AntD) administration restored this effect. Overall, a minor effect was noted on 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 and expression and Lh secretion Together with the stimulatory effect of Nkb/f on Lh/Fsh secretion from pituitary cells, , 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.

Mammalian pituitaries exhibit a high degree of intercellular coordination; this enables them to mount large-scale coordinated responses to various physiological stimuli. This type of communication has not been adequately demonstrated in teleost pituitaries, which exhibit direct hypothalamic innervation and expression of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in distinct cell types. We found that in two fish species, namely tilapia and zebrafish, LH cells exhibit close cell-cell contacts and form a continuous network throughout the gland. FSH cells were more loosely distributed but maintained some degree of cell-cell contact by virtue of cytoplasmic processes. These anatomical differences also manifest themselves at the functional level as evidenced by the effect of gap-junction uncouplers on gonadotropin release. These substances abolished the LH response to gonadotropin-releasing hormone stimulation but did not affect the FSH response to the same stimuli. Dye transfer between neighboring LH cells provides further evidence for functional coupling. The two gonadotropins were also found to be differently packaged within their corresponding cell types. Our findings highlight the evolutionary origin of pituitary cell networks and demonstrate how the different levels of cell-cell coordination within the LH and FSH cell populations are reflected in their distinct secretion patterns.

In vertebrates, gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa-R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa-R has not been studied in teleost species, partially because of the lack of fish-specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa-immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa-R-immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa-immunoreactive fibers nor LPXRFa-R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α-melanotropin (α-MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa-R are expressed only in LH, ACTH, and α-MSH cells. These results suggest that LPXRFa and LPXRFa-R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753-2775, 2016. © 2016 Wiley Periodicals, Inc.

In the reproduction process of male and female fish, pituitary derived gonadotropins (GTHs) play a key role. To be able to specifically investigate certain functions of Luteinizing (LH) and Follicle stimulating hormone (FSH) in Russian sturgeon (Acipenser gueldenstaedtii; st), we produced recombinant variants of the hormones using the yeast Pichia pastoris as a protein production system. We accomplished to create in vitro biologically active heterodimeric glycoproteins consisting of two associated α- and β-subunits in sufficient quantities. Three dimensional modelling of both GTHs was conducted in order to study the differences between the two GTHs. Antibodies were produced against the unique β-subunit of each of the GTHs, in order to be used for immunohistochemical analysis and to develop an ELISA for blood and pituitary hormone quantification. This detection technique revealed the specific localization of the LH and FSH cells in the sturgeon pituitary and pointed out that both cell types are present in substantially higher numbers in mature males and females, compared to immature fish. With the newly attained option to prevent cross-contamination when investigating on the effects of GTH administration, we compared the steroidogeneic response (estradiol and 11-Keto testosterone (11-KT) in female and males, respectively) of recombinant stLH, stFSH, and carp pituitary extract in male and female sturgeon gonads at different developmental stages. Finally, we injected commercially available gonadotropin releasing hormones analog (GnRH) to mature females, and found a moderate effect on the development of ovarian follicles. Application of only testosterone (T) resulted in a significant increase in circulating levels of 11-KT whereas the combination of GnRH + T did not affect steroid levels at all. The response pattern for estradiol demonstrated a similar situation. FSH levels showed significant increases when GnRH + T was administered, while no changes were present in LH levels.

The folliculostellate cells of the mammalian pituitary are non-endocrine cells that are implicated in long-distance communication and paracrine signaling, but to date, these cells have yet to be characterized in teleosts. We found that the stellate cells of the teleost pituitary share many common attributes with mammalian folliculostellate cells. By labeling of stellate cells in live preparations of tilapia pituitaries we investigated their distribution, association with other endocrine cells and their anatomical and functional coupling. In the pars intermedia, stellate cells were arranged around neuronal bundles and their processes extended into the pars distalis. Within the pars distalis, stellate cells formed close associations with FSH cells and, to a lesser degree, with GH and LH cells, suggesting differential paracrine regulation of the two gonadotrope populations. The production of follistatin by stellate cells further corroborates the notion of a paracrine role on FSH release. We also found stellate cells to form gap junctions that enabled dye transfer to neighboring stellate cells, implicating that these cells form a large-scale network that connects distant parts of the pituitary. Our findings represent the first wide-scale study of stellate cells in teleosts and provide valuable information regarding their functional roles in pituitary function.

The function and components of the hypothalamic-pituitary axis are conserved among vertebrates; however, in fish, a neuroglandular mode of delivery (direct contact between axons and endocrine cells) was considered dominant, whereas in tetrapods hypothalamic signals are relayed to their targets via the hypophysial portal blood system (neurovascular delivery mode). By using a transgenic zebrafish model we studied the functional and anatomical aspects of gonadotrope regulation thus revisiting the existing model. FSH cells were found to be situated close to the vasculature whereas the compact organization of LH cells prevented direct contact of all cells with the circulation. GnRH3 fibers formed multiple boutons upon reaching the pituitary, but most of these structures were located in the neurohypophysis rather than adjacent to gonadotropes. A close association was observed between FSH cells and GnRH3 boutons, but only a fifth of the LH cells were in direct contact with GnRH3 axons, suggesting that FSH cells are more directly regulated than LH cells. GnRH3 fibers closely followed the vasculature in the neurohypophysis and formed numerous boutons along these tracts. These vessels were found to be permeable to relatively large molecules, suggesting the uptake of GnRH3 peptides. Our findings have important implications regarding the differential regulation of LH and FSH and contradict the accepted notion that fish pituitary cells are mostly regulated directly by hypothalamic fibers. Instead, we provide evidence that zebrafish apply a dual mode of gonadotrope regulation by GnRH3 that combines both neuroglandular and neurovascular components.

The gnathostome (jawed vertebrates) classical pituitary glycoprotein hormones, FSH, LH, and TSH, consist of a common α-subunit (GpA1) and unique β-subunits (Gpβ1, -2, and -3), whereas a recently identified pituitary glycoprotein hormone, thyrostimulin, consists of GpA2 and GpB5. This paper reports the identification, expression, and function of an ancestral, nonclassical, pituitary heterodimeric glycoprotein hormone (GpH) consisting of the thyrostimulin A2 subunit with the classical β-subunit in the sea lamprey, Petromyzon marinus, a jawless basal vertebrate. Lamprey (l) GpA2, and lGpHβ were shown to form a heterodimer by coimmunoprecipitation of lGpA2 with FLAG-tagged lGpHβ after the overexpression in transiently transfected COS7 cells using a bipromoter vector. Dual-label fluorescent in situ hybridization and immunohistochemistry showed the coexpression of individual subunits in the proximal pars distalis of the pituitary. GnRH-III (1μΜ) significantly increased the expression of lGpHβ and lGpA2 in in vitro pituitary culture. Recombinant lamprey GpH was constructed by tethering the N terminal of lGpA2 to the C terminal of lGpHβ with a linker region composed of six histidine residues followed by three glycine-serine repeats. This recombinant lamprey GpH activated the lamprey glycoprotein hormone receptor I as measured by increased cAMP/luciferase activity. These data are the first to demonstrate a functional, unique glycoprotein heterodimer that is not found in any other vertebrate. These data suggest an intermediate stage of the structure-function of the gonadotropin/thyroid-stimulating hormone in a basal vertebrate, leading to the emergence of the highly specialized gonadotropin hormones and thyroid stimulating hormones in gnathostomes.

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.

Estrogens have a pivotal role in natural female sexual differentiation of tilapia while lack of steroids results in testicular development. Despite the fact that androgens do not participate in natural sex differentiation, synthetic androgens, mainly 17-α-methyltestosterone (MT) are effective in the production of all-male fish in aquaculture. The sex inversion potency of synthetic androgens may arise from their androgenic activity or else as inhibitors of aromatase activity. The current study is an attempt to differentiate between the two alleged activities in order to evaluate their contribution to the sex inversion process and aid the search for novel sex inversion agents. In the present study, MT inhibited aromatase activity, when applied in vitro as did the non-aromatizable androgen dihydrotestosterone (DHT). In comparison, exposure to fadrozole, a specific aromatase inhibitor, was considerably more effective. Androgenic activity of MT was evaluated by exposure of Sciaenochromis fryeri fry to the substance and testing for the appearance of blue color. Flutamide, an androgen antagonist, administered concomitantly with MT, reduced the appearance of the blue color and the sex inversion potency of MT in a dose-dependent manner. In tilapia, administration of MT, fadrozole or DHT resulted in efficient sex inversion while flutamide reduced the sex inversion potency of all three compounds. In the case of MT and DHT the decrease in sex inversion efficiency caused by flutamide is most likely due to the direct blocking of the androgen binding to its cognate receptor. The negative effect of flutamide on the efficiency of the fadrozole treatment may indicate that the masculinizing activity of fadrozole may be attributed to excess, un-aromatized, androgens accumulated in the differentiating gonad. The present study shows that when androgen receptors are blocked, there is a reduction in the efficiency of sex inversion treatments. Our results suggest that in contrast to natural sex differentiation, during sex inversion treatments, androgens, either endogenous or exogenous, participate in inducing testicular differentiation.

In fish, both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in reproduction. Here we explored the structure and differential specificity of tilapia (t) gonadotropins (GTHs) to delineate their physiological relevance and the nature of their regulation. We generated structural models of tGTHs and GTH receptors (R) that enabled us to better understand the hormone-receptor interacting region. In tilapia, FSH release is under the control of the hypothalamic decapeptide GnRH, an effect that was abolished by specific bioneutralizing antisera [anti-recombinant (r) tFSHβ]. These antisera also reduced the basal secretion and delayed GnRH-stimulated production of 11-ketotestosterone (11KT), and dramatically reduced LH levels. Immunoneutralization of tLH using anti-rtLHβ significantly reduced its GnRH-stimulated levels. Basal 11KT and FSH levels were also reduced. Taken together, these results suggest a feedback mechanism between FSH and LH release in tilapia.

Neurokinin B (NKB) was recently identified as a key regulator of reproduction in mammals and fish. Fish were found to possess a specific novel neurokinin termed NKF. To study the role of NKB/NKF in the regulation of fish reproduction and to investigate the role of NKB/NKF and their receptors in the piscine pituitary, we have identified the NKB/tachikinin 3 receptor (tac3r) system in tilapia. Bioinformatics and phylogenetic analyses have demonstrated that the tilapia holds 1 putative tac3 gene and 2 NKB receptor genes (tac3ra and tac3rb) that clustered with other piscine Tac3 and NKB receptor lineages. Furthermore, we found that in African cichlids, NKB peptides differ from other vertebrate NKBs in their C-terminal sequence, possessing isoleucine instead of valine as the X in the NKB FXGLM-NH2-terminal consensus sequence. Signal transduction analysis demonstrated that tilapia NKB (tiNKB), tiNKF, and human NKB activated both CRE-luc and SRE-luc transcriptional activity of both tilapia and human NKB receptors. Two hours after ip injection of tiNKB, the plasma levels of both FSH and LH were increased, whereas tiNKF was more effective in increasing LH levels. However, tiNKB was more effective than tiNKF in increasing both FSH and LH from tilapia pituitary dispersed cells. Using in situ hybridization and fluorescent immunohistochemistry, we have shown that LH cells possess tac3, tac3ra, and tac3rb mRNAs, whereas FSH cells possess mainly tac3rb and tac3ra and tac3 to a much lesser extent. These results suggest that the members of the NKB/tac3r system may serve as paracrine/autocrine regulators of gonadotropin release in fish pituitary.

Organotypic cultures, prepared from hypothalamo-pituitary slices of tilapia, were developed to enable long-term study of secretory cells in the pituitary of a teleost. Values of membrane potential at rest were similar to those recorded from acute slices, and cells presented similar spontaneous spikes and spikelets. Some cells also exhibited slow spontaneous oscillations in membrane potential, which may be network-driven. Long-term (6days) continuous exposure to GnRH induced increases in LH and FSH secretion. FSH levels reached the highest levels after 24h of exposure to GnRH, and the highest secretion of LH was observed in days 4 and 5 of the experiment. Since slices were viable for several weeks in culture, maintaining the original cytoarchitecture, electrical membrane properties and the ability to secrete hormones in response to exogenous GnRH, this technique is ideal for studying the mechanisms regulating cell-to-cell communication under conditions resembling the in vivo tissue organization.

LPXRFamide (LPXRFa) peptides have been characterized for their ability to inhibit gonadotropin (GTH) release in birds and stimulate growth hormone (GH) release in frogs. However, their involvement in regulating the reproductive hypothalamo-pituitary-gonadal axis in mammals and fish is inconclusive. To study the role of LPXRFa peptides in the regulation of GTH secretion, we cloned tilapia LPXRFa and LPXRF receptor (LPXRF-R). Processing of the tilapia preproLPXRFa liberated three mature LPXRFa peptides that varied in size and post-translational modifications. Phylogenetic analysis of LPXRFa and the closely related RFamide peptide PQRFa showed clear clustering of each peptide sequence with its orthologs from various vertebrates. Signal-transduction analysis of the tilapia LPXRF-R in COS-7 cells showed clear stimulation of CRE-dependent luciferase activity, whereas the human NPFFR1 showed suppression of forskolin-induced CRE-dependent activity in this system. Administration of the tilapia pyroglutaminated LPXRFa-2 peptide to primary cell culture of tilapia pituitaries, or to reproductive female tilapia by ip injection, positively regulated both LH and FSH release in vivo and in vitro. Using double-labeled fluorescent in-situ hybridization and immunofluorescence, βLH cells were found to co-express both tilapia lpxrf and tilapia lpxrf-r mRNA, whereas some of the βFSH cells coexpressed only lpxrf-r mRNA. No coexpression of tilapia lpxrf-r was identified in GH-positive cells. These findings suggest that the LPXRFa system is a potent positive regulator of the reproductive neuroendocrine axis of tilapia.

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.