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 lhβ 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 post-treatment, 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 E2 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.

Transcriptome of Distinct LH and FSH Cells Reveals Different Regulation Unique to Each Cell Type

From mammals to fish, gametogenesis and sexual maturation are driven by LH and FSH, the two gonadotropic hormones temporally secreted from the pituitary. Teleost fish are an excellent model for addressing the unique regulation and function of each gonadotropin hormone since, unlike mammals; they synthesize and secrete LH and FSH from distinct cells. By performing cell specific transcriptome analysis of double-labelled transgenic Nile tilapia (Oreochromis niloticus) expressing GFP and RFP in LH or FSH cells, respectively, we identified genes specifically enriched in each cell type. Though GnRH is considered the main neuropeptide regulating LH and FSH, we found that each LH and FSH cell express unique GPCR signature that reveals the direct regulation of additional metabolic and homeostatic hormones (like cholecystokinin, somatostatin and glutamate). Moreover, some of those GPCRs were conserved also in gonadotrophs of mammals (like PACAP receptor, Adropin receptor and GABBA receptor). Next, we had exploited the unique behavior of Nile tilapia where a behavioral hierarchy is created between males, to compare the gene expression in the pituitary and brain of dominant (reproducing) males to a subordinate (non-reproducing) males. By combining the two transcriptome sets we had identified novel players in the hypothalamic regulation of the HPG axis, and revealed how brain aromatase (cyp19a1b), that is enriched specifically in LH cells, is the key factor in regulating the activity of LH and FSH cells in dominant reproducing fish. Thereby, unraveling novel mechanisms in the differential regulation of LH and FSH. The research was funded by the Israel Science Foundation (ISF) no. 1540/17.

Somatostatin, as a Bridge Between the GH-Axis and the Gth-Axis

Somatostatin (SST) is a 14-amino acid peptide produced in the hypothalamus of vertebrates, including fish. It regulates many physiological processes such as growth development and metabolic processes in the animal’s body. Negative control of growth hormone in vivo and in vitro was characterized in several fish species such as salmon, goldfish, rainbow trout and tilapia. Although very important, the SST/SST-R system in Nile tilapia (Oreochromis niloticus) was not deeply characterized. The somatostatin system in tilapia possess two ligands (Somatostatin1b and Somatostatin 2), and five receptors (SST-R 1-5). Unlike mammals, in fish, FSH and LH are secreted from different cell populations in the pituitary. By performing cell specific transcriptome analysis of double-labelled transgenic tilapia expressing GFP and RFP in LH or FSH cells, respectively, we identified genes specifically enriched in each cell type. Analysis of the RNA-seq discovered 4 types of SST-Rs: sstr2, sstr3, sstr5 and sstr5x3. The specific localization of each SST-R was identified by In Situ hybridization with specific probes for each of the SST-Rs. SST-R2 and SST-R5x3 were expressed on LH and FSH cells, while SST-R5 was exclusively expressed on LH cells. Interestingly, SST-R3, which was expressed on GH secreting cells, was also expressed on both gonadotropin-secreting cells. Transactivation assays, using COS7 cell line transfected with tilapia SST-Rs together with the reporter plasmid CRE-luc, demonstrated an effect through the cAMP/PKA pathway. Signal transduction analysis demonstrated that SST agonist (Octreotide; IC50 = 0.8-60nM) decreased the cAMP/PKA pathway, while an opposite effect was found when SST antagonist (Cyclosomatostatin; EC50 = 0.1 - 188 nM) was used. To understand the physiological effects of somatostatin on gonadotropins and GH release, we examined the effect of ip injection (100 μg/kg BW) of somatostatin agonist and antagonist on plasma FSH, LH and GH levels. SST agonist decreased plasma GH and FSH levels, as fast as two hours post injection and their levels remained low until the end of the experiment. On the other hand, SST antagonist increased LH and FSH levels two hours post injection, but while FSH levels remained high during the entire experiment, LH levels went back to basal levels afterwards. Our results show - for the first time in fish - a direct effect of SST on gonadotropin release, that could serve as a bridge between the GH-axis and the GTH-axis. The research was funded by the Israel Science Foundation (ISF) no. 1540/17.

Long extensions with varicosity-like structures in gonadotrope Lh cells facilitate clustering in medaka pituitary culture

Accumulating evidence indicates that some pituitary cell types are organized in complex networks in both mammals and fish. In this study, we have further investigated the previously described cellular extensions formed by the medaka (Oryzias latipes) luteinizing hormone gonadotropes (Lh cells). Extensions, several cell diameters long, with varicosity-like swellings, were common both in vitro and in vivo. Some extensions approached other Lh cells, while others were in close contact with blood vessels in vivo. Gnrh further stimulated extension development in vitro. Two types of extensions with different characteristics could be distinguished, and were classified as major or minor according to size, origin and cytoskeleton protein dependance. The varicosity-like swellings appeared on the major extensions and were dependent on both microtubules and actin filaments. Immunofluorescence revealed that Lhβ protein was mainly located in these swellings and at the extremity of the extensions. We then investigated whether these extensions contribute to network formation and clustering, by following their development in primary cultures. During the first two days in culture, the Lh cells grew long extensions that with time physically attached to other cells. Successively, tight cell clusters formed as cell somas that were connected via extensions migrated towards each other, while shortening their extensions. Laser photolysis of caged Ca2+ showed that Ca2+ signals originating in the soma propagated from the soma along the major extensions, being particularly visible in each swelling. Moreover, the Ca2+ signal could be transferred between densely clustered cells (sharing soma-soma border), but was not transferred via extensions to the connected cell. In summary, Lh gonadotropes in medaka display a complex cellular structure of hormone-containing extensions that are sensitive to Gnrh, and may be used for clustering and possibly hormone release, but do not seem to contribute to communication between cells themselves.

Oxytocin-like signal regulates Lh cells directly but not Fsh cells in the ricefield eel Monopterus albus†

The synthesis and release of LH and FSH in the pituitary of vertebrates are differentially regulated during gonadal development and maturation. However, the underlying neuroendocrine mechanisms remain to be fully elucidated. The present study examined the possible involvement of isotocin (Ist), an oxytocin-like neuropeptide, in the regulation of Lh and Fsh in a teleost, the ricefield eel Monopterus albus. The immunoreactive isotocin receptor 2 (Istr2) was shown to be localized to Lh but not Fsh cells. In contrast, immunoreactive isotocin receptor 1 (Istr1) was not observed in either Lh or Fsh cells in the pituitary. Interestingly, Lh cells in female ricefield eels expressed Istr2 and secreted Lh in response to Ist challenge stage-dependently and in correlation with ovarian vitellogenesis. Moreover, Ist decreased Lh contents in the pituitary of female fish, indicating its stimulatory roles on Lh release in vivo. The induction of Lh release by Ist in dispersed pituitary cells was blocked by a PLC or IP3R inhibitor but not by a PKA or PKC inhibitor, indicating the involvement of the IP3/Ca2+ pathway. Collectively, the above results indicate that isotocin may bind to Istr2 to stimulate Lh release via the IP3/Ca2+ pathway, and play important roles in the ovarian maturation in ricefield eels. Furthermore, the present study suggests a novel neuroendocrine mechanism underlying the differential regulation of Lh and Fsh in vertebrates.

Plasticity in medaka gonadotropes via cell proliferation and phenotypic conversion

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) produced by the gonadotropes play a major role in control of reproduction. Contrary to mammals and birds, Lh and Fsh are mostly produced by two separate cell types in teleost. Here, we investigated gonadotrope plasticity, using transgenic lines of medaka (Oryzias latipes) where DsRed2 and hrGfpII are under the control of the fshb and lhb promotors respectively. We found that Fsh cells appear in the pituitary at 8 dpf, while Lh cells were previously shown to appear at 14 dpf. Similar to Lh cells, Fsh cells show hyperplasia from juvenile to adult stages. Hyperplasia is stimulated by estradiol. Both Fsh and Lh cells show hypertrophy during puberty with similar morphology. They also share similar behavior, using their cellular extensions to make networks. We observed bi-hormonal gonadotropes in juveniles and adults but not in larvae where only mono-hormonal cells are observed, suggesting the existence of phenotypic conversion between Fsh and Lh in later stages. This is demonstrated in cell culture, where some Fsh cells start to produce Lhβ, a phenomenon enhanced by gonadotropin-releasing hormone (Gnrh) stimulation. We have previously shown that medaka Fsh cells lack Gnrh receptors, but here we show that with time in culture, some Fsh cells start responding to Gnrh, while fshb mRNA levels are significantly reduced, both suggestive of phenotypic change. All together, these results reveal high plasticity of gonadotropes due to both estradiol-sensitive proliferation and Gnrh promoted phenotypic conversion, and moreover, show that gonadotropes lose part of their identity when kept in cell culture.

Representation of distinct reward variables for self and other in primate lateral hypothalamus

The lateral hypothalamus (LH) has long been implicated in maintaining behavioral homeostasis essential for the survival of an individual. However, recent evidence suggests its more widespread roles in behavioral coordination, extending to the social domain. The neuronal and circuit mechanisms behind the LH processing of social information are unknown. Here, we show that the LH represents distinct reward variables for “self” and “other” and is causally involved in shaping socially motivated behavior. During a Pavlovian conditioning procedure incorporating ubiquitous social experiences where rewards to others affect one’s motivation, LH cells encoded the subjective value of self-rewards, as well as the likelihood of self- or other-rewards. The other-reward coding was not a general consequence of other’s existence, but a specific effect of other’s reward availability. Coherent activity with and top-down information flow from the medial prefrontal cortex, a hub of social brain networks, contributed to signal encoding in the LH. Furthermore, deactivation of LH cells eliminated the motivational impact of other-rewards. These results indicate that the LH constitutes a subcortical node in social brain networks and shapes one’s motivation by integrating cortically derived, agent-specific reward information.

Spexin and a novel cichlid-specific spexin paralog both inhibit FSH and LH through a specific galanin receptor (Galr2b) in tilapia

Spexin (SPX) is a 14 amino acid peptide hormone that has pleiotropic functions across vertebrates, one of which is involvement in the brain-pituitary-gonad axis of fish. SPX(1) has been identified in each class of vertebrates, and a second SPX (named SPX2) has been found in some non-mammalian species. We have cloned two spexin paralogs, designated as Spx1a and Spx1b, from Nile tilapia (Oreochromis niloticus) that have varying tissue distribution patterns. Spx1b is a novel peptide only identified in cichlid fish, and is more closely related to Spx1 than Spx2 homologs as supported by phylogenetic, synteny, and functional analyses. Kisspeptin, Spx, and galanin (Gal) peptides and their corresponding kiss receptors and Gal receptors (Galrs), respectively, are evolutionarily related. Cloning of six tilapia Galrs (Galr1a, Galr1b, Galr2a, Galr2b, Galr type 1, and Galr type 2) and subsequent in vitro second-messenger reporter assays for Gαs, Gαq, and Gαi suggests that Gal and Spx activate Galr1a/Galr2a and Galr2b, respectively. A decrease in plasma follicle stimulating hormone and luteinizing hormone concentrations was observed with injections of Spx1a or Spx1b in vivo. Additionally, application of Spx1a to pituitary slices decreased the firing rate of LH cells, suggesting direct inhibition at the pituitary level. These data collectively suggest an inhibitory mechanism of action against the secretion of gonadotropins for a traditional and a novel spexin paralog in cichlid species.

Gnrh1-Induced Responses Are Indirect in Female Medaka Fsh Cells, Generated Through Cellular Networks

Reproductive function in vertebrates is stimulated by GnRH that controls the synthesis and release of the two pituitary gonadotropins, FSH and LH. FSH and LH, which regulate different stages of gonadal development, are produced by two different cell types in the fish pituitary. This is in contrast to the situation in mammals and birds, and it enables investigation of their differential regulation. In the present study, we used fluorescence in situ hybridization to show that Lh cells in adult female medaka express Gnrh receptors, whereas Fsh cells do not. This result was confirmed by patch-clamp recordings and by cytosolic Ca2+ measurements on dispersed pituitary cells, where Lh cells, but not Fsh cells, responded to Gnrh1 by biphasic alteration in action-potential frequencies and cytosolic Ca2+ levels. In contrast, both Fsh and Lh cells are able to respond to Gnrh1 in brain-pituitary tissue slices both electrically and by elevating the cytosolic Ca2+ levels. Using Ca2+ uncaging in combination with patch-clamp recordings and cytosolic Ca2+ measurements, we show that Fsh and Lh cells form homotypic and heterotypic networks in the pituitary. Taken together, these results show that the effects of Gnrh1 on Fsh release in adult female medaka are indirect and probably mediated via Lh cells.

Gnrh1-induced responses are indirect in female medaka Fsh cells

ABSTRACTReproductive function in vertebrates is stimulated by gonadotropin-releasing hormone (GnRH) that controls the synthesis and release of the two pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH, which regulates different stages of gonadal development, are produced by two different cell types in the fish pituitary, in contrast to mammals and birds, thus allowing the investigation of their differential regulation. In the present work, we show by fluorescentin situhybridization that Lh cells in adult female medaka express Gnrh receptors, whereas Fsh cells do not. This is confirmed by patch clamp recordings and cytosolic Ca2+measurements on dispersed pituitary cells, where Lh cells, but not Fsh cells, respond to Gnrh1 by increased action potential frequencies and cytosolic Ca2+levels. In contrast, both Fsh and Lh cells are able to respond electrically and by elevating the cytosolic Ca2+levels to Gnrh1 in brain-pituitary tissue slices. Using Ca2+uncaging in combination with patch clamp recordings and cytosolic Ca2+measurements, we show that Fsh and Lh cells form homo- and heterotypic networks in the pituitary. Taken together, these results show that the effects of Gnrh1 on Fsh release in adult female medaka is indirect, likely mediated via Lh cells.