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Melatonin is a key hormone involved in the photoperiodic signaling pathway. In both teleosts and mammals, melatonin produced in the pineal gland at night is released into the blood and cerebrospinal fluid, providing rhythmic information to the whole organism. Melatonin acts via specific receptors, allowing the synchronization of daily and annual physiological rhythms to environmental conditions. The pituitary gland, which produces several hormones involved in a variety of physiological processes such as growth, metabolism, stress and reproduction, is an important target of melatonin. Melatonin modulates pituitary cellular activities, adjusting the synthesis and release of the different pituitary hormones to the functional demands, which changes during the day, seasons and life stages. It is, however, not always clear whether melatonin acts directly or indirectly on the pituitary. Indeed, melatonin also acts both upstream, on brain centers that control the pituitary hormone production and release, as well as downstream, on the tissues targeted by the pituitary hormones, which provide positive and negative feedback to the pituitary gland. In this review, we describe the known pathways through which melatonin modulates anterior pituitary hormonal production, distinguishing indirect effects mediated by brain centers from direct effects on the anterior pituitary. We also highlight similarities and differences between teleosts and mammals, drawing attention to knowledge gaps, and suggesting aims for future research.

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.

We have previously characterized the response to gonadotropin-releasing hormone (Gnrh) 2 in luteinizing hormone ( lhb )-expressing cells from green fluorescent protein (Gfp)-transgenic medaka ( Oryzias latipes ), with regard to changes in the cytosolic Ca 2+ concentration. In the current study we present the corresponding responses to Gnrh1 and Gnrh3. Ca 2+ imaging revealed three response patterns to Gnrh1 and Gnrh3, one monophasic and two types of biphasic patterns. There were few significant differences in the shape of the response patterns between the three Gnrh forms, although the amplitude of the Ca 2+ signal was considerably lower for Gnrh1 and Gnrh3 than for Gnrh2, and the distribution between the two different biphasic patterns differed. The different putative Ca 2+ sources were examined by depleting intracellular Ca 2+ stores with thapsigargin, or preventing influx of extracellular Ca 2+ by either extracellular Ca 2+ depletion or the L-type Ca 2+ -channel blocker verapamil. Both Gnrh1 and 3 relied on Ca 2+ from both intracellular and extracellular sources, with some unexpected differences in the relative contribution. Furthermore, gene expression of Gnrh-receptors ( gnrhr ) in whole pituitaries was studied during development from juvenile to adult. Only two of the four identified medaka receptors were expressed in the pituitary, gnrhr1b and gnrhr2a , with the newly discovered gnrhr2a showing the highest expression level at all stages as analyzed by quantitative PCR. While both receptors differed in expression level according to developmental stage, only the expression of gnrhr2a showed a clear-cut increase with gonadal maturation. RNA sequencing analysis of FACS-sorted Gfp-positive lhb -cells revealed that both gnrhr1b and gnrhr2a were expressed in lhb -expressing cells, and confirmed the higher expression of gnrhr2a compared to gnrhr1b . These results show that although lhb -expressing gonadotropes in medaka show similar Ca 2+ response patterns to all three endogenous Gnrh forms through the activation of two different receptors, gnrhr1b and gnrhr2a , the differences observed between the Gnrh forms indicate activation of different Ca 2+ signaling pathways.

Through the action of cortisol, stress can affect reproductive biology with behavioural and physiological alterations. Using mixed sex primary pituitary cultures from Atlantic cod (Gadus morhua), the present study aimed to investigate potential direct effects of basal and stress level cortisol on the pituitary in terms of cell viability and reproduction-related gene expression at different stages of sexual maturity. Stress level of cortisol stimulated cell viability in cells derived from sexually maturing and mature fish. In cells from spent fish, high cortisol levels did not affect cell viability in terms of metabolic activity, but did stimulate viability in terms of membrane integrity. Basal cortisol levels did not affect cell viability. Ethanol, used as solvent for cortisol, decreased cell viability at all maturity stages, but did generally not affect gene expression. Genes investigated were fshb, lhb and two Gnrh receptors expressed in cod gonadotropes (gnrhr1b and gnrhr2a). Cortisol had dual effects on fshb expression; stimulating expression in cells from mature fish at stress dose, while inhibiting expression in cells from spent fish at both doses. In contrast, cortisol had no direct effect on lhb expression. While gnrhr2a transcript levels largely increased following cortisol treatment, gnrhr1b expression decreased in cells from spent fish and was unaffected at other maturity stages. These findings demonstrate that cortisol can act directly and differentially at the pituitary level in Atlantic cod and that factors facilitating these actions are dose-dependently activated and vary with level of sexual maturity.

Pituitary endocrine cells fire action potentials (APs) to regulate their cytosolic Ca2+ concentration and hormone secretion rate. Depending on animal species, cell type, and biological conditions, pituitary APs are generated either by TTX-sensitive Na+ currents (INa), high-voltage activated Ca2+ currents (ICa), or by a combination of the two. Previous computational models of pituitary cells have mainly been based on data from rats, where INa is largely inactivated at the resting potential, and spontaneous APs are predominantly mediated by ICa. Unlike in rats, spontaneous INa-mediated APs are consistently seen in pituitary cells of several other animal species, including several species of fish. In the current work we develop a computational model of gonadotropin releasing cells in the teleost fish medaka (Oryzias latipes). The model stands out from previous modeling efforts by being (1) the first model of a pituitary cell in teleosts, (2) the first pituitary cell model that fires sponateous APs that are predominantly mediated by INa, and (3) the first pituitary cell model where the kinetics of the depolarizing currents, INa and ICa, are directly fitted to voltage-clamp data. We explore the firing properties of the model, and compare it to the properties of previous models that fire ICa-based APs. We put a particular focus on how the big conductance K+ current (IBK) modulates the AP shape. Interestingly, we find that IBK can prolong AP duration in models that fire ICa-based APs, while it consistently shortens the duration of the predominantly INa-mediated APs in the medaka gonadotroph model. Although the model is constrained to experimental data from gonadotroph cells in medaka, it may likely provide insights also into other pituitary cell types that fire INa-mediated APs., Author summary Excitable cells elicit electrical pulses called action potentials (APs), which are generated and shaped by a combination of ion channels in the cell membrane. Since one type of ion channels is permeable to Ca2+ ions, there is typically an influx of Ca2+ during an AP. Pituitary cells therefore use AP firing to regulate their cytosolic Ca2+ concentration, which in turn controls their hormone secretion rate. The amount of Ca2+ that enters during an AP depends strongly on how long it lasts, and it is therefore important to understand the mechanisms that control this. Pituitary APs are generally mediated by a combination of Ca2+ channels and Na+ channels, and the relative contributions of from the two vary between cell types, animal species and biological conditions. Previous computer models have predominantly been adapted to data from pituitary cells which tend to fire Ca2+-based APs. Here we develop a new model, adapted to data from pituitary cells in the fish medaka, which APs that are predominantly Na+-based, and compare its dynamical properties to the previous models that fire Ca2+-based APs.

Bisphenol A (BPA) and tetrabromobisphenol A (TBBPA) are widely used industrial chemicals, ubiquitously present in the environment. While BPA is a well-known endocrine disruptor and able to affect all levels of the teleost reproductive axis, information regarding TBBPA on this subject is very limited. Using primary cultures from Atlantic cod (Gadus morhua), the present study was aimed at investigating potential direct effects of acute (72 h) BPA and TBBPA exposure on cell viability and the expression of reproductive-relevant genes in the pituitary. The results revealed that both bisphenols stimulate cell viability in terms of metabolic activity and membrane integrity at environmentally relevant concentrations. BPA had no direct effects on gonadotropin gene expression, but enhanced the expression of gonadotropin-releasing hormone (GnRH) receptor 2a, the main gonadotropin modulator in Atlantic cod. In contrast, TBBPA increased gonadotropin transcript levels but had no effect on GnRH receptor mRNA. In conclusion, both anthropogenic compounds display endocrine disruptive properties and are able to directly interfere with gene expression related to reproductive function in cod pituitary cells at environmentally relevant concentrations in vitro.

The onset of puberty and reproduction are tightly controlled by extrinsic and intrinsic inputs combined with genetically determined biological blueprints. Environmental inputs are then mediated by the brain– pituitary–gonad endocrine axis resulting in a unified output. In fish, one of the primary factors controlling the timing of sexual maturation is light, although how these signals are mediated in the brain and pituitary is not well understood. We therefore aimed to elucidate the molecular basis of the control of reproduction during the first spawning season in two year old female Atlantic cod. To this end, we measured GnRH and GnRH-R variant gene expression in brains and pituitaries collected from cod kept under four different photoperiod regimes: natural light (NL), continuous light (LL) and combined treatment of NL–LL and LL–NL. LL inhibited sexual development and spawning and LL–NL delayed sexual development and spawning. LL inhibited the spawning-related increase in brain GnRH3 and pituitary GnRH-R2a gene expression found under NL conditions, and the expression of these genes were delayed in concert with spawning of LL–NL cod. This study indicates that regulation of brain GnRH3 and pituitary GnRH-R2a genes likely mediates photoperiod induced changes in cod gonadal maturation.

Aims: Thyrotropin-releasing hormone (TRH) induces biphasic changes in electrical activity, cytosolic free Ca 2 + level ([Ca 2 + ] i ), and prolactin secretion from both clonal GH cells and native lactotrophs. The first phase of the TRH response is characterized by hyperpolarization because of activation of Ca 2 + -activated K + channels (K C a ). In the present study, the relative contribution of BK, SK, and IK channels to the first phase of the TRH response in GH 4 cells was assessed. Methods: The expression of IK channels was confirmed by PCR with specific primers for SK4 (IK). The response to TRH was studied using the perforated patch technique and Ca 2 + microfluoromety (fura-2). The involvement of different K C a channels was estimated by employing the specific channel blockers iberiotoxin (BK), apamin (SK) and clotrimazole (IK). Results: Application of 100 nM iberiotoxin, 1 μM apamin, and 10 μM clotrimazole reduced the peak value of the outward K + current during the first phase of the TRH response by 33, 26, and 33%, respectively. Clotrimazole also shortened the duration of the outward current response by 60%, causing a reduction of total charge movement by 73%. All these toxin-induced reductions were significant (P < 0.05). A combination of all three toxins abolished the current response almost completely. Conclusion: All the three main types of K C a channels are involved in the first phase of the TRH response, with IK as the major contributor. This is the first demonstration of a dominant role of IK compared with BK and SK channels in excitable cells.

Aim: Thyrotropin-releasing hormone (TRH) induces biphasic changes in the electrical activity, the cytosolic free Ca 2+ concentration ([Ca 2+ ] i ), and prolactin secretion from both GH cells and native lactotrophs. It is well established that inhibition of erg channels contributes to the second phase of the TRH response. We have investigated if BK channels are also involved. Results: The BK channels may be active at the resting membrane potential (open probability, P o = 0.01) in clonal rat anterior pituitary cells (GH 4 ), which makes it possible that inhibition of these channels may contribute to the reduced K + conductance during the TRH response. The specific BK channel blocker iberiotoxin (IbTx, 100 nM) had no effect on the resting conductance at holding potentials negative to -40 mV, but significantly reduced the conductance at shallower membrane potentials. This corresponds to the voltage dependency of the sustained [Ca 2+ ] i . Furthermore, IbTx increased the action potential frequency by 36% in spontaneously firing cells. During the second phase of the TRH response, the action potential frequency increased by 34%, concomitantly with 61% reduction of the P o of single BK channels. The protein kinase C (PKC)-activating phorbol ester TPA had no significant effect on BK channel P o within the normal range of the resting potential. Conclusion: The BK channels may contribute to the resting membrane conductance, and they are partially inhibited by TRH during the second phase. This modulation seems not to depend on PKC. We propose that inhibition of erg and BK channels acts in concert to enhance the cell excitability during the second phase of the response to TRH.

Pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. The different cell types in the vertebrate pituitary develop from common progenitor cells just after gastrulation. Proper development and merging of the anterior and posterior pituitary is dependent upon carefully regulated cell-to-cell interactions, and a suite of signaling pathways with precisely organized temporal and spatial expression patterns, which include transcription factors and their co-activators and repressors. Among the pituitary endocrine cell types, the gonadotropes are the last to develop and become functional. Although much progress has been made during the last decade regarding details of gonadotrope development, the coordinated program for their maturation is not well described. FSH and LH form an integral part of the hypothalamo-pituitary-gonad axis, the main regulator of gonad development and reproduction. Besides regulating gonad development, pre- and early post-natal activity in this axis is thought to be essential for proper development, especially of the central nervous system in mammals. As a means to investigate early functions of FSH and LH in more detail, we have developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promoter driving green fluorescent protein (Gfp) expression to characterize development of lhb-expressing gonadotropes. The lhb gene is maternally expressed early during embryogenesis. lhb-Expressing cells are initially localized outside the primordial pituitary in the developing gut tube as early as 32 hpf. At hatching, lhb-Gfp is clearly detected in the gut epithelium and in the anterior digestive tract. lhb-Gfp expression later consolidates in the developing pituitary by 2 weeks post-fertilization. This review discusses status of knowledge regarding pituitary morphology and development, with emphasis on gonadotrope cells and gonadotropins during early development, comparing main model species like mouse, zebrafish and medaka, including possible developmental functions of the observed extra pituitary expression of lhb in medaka.

Synthesis and release of FSH and LH are differentially regulated by GnRH, but the mechanisms by which this regulation is achieved are not well understood. Teleost fish are powerful models for studyingthisdifferentialregulationbecausetheyhavedistinctpituitarycellsproducingeitherFSH or LH. By using pituitary cultures from Atlantic cod (Gadus morhua), we were able to investigate and compare the electrophysiological properties of fshb- and lhb-expressing cells, identified by single-cell quantitative PCR after recording. Both cell types fired action potentials spontaneously. The relative number of excitable cells was dependent on reproductive season but varied in opposing directions according to season in the 2 cell types. Excitable and quiescent gonadotropes displayed different ion channel repertoires. The dynamics of outward currents and GnRH-induced membrane responses differed between fshb- and lhb-expressing cells, whereas GnRH-induced cytosolic Ca 2 responses were similar. Expression of Ca 2 -activated K channels also differed with cell type and showed seasonal variation when measured in whole pituitary. The differential presence of these channels corresponds to the differences observed in membrane response to GnRH. We speculate that differences in ion channel expression levels may be involved in seasonal regulation of hormone secretion as well as the differential response to GnRH in LHand FSH-producing gonadotropes, through differences in excitability and Ca 2 influx. (Endocrinology 154: 3319–3330, 2013)

Background: Luteinizing hormone (LH) and follicle stimulating hormone (FSH), produced in gonadotrope cells in the adenohypophysis are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. We developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promotor driving green fluorescent protein (gfp) expression to characterize development of LH-producing gonadotropes in whole larvae and histological sections. Additionally, developmental and tissue-specific gene expression was examined. Results: The lhb gene is maternally expressed during early embryogenesis. Transcript levels increase by stage 21 (36 hours post fertilization [hpf]) and then decrease during continued larval development. Examination of the expression of pituitary marker genes show that LH-producing cells are initially localized outside the primordial pituitary, and they were localized to the developing gut tube by 32 hpf. At hatching, lhb-GFP is clearly detected in the gut epithelium and in the anterior digestive tract. lhb-GFP expression later consolidate in the developing pituitary by 2 weeks postfertilization. Conclusions: During embryonic development, lhb is primarily expressed outside the central nervous system and pituitary. The novel expression of lhb in the embryonic gut suggests that LH has a hitherto unidentified developmental function. Developmental Dynamics 241:1665–1677, 2012. © 2012 Wiley Periodicals, Inc.

Gonadotropin releasing hormones (GnRH) are an important part of the brain–pituitary–gonad axis in vertebrates. GnRH binding to its receptors (GnRH-R) stimulates synthesis and release of gonadotropins in the pituitary. GnRH-Rs also mediate other processes in the central nervous system such as reproductive behavior and neuromodulation. As many as five GnRH-R genes have been identified in two teleost fish species, but the function and phylogenetic relationship of these receptors is not fully understood. To gain a better understanding of the functional relationship between multiple GnRH-Rs in an important aquaculture species, the Atlantic cod (Gadus morhua), we identified four GnRH-Rs (gmGnRH-R) by RT-PCR, followed by full-length cloning and sequencing. The deduced amino acid sequences were used for phylogenetic analysis to identify conserved functional motifs and to clarify the relationship of gmGnRH-Rs with other vertebrate GnRH-Rs. The function of GnRH-R variants was investigated by quantitative PCR gene expression analysis in the brain and pituitary of female cod during a full reproductive cycle and in various peripheral tissues in sexually mature fish. Phylogenetic analysis revealed two types of teleost GnRH-Rs: Type I including gmGnRH-R1b and Type II including gmGnRH-R2a, gmGnRH-R2b and gmGnRH-R2c. All four gmGnRH-Rs are expressed in the brain, and gmGnRH-R1b, gmGnRH-R2a and gmGnRH-R2c are expressed in the pituitary. The only GnRH-R differentially expressed in the pituitary during the reproductive cycle is gmGnRH-R2a such that its expression is significantly increased during spawning. These data suggest that gmGnRH-R2a is the most likely candidate to mediate the hypophysiotropic function of GnRH in Atlantic cod.

ATP-sensitive potassium (K ATP ) channels in the pancreatic β cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca 2+ influx and exocytosis. Metabolic regulation of K ATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The β cell K ATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate K ATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg 2+ caused an apparent reduction in the inhibitory action of ATP on wild-type K ATP channels, and MgATP actually activated K ATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates K ATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg 2+ .