Day length regulates gonadotrope proliferation and reproduction via an intra-pituitary pathway in the model vertebrate Oryzias latipes.

In seasonally breeding mammals and birds, the production of the hormones that regulate reproduction (gonadotropins) is controlled by a complex pituitary-brain-pituitary pathway. Indeed, the pituitary thyroid-stimulating hormone (TSH) regulates gonadotropin expression in pituitary gonadotropes, via dio2-expressing tanycytes, hypothalamic Kisspeptin, RFamide-related peptide, and gonadotropin-releasing hormone neurons. However, in fish, how seasonal environmental signals influence gonadotropins remains unclear. In addition, the seasonal regulation of gonadotrope (gonadotropin-producing cell) proliferation in the pituitary is, to the best of our knowledge, not elucidated in any vertebrate group. Here, we show that in the vertebrate model Japanese medaka (Oryzias latipes), a long day seasonally breeding fish, photoperiod (daylength) not only regulates hormone production by the gonadotropes but also their proliferation. We also reveal an intra-pituitary pathway that regulates gonadotrope cell number and hormone production. In this pathway, Tsh regulates gonadotropes via folliculostellate cells within the pituitary. This study suggests the existence of an alternative regulatory mechanism of seasonal gonadotropin production in fish.

Functional and developmental heterogeneity of pituitary lactotropes in medaka.

In fish, prolactin-producing cells (lactotropes) are located in the anterior part of the pituitary and play an essential role in osmoregulation. However, small satellite lactotrope clusters have been described in other parts of the pituitary in several species. The functional and developmental backgrounds of these satellite clusters are not known. We recently discovered two distinct prolactin-expressing cell types in Japanese medaka (Oryzias latipes), a euryhaline species, using single cell transcriptomics. In the present study, we characterize these two transcriptomically distinct lactotrope cell types and explore the hypothesis that they represent spatially distinct cell clusters, as found in other species. Single cell RNA sequencing shows that one of the two lactotrope cell types exhibits an expression profile similar to that of stem cell-like folliculo-stellate cell populations. Using in situ hybridization, we show that the medaka pituitary often develops additional small satellite lactotrope cell clusters, like in other teleost species. These satellite clusters arise early during development and grow in cell number throughout life regardless of the animal's sex. Surprisingly, our data do not show a correspondence between the stem cell-like lactotropes and these satellite lactotrope clusters. Instead, our data support a scenario in which the stem cell-like lactotropes are an intrinsic stage in the development of every spatially distinct lactotrope cluster. In addition, lactotrope activity in both spatially distinct lactotrope clusters decreases when environmental salinity increases, supporting their role in osmoregulation. However, this decrease appears weaker in the satellite lactotrope cell clusters, suggesting that these lactotropes are regulated differently.

Characterization of gonadotropin receptors Fshr and Lhr in Japanese medaka, Oryzias latipes.

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshßα, mdLhßα, tiFshßα, tiLhßα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshßα was able to activate the mdLhr, and mdLhßα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhßα, tiFshßα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshßα, tiLhßα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshßα, mdLhßα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia.

Functional divergence of thyrotropin beta-subunit paralogs gives new insights into salmon smoltification metamorphosis.

Smoltification is a metamorphic event in salmon life history, which initiates downstream migration and pre-adapts juvenile salmon for seawater entry. While a number of reports concern thyroid hormones and smoltification, few and inconclusive studies have addressed the potential role of thyrotropin (TSH). TSH is composed of a α-subunit common to gonadotropins, and a ß-subunit conferring hormone specificity. We report the presence and functional divergence of duplicated TSH ß-subunit paralogs (tshßa and tshßb) in Atlantic salmon. Phylogeny and synteny analyses allowed us to infer that they originated from teleost-specific whole genome duplication. Expression profiles of both paralogs in the pituitary were measured by qPCR throughout smoltification in Atlantic salmon from the endangered Loire-Allier population raised in a conservation hatchery. This revealed a striking peak of tshßb expression in April, concomitant with downstream migration initiation, while tshßa expression remained relatively constant. In situ hybridization showed two distinct pituitary cell populations, tshßa cells in the anterior adenohypophysis, and tshßb cells near to the pituitary stalk, a location comparable to the pars tuberalis TSH cells involved in seasonal physiology and behaviour in birds and mammals. Functional divergence of tshß paralogs in Atlantic salmon supports a specific role of tshßb in smoltification.

Medaka follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression levels.

The two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are of particular importance within the hypothalamic-pituitary-gonadal (HPG) axis of vertebrates. In the current study, we demonstrate the production and validation of Japanese medaka (Oryzias latipes) recombinant (md) gonadotropins Fshß (mdFshß), Lhß (mdLhß), Fshßα (mdFshßα), and Lhßα (mdLhßα) by Pichia pastoris, the generation of specific rabbit antibodies against their respective ß subunits, and their use within the development and validation of competitive enzyme-linked immunosorbent assays (ELISAs) for quantification of medaka Fsh and Lh. mdFsh and mdLh were produced as single-chain polypeptides by linking the α subunit with mdFshß or mdLhß mature protein coding sequences to produce a "tethered" polypeptide with the ß-chain at the N-terminal and the α-chain at the C-terminal. The specificity of the antibodies raised against mdFshß and mdLhß was determined by immunofluorescence (IF) for Fshß and Lhß on medaka pituitary tissue, while comparison with fluorescence in situ hybridization (FISH) for fshb and lhb mRNA was used for validation. Competitive ELISAs were developed using antibodies against mdFshß or mdLhß, and the tethered proteins mdFshßα or mdLhßα for standard curves. The standard curve for the Fsh ELISA ranged from 97.6 pg/ml to 50 ng/ml, and for the Lh ELISA from 12.21 pg/ml to 6.25 ng/ml. The sensitivity of the assays for Fsh and Lh was 44.7 and 70.8 pg/ml, respectively. A profile of pituitary protein levels of medaka Fsh and Lh comparing juveniles with adults showed significant increase of protein amount from juvenile group (body length from 12 mm to 16.5 mm) to adult group (body length from 21 mm to 26.5 mm) for both hormones in male medaka. Comparing these data to a developmental profile of pituitary mRNA expression of medaka fshb and lhb, the mRNA expression of lhb also increased during male maturation and a linear regression analysis revealed a significant increase of lhb expression with increased body length that proposes a linear model. However, fshb mRNA expression did not change significantly during male development and therefore was not correlated with body length. In summary, we have developed and validated homologous ELISA assays for medaka Fsh and Lh based on proteins produced in P. pastoris, assays that will be used to study the functions and regulations of Fsh and Lh in more detail.

Sex steroids differentially regulate fshb, lhb and gnrhr expression in Atlantic cod (Gadus morhua).

Depending on the stage of gonad maturation, as well as other factors, gonadal steroids can exert either a positive or negative feedback at the brain and pituitary level. While this has been demonstrated in many teleost species, little is known about the nature of steroid feedback in Gadiform fish. Using an optimized in vitro model system of the Atlantic cod pituitary, the present study investigated the potential effects of two physiologically relevant doses of estradiol, testosterone (TS) or dihydrotestosterone (DHTS) on cell viability and gene expression of gonadotropin subunits (fshb/lhb) and two suggested reproduction-relevant gonadotropin-releasing hormone receptors (gnrhr1b/gnrhr2a) during three stages of sexual maturity. In general, all steroids stimulated cell viability in terms of metabolic activity and membrane integrity. Furthermore, all steroids affected fshb expression, with the effect depending on both the specific steroid, dose and maturity status. Conversely, only DHTS exposure affected lhb levels, and this occurred only during the spawning season. Using single-cell qPCR, co-transcription of gnrhr1b and gnrhr2a was confirmed to both fshb- and lhb- expressing gonadotropes, with gnrhr2a being the most prominently expressed isoform. While steroid exposure had no effect on gnrhr1b expression, all steroids affected gnrhr2a transcript levels in at least one maturity stage. These and previous results from our group point to Gnrhr2a as the main modulator of gonadotropin regulation in cod and that regulation of its gene expression level might function as a direct mechanism for steroid feedback at the pituitary level.

Dose-dependent effects of gamma radiation on the early zebrafish development and gene expression.

Ionizing radiation from natural sources or anthropogenic activity has the potential to cause oxidative stress or genetic damage in living organisms, through the ionization and excitation of molecules and the subsequent production of free radicals and reactive oxygen species (ROS). The present work focuses on radiation-induced biological effects using the zebrafish (Danio rerio) vertebrate model. Changes in developmental traits and gene expression in zebrafish were assessed after continuous external gamma irradiation (0.4, 3.9, 15 and 38 mGy/h) with corresponding controls, starting at 2.5 hours post fertilization (hpf) and lasting through embryogenesis and the early larval stage. The lowest dose rate corresponded to recommended benchmarks at which adverse effects are not expected to occur in aquatic ecosystems (2-10 mGy/day). The survival observed at 96 hours post fertilization (hpf) in the 38 mGy/h group was significantly lower, while other groups showed no significant difference compared to controls. The total hatching was significantly lower from controls in the 15 mGy/h group and a delay in hatching onset in the 0.4 mGy/h group was observed. The deformity frequency was significantly increased by prolonged exposure duration at dose rates ≥ 0.4 mGy/h. Molecular responses analyzed by RNA-seq at gastrulation (5.5 hpf transcriptome) indicate that the radiation induced adverse effects occurred during the earliest stages of development. A dose-response relationship was found in the numbers of differentially regulated genes in exposure groups compared to controls at a total dose as low as 1.62 mGy. Ingenuity Pathway Analysis identified retinoic acid receptor activation, apoptosis, and glutathione mediated detoxification signaling as the most affected pathways in the lower dose rate (0.54 mGy/h), while eif2 and mTOR, i.e., involved in the modulation of angiogenesis, were most affected in higher dose rates (5.4 and 10.9 mGy/h). By comparing gene expression data, myc was found to be the most significant upstream regulator, followed by tp53, TNF, hnf4a, TGFb1 and cebpa, while crabp2b and vegfab were identified as most frequent downstream target genes. These genes are associated with various developmental processes. The present findings show that continuous gamma irradiation (≥ 0.54 mGy/h) during early gastrula causes gene expression changes that are linked to developmental defects in zebrafish embryos.

Identified lhb-expressing cells from medaka (Oryzias latipes) show similar Ca(2+)-response to all endogenous Gnrh forms, and reveal expression of a novel fourth Gnrh receptor.

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.

Electrophysiological differences between fshb- and lhb-expressing gonadotropes in primary culture.

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 studying this differential regulation because they have distinct pituitary cells producing either FSH 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 Ca²âº responses were similar. Expression of Ca²âº-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 LH- and FSH-producing gonadotropes, through differences in excitability and Ca²âº influx.

Parental exposure to natural mixtures of POPs reduced embryo production and altered gene transcription in zebrafish embryos.

Determination of toxicity of complex mixtures has been proposed to be one of the most important challenges for modern toxicology. In this study we performed genome wide transcriptome profiling to assess potential toxicant induced changes in gene regulation in zebrafish embryos following parental exposure to two natural mixtures of persistent organic pollutants (POPs). The mixtures used were extracted from burbot (Lota lota) liver originating from two lakes (Lake Mjøsa and Lake Losna) belonging to the same freshwater system in Norway. The dominating groups of contaminants were polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane metabolites (DDTs). Because both mixtures used in the present study induced similar effects, it is likely that the same toxicants are involved. The Mjøsa mixture contains high levels of PBDEs while this group of pollutants is low in the Losna mixture. However, both mixtures contain substantial concentrations of PCB and DDT suggesting these contaminants as the predominant contributors to the toxicity observed. The observed effects included phenotypic traits, like embryo production and survival, and gene transcription changes corresponding with disease and biological functions such as cancer, reproductive system disease, cardiovascular disease, lipid and protein metabolism, small molecule biochemistry and cell cycle. The changes in gene transcription included genes regulated by HNF4A, insulin, LH, FSH and NF-κB which are known to be central regulators of endocrine signaling, metabolism, metabolic homeostasis, immune functions, cancer development and reproduction. The results suggest that relative low concentrations of the natural mixtures of POPs used in the present study might pose a threat to wild freshwater fish living in the lakes from which the POPs mixtures originated.

Natural mixtures of POPs affected body weight gain and induced transcription of genes involved in weight regulation and insulin signaling.

Obesity is reaching epidemic proportions worldwide, and is associated with chronic illnesses such as diabetes, cardiovascular disease, hypertension and dyslipidemias (metabolic syndrome). Commonly held causes of obesity are overeating coupled with a sedentary lifestyle. However, it has also been postulated that exposure to endocrine disrupting chemicals (EDCs) may be related to the significant increase in the prevalence of obesity and associated diseases. In the present study, developmental and reproductive effects of lifelong exposure to environmentally relevant concentrations of two natural mixtures of persistent organic pollutants (POPs) were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway (Lake Mjøsa and Lake Losna). The concentration of POPs in the zebrafish ranged from levels detected in wild fish (Lake Mjøsa and Lake Losna), to concentrations reported in human and wildlife populations. Phenotypic effects observed in both exposure groups included (1) earlier onset of puberty, (2) elevated male/female sex ratio, and (3) increased body weight at 5 months of age. Interestingly, genome-wide transcription profiling identified functional networks of genes, in which key regulators of weight homeostasis (PPARs, glucocoricoids, CEBPs, estradiol), steroid hormone functions (glucocoricoids, estradiol, NCOA3) and insulin signaling (HNF4A, CEBPs, PPARG) occupied central positions. The increased weight and the regulation of genes associated with weight homeostasis and insulin signaling observed in the present study suggest that environmental pollution may affect the endocrine regulation of the metabolism, possibly leading to increased weight gain and obesity.