Endocrine patterns associated with ovarian development in female Pacific halibut (Hippoglossus stenolepis).

The Pacific halibut (Hippoglossus stenolepis) is a large migratory demersal flatfish species that occupies a top trophic role in the North Pacific Ocean and Bering Sea ecosystems, where it also supports various fisheries. As a first attempt to characterize the endocrine mechanisms driving sexual maturation in this important species, we collected pituitary, ovarian and blood samples from Pacific halibut females captured in the wild that were classified histologically into various female developmental stages. We conducted gene expression analyses of gonadotropin beta subunits in the pituitary and observed that mRNA expression levels of fshb gradually increased throughout vitellogenesis, remained elevated until before ovulation and declined after spawning. In contrast, the mRNA expression levels of lhb markedly increased during oocyte maturation and remained elevated until after spawning. Ovarian mRNA expression levels of the gonadotropin receptor genes fshr and lhr peaked during oocyte maturation and before spawning, respectively, immediately following the developmental stage at which pituitary fshb and lhb mRNA expression first reached maximum levels. The ovarian gene expression patterns of steroidogenic enzyme genes cyp19a1 and hsd20b2 paralleled those of fshr and lhr, respectively. Testosterone and 17ß-estradiol (E2) plasma levels increased concomitantly with fshr and cyp19a1 mRNA expression levels, and vitellogenin plasma levels increased throughout vitellogenesis and reached maximum levels prior to spawning. These results are consistent with the notion that in female Pacific halibut, as in other teleosts, vitellogenesis and oocyte maturation and ovulation are likely under the control of pituitary gonadotropic hormones Fsh and Lh, respectively.

Regulation of steroid production and key genes in catfish Heteropneustes fossilis using recombinant gonadotropins.

The two gonadotropins, FSH and LH, stimulate growth and development of the gonads through gonadal biosynthesis of steroid hormones and growth factors. To date, cDNA sequences encoding gonadotropin subunits have been isolated and characterized from a large number of fish species. Recently, we successfully cloned and characterized gonadotropins (LHß, FSHß, and GPα) from the pituitary glands of the catfish, Heteropneustes fossilis. In the present study, we describe herein the production of recombinant stinging catfish, H. fossilis (hf) FSH (rhfFSH) and LH (rhfLH) using the methylotrophic yeast P. pastoris expression system. We further explored the hypothesis that the recombinant gonadotropins can modulate the hypothalamus-pituitary-ovarian (HPO) axis genes (avt, it, gnrh2, kiss2, and cyp19a1a) and regulate their transcriptional profile and steroid levels in relation to their annual developmental stage during preparatory and pre-spawning phases under in-vitro conditions. We found that the different concentrations of recombinant rhfFSH and rhfLH significantly stimulated E2 levels in the preparatory and prespawning season, and also upregulated gonadal aromatase gene expression in a dose dependent manner. Our results demonstrate that the yeast expression system produced biologically active recombinant catfish gonadotropins, enabling the study of their function in the catfish.

Molecular cloning and bioinformatic characterization of Gonadotropin Inhibitory Hormone (GnIH) and its receptors in the freshwater murrel, Channa punctatus (Bloch, 1793).

Gonadotropin inhibitory hormone belonging to the RFamide peptide family, a hypothalamic neuropeptide, regulates Hypothalamus-pituitary-gonadal (HPG) axis and inhibits gonadal development. GnIH polypeptide precursor has an Arg-Phe-NH2 (RFamide) motif at the C-terminal, which has LPXRF (X = Q or L) domain. The actions of GnIH are mediated through G-protein coupled receptors and upto three receptors have been characterized in many teleosts. GnIH exerts its inhibitory effect on the HPG axis through direct interaction with GnRH and Kisspeptin neurons in the brain and acts directly on the pituitary gonadotrophs. To decipher the role of GnIH in Indian freshwater murrel, Channa punctatus, we sequenced the cDNA encoding GnIH and its two receptors. The identified GnIH mRNA encodes three RFamide peptides having -MPMRF, -MPQRF, and -LPQRFamide motifs. In silico analysis of the amino acid sequence of GnIH exhibits its molecular and functional properties and the protein-protein interaction with significant factors regulating the HPG axis. The 3-D structure of GnIH and its receptors, provides more relevant information about the active residues of these proteins which might be involved in their functioning and interaction with other proteins. Molecular dynamic simulation of GnIH protein has provided more insight into its dynamic behavior. The expression of GnIH and its receptors, shows an inverse correlation with gonadal development during the annual reproductive cycle.

Transcriptomes of testis and pituitary from male Nile tilapia (O. niloticus L.) in the context of social status.

African cichlids are well established models for studying social hierarchies in teleosts and elucidating the effects social dominance has on gene expression. Ascension in the social hierarchy has been found to increase plasma levels of steroid hormones, follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) as well as gonadosomatic index (GSI). Furthermore, the expression of genes related to gonadotropins and steroidogenesis and signaling along the brain-pituitary-gonad axis (BPG-axis) is affected by changes of an animal's social status. In this study, we use RNA-sequencing to obtain an in-depth look at the transcriptomes of testes and pituitaries from dominant and subordinate male Nile tilapia living in long-term stable social hierarchies. This allows us to draw conclusions about factors along the brain-pituitary-gonad axis that are involved in maintaining dominance over weeks or even months. We identify a number of genes that are differentially regulated between dominant and subordinate males and show that in high-ranking fish this subset of genes is generally upregulated. Genes differentially expressed between the two social groups comprise growth factors, related binding proteins and receptors, components of Wnt-, Tgfß- and retinoic acid-signaling pathway, gonadotropin signaling and steroidogenesis pathways. The latter is backed up by elevated levels of 11-ketotestosterone, testosterone and estradiol in dominant males. Luteinizing hormone (Lh) is found in higher concentration in the plasma of long-term dominant males than in subordinate animals. Our results both strengthen the existing models and propose new candidates for functional studies to expand our understanding of social phenomena in teleost fish.

Epigenetic repression of gonadotropin gene expression via a GnRH-mediated DNA delivery system.

The reproductive axis is activated by gonadotropin-releasing hormone (GnRH), which stimulates the pituitary gonadotropes to secrete hormones that drive gonadal function and steroidogenesis. Thus repression of this axis, which is conserved across mammals and sexes, can reduce steroid levels and/or prevent reproduction. Steroid-dependent pathologies, including various cancers, are commonly treated with GnRH super-analogs which have long-term side-effects, while humane solutions for controlling reproduction in domestic and wild animal populations are lacking. GnRH-conjugated toxins are undergoing clinical trials for GnRHR-expressing cancer cells, and have been examined for gonadotrope ablation in animals, but showed low and/or transient effects and administration of toxins has many potential complications. Here we exploit GnRH targeting to gonadotropes to deliver DNA encoding an effector that induces gonadotropin gene repressive epigenetic modifications which are perpetuated over time. Several layers of specificity are endowed through targeting to GnRHR-expressing cells and due to local cleavage of the peptide packaging the DNA; the DNA-encoded effector is expressed and directed to the target genes by the DNA binding domain of a highly specific transcription factor. This design has multiple advantages over existing methods of shutting down the reproductive axis, and its modular design should allow adaptation for broad applications.

Deletion of Gαq/11 or Gαs Proteins in Gonadotropes Differentially Affects Gonadotropin Production and Secretion in Mice.

Gonadotropin-releasing hormone (GnRH) regulates gonadal function via its stimulatory effects on gonadotropin production by pituitary gonadotrope cells. GnRH is released from the hypothalamus in pulses and GnRH pulse frequency differentially regulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis and secretion. The GnRH receptor (GnRHR) is a G protein-coupled receptor that canonically activates Gα q/11-dependent signaling on ligand binding. However, the receptor can also couple to Gα s and in vitro data suggest that toggling between different G proteins may contribute to GnRH pulse frequency decoding. For example, as we show here, knockdown of Gα s impairs GnRH-stimulated FSH synthesis at low- but not high-pulse frequency in a model gonadotrope-derived cell line. We next used a Cre-lox conditional knockout approach to interrogate the relative roles of Gα q/11 and Gα s proteins in gonadotrope function in mice. Gonadotrope-specific Gα q/11 knockouts exhibit hypogonadotropic hypogonadism and infertility, akin to the phenotypes seen in GnRH- or GnRHR-deficient mice. In contrast, under standard conditions, gonadotrope-specific Gα s knockouts produce gonadotropins at normal levels and are fertile. However, the LH surge amplitude is blunted in Gα s knockout females and postgonadectomy increases in FSH and LH are reduced both in males and females. These data suggest that GnRH may signal principally via Gα q/11 to stimulate gonadotropin production, but that Gα s plays important roles in gonadotrope function in vivo when GnRH secretion is enhanced.

Differential Regulation of Gonadotropins as Revealed by Transcriptomes of Distinct LH and FSH Cells of Fish Pituitary.

From mammals to fish, reproduction is driven by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) temporally secreted from the pituitary gland. Teleost fish are an excellent model for addressing the unique regulation and function of each gonadotropin cell since, unlike mammals, they synthesize and secrete LH and FSH from distinct cells. Only very distant vertebrate classes (such as fish and birds) demonstrate the mono-hormonal strategy, suggesting a potential convergent evolution. Cell-specific transcriptome analysis of double-labeled transgenic tilapia expressing GFP and RFP in LH or FSH cells, respectively, yielded genes specifically enriched in each cell type, revealing differences in hormone regulation, receptor expression, cell signaling, and electrical properties. Each cell type expresses a unique GPCR signature that reveals the direct regulation of metabolic and homeostatic hormones. Comparing these novel transcriptomes to that of rat gonadotrophs revealed conserved genes that might specifically contribute to each gonadotropin activity in mammals, suggesting conserved mechanisms controlling the differential regulation of gonadotropins in vertebrates.

Acute social defeat stress upregulates gonadotrophin inhibitory hormone and its receptor but not corticotropin-releasing hormone and ACTH in the Male Nile Tilapia (Oreochromis niloticus).

Stress impairs the hypothalamic-pituitary-gonadal (HPG) axis, probably through its influence on the hypothalamic-pituitary-adrenal (= interrenals in the teleost, HPI) axis leading to reproductive failures. In this study, we investigated the response of hypothalamic neuropeptides, gonadotropin-inhibitory hormone (GnIH), a component of the HPG axis, and corticotropin-releasing hormone (CRH) a component of the HPI axis, to acute social defeat stress in the socially hierarchical male Nile tilapia (Oreochromis niloticus). Localization of GnIH cell bodies, GnIH neuronal processes, and numbers of GnIH cells in the brain during acute social defeat stress was studied using immunohistochemistry. Furthermore, mRNA levels of GnIH and CRH in the brain together with GnIH receptor, gpr147, and adrenocorticotropic hormone (ACTH) in the pituitary were quantified in control and socially defeated fish. Our results show, the number of GnIH-immunoreactive cell bodies and GnIH mRNA levels in the brain and the levels of gpr147 mRNA in the pituitary significantly increased in socially defeated fish. However, CRH and ACTH mRNA levels did not change during social defeat stress. Further, we found glucocorticoid type 2b receptor mRNA in laser captured immunostained GnIH cells. These results show that acute social defeat stress activates GnIH biosynthesis through glucocorticoid receptors type 2b signalling but does not change the CRH and ACTH mRNA expression in the tilapia, which could lead to temporary reproductive dysfunction.

HSP70 inhibits pig pituitary gonadotrophin synthesis and secretion by regulating the corticotropin-releasing hormone signaling pathway and targeting SMAD3.

High levels or long periods of stress have been shown to negatively impact cell homeostasis, including with respect to abnormalities in domestic animal reproduction, which are typically activated through the hypothalamus-pituitary-adrenal axis, in which corticotropin-releasing hormone (CRH) and heat shock protein 70 (HSP70) are involved. In addition, CRH has been reported to inhibit pituitary gonadotrophin synthesis, and HSP70 is expressed in the pituitary gland. The aim of this study was to determine whether HSP70 was involved in regulating gonadotrophin synthesis and secretion by mediating the CRH pathway in the porcine pituitary gland. Our results showed that HSP70 was highly expressed in the porcine pituitary gland, with over 90% of gonadotrophic cells testing HSP70 positive. The results of functional studies demonstrated that the HSP70 inducer decreased FSH and LH levels in cultured porcine primary pituitary cells, whereas an HSP70 inhibitor blocked the negative effect of CRH on gonadotrophin synthesis and secretion. Furthermore, our results demonstrated that HSP70 inhibited gonadotrophin synthesis and secretion by blocking GnRH-induced SMAD3 phosphorylation, which acts as the targeting molecule of HSP70, while CRH upregulated HSP70 expression through the PKC and ERK pathways. Collectively, these data demonstrate that HSP70 inhibits pituitary gonadotrophin synthesis and secretion by regulating the CRH signaling pathway and inhibiting SMAD3 phosphorylation, which are important for our understanding the mechanisms of the stress affects domestic animal reproductive functions.

A novel G protein-coupled receptor for starfish gonadotropic hormone, relaxin-like gonad-stimulating peptide.

Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP.

Genetic evidence for Amh modulation of gonadotropin actions to control gonadal homeostasis and gametogenesis in zebrafish and its noncanonical signaling through Bmpr2a receptor.

Anti-Müllerian hormone (Amh) plays an important role in gonadal function. Amh deficiency causes severe gonadal dysgenesis and dysfunction in zebrafish, with gonadal hypertrophy in both sexes. However, its mechanism of action remains unknown. Intriguingly, the Amh cognate type II receptor (Amhr2) is missing in the zebrafish genome, in sharp contrast to other species. Using a series of zebrafish mutants (amh, fshb, fshr and lhcgr), we provided unequivocal evidence for actions of Amh, via modulation of gonadotropin signaling, on both germ cell proliferation and differentiation. The gonadal hypertrophy in amh mutants was abolished in the absence of Fshr in females or Fshr/Lhcgr in males. Furthermore, we demonstrated that knockout of bmpr2a, but not bmpr2b, phenocopied all phenotypes of the amh mutant in both sexes, including gonadal hypertrophy, hyperproliferation of germ cells, retarded gametogenesis and reduced fshb expression. In summary, the present study provided comprehensive genetic evidence for an intimate interaction of gonadotropin and Amh pathways in gonadal homeostasis and gametogenesis and for Bmpr2a as the possible missing link for Amh signaling in zebrafish.

The role of miR-7 as a potential switch in the mouse hypothalamus-pituitary-ovary axis through regulation of gonadotropins.

The hypothalamus-pituitary-ovary (HPO) axis plays fundamental roles in female neuroendocrinology and reproduction. Pituitary gonadotropins are located in the center of this axis. Previous investigation suggested that miR-7 is closely linked with gonadotropins. However, the interaction between miR-7 and the HPO axis remains unclear. This study aims to determine whether and how miR-7 functions in this axis. A mouse ovariectomy model and mouse primary pituitary cells were used in this study. The results showed that miR-7 is localized to gonadotrophs and somatotrophs. miR-7 can inhibit the expression, synthesis and secretion of gonadotropins, but not growth hormones. Gonadotropin-releasing hormone (GnRH) has inhibitory effects on miR-7, while estrogen enhances miR-7 expression. miR-7 is vital for the pathway by which GnRH and estrogen regulate gonadotropins by targeting v-raf-leukemia viral oncogene 1 (Raf1). Together, these results indicate that miR-7 acts as a potential switch in the feedback loop of the HPO axis by regulating gonadotropins.

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

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

Expression of additional transcription factors is of prognostic value for aggressive behavior of pituitary adenomas.

OBJECTIVE: According to the latest WHO classification of tumors of endocrine organs in 2017, plurihormonal adenomas are subclassified by their transcription factor (TF) expression. In the group of plurihormonal adenomas with unusual immunohistochemical combinations (PAWUC), the authors identified a large fraction of adenomas expressing TFs for gonadotroph adenoma (TFGA) cells in addition to other TFs. The aim of this study was to compare clinicopathological parameters of PAWUC with TFGA expression to gonadotroph adenomas that only express TFGA. METHODS: This retrospective single-center series comprises 73 patients with TFGA-positive pituitary adenomas (SF1, GATA3, estrogen receptor α): 22 PAWUC with TFGA (TFGA-plus group) and 51 with TFGA expression only (TFGA-only group). Patient characteristics, outcome parameters, rate of invasiveness (assessed by direct endoscopic inspection), and MIB1 and MGMT status were compared between groups. RESULTS: Patients in the TFGA-plus group were significantly younger than patients in the TFGA-only group (age 46 vs 56 years, respectively; p = 0.007). In the TFGA-only group, pituitary adenomas were significantly larger (diameter 25 vs 18.3 mm, p = 0.002). Intraoperatively, signs of invasiveness were significantly more common in the TFGA-plus group than in the TFGA-only group (50% vs 16%, p = 0.002). Gross-total resection was significantly lower in the nonfunctioning TFGA-plus group than in the TFGA-only group (44% vs 86%, p = 0.004). MIB1 and MGMT status showed no significant difference between groups. CONCLUSIONS: These data suggest a more aggressive behavior of TFGA-positive adenomas if an additional TF is expressed within the tumor cells. Shorter radiographic surveillance and earlier consideration for retreatment should be recommended in these adenoma types.

Does the presence of AGG interruptions within the CGG repeat tract have a protective effect on the fertility phenotype of female FMR1 premutation carriers?

PURPOSE: While FMR1 premutation carriers (CGG 55-200) were shown to have reduced success with IVF treatment (lower oocyte yield), studies reporting on the association between the number of CGG repeats and patients' response to controlled ovarian hyperstimulation (COH) are inconsistent. In the present study, we aim to explore whether the number of CGG repeats in women with premutation in FMR1 gene, undergoing COH for IVF, correlates with COH variables and whether the number of AGG interruptions may function as a "protective factor" by improving the ovarian response to COH. METHODS: Retrospective study, in an academic IVF-PGD unit. Fifty-seven consecutive FMR1 premutation carriers who underwent 285 IVF treatment cycles were included. The numbers of CGG repeats and AGG interruptions were retrieved and correlated to the demographics and COH variables. RESULTS: There were no significant association between the numbers of CGG or the AGG interruptions and the number of oocyte retrieved or the peak estradiol levels. The lack of association was also observed when including all the IVF treatment cycles or only the first or last IVF treatment cycle. Moreover, no associations were found between the number of CGG repeats or AGG interruptions and other COH variables, i.e., duration of stimulation, the total dose of gonadotropin used, or the number of top-quality embryos. CONCLUSIONS: No associations were observed between the number of CGG repeats or AGG interruptions and any of the COH variables. Further studies are required to identify early biomarkers of POI to empower FMR1 premutation carriers with risk assessment tools to consider procedures such as fertility preservation.

Origin and Evolution of the Neuroendocrine Control of Reproduction in Vertebrates, With Special Focus on Genome and Gene Duplications.

In humans, as in the other mammals, the neuroendocrine control of reproduction is ensured by the brain-pituitary gonadotropic axis. Multiple internal and environmental cues are integrated via brain neuronal networks, ultimately leading to the modulation of the activity of gonadotropin-releasing hormone (GnRH) neurons. The decapeptide GnRH is released into the hypothalamic-hypophysial portal blood system and stimulates the production of pituitary glycoprotein hormones, the two gonadotropins luteinizing hormone and follicle-stimulating hormone. A novel actor, the neuropeptide kisspeptin, acting upstream of GnRH, has attracted increasing attention in recent years. Other neuropeptides, such as gonadotropin-inhibiting hormone/RF-amide related peptide, and other members of the RF-amide peptide superfamily, as well as various nonpeptidic neuromediators such as dopamine and serotonin also provide a large panel of stimulatory or inhibitory regulators. This paper addresses the origin and evolution of the vertebrate gonadotropic axis. Brain-pituitary neuroendocrine axes are typical of vertebrates, the pituitary gland, mediator and amplifier of brain control on peripheral organs, being a vertebrate innovation. The paper reviews, from molecular and functional perspectives, the evolution across vertebrate radiation of some key actors of the vertebrate neuroendocrine control of reproduction and traces back their origin along the vertebrate lineage and in other metazoa before the emergence of vertebrates. A focus is given on how gene duplications, resulting from either local events or from whole genome duplication events, and followed by paralogous gene loss or conservation, might have shaped the evolutionary scenarios of current families of key actors of the gonadotropic axis.

Trpc5 deficiency causes hypoprolactinemia and altered function of oscillatory dopamine neurons in the arcuate nucleus.

Dopamine neurons of the hypothalamic arcuate nucleus (ARC) tonically inhibit the release of the protein hormone prolactin from lactotropic cells in the anterior pituitary gland and thus play a central role in prolactin homeostasis of the body. Prolactin, in turn, orchestrates numerous important biological functions such as maternal behavior, reproduction, and sexual arousal. Here, we identify the canonical transient receptor potential channel Trpc5 as an essential requirement for normal function of dopamine ARC neurons and prolactin homeostasis. By analyzing female mice carrying targeted mutations in the Trpc5 gene including a conditional Trpc5 deletion, we show that Trpc5 is required for maintaining highly stereotyped infraslow membrane potential oscillations of dopamine ARC neurons. Trpc5 is also required for eliciting prolactin-evoked tonic plateau potentials in these neurons that are part of a regulatory feedback circuit. Trpc5 mutant females show severe prolactin deficiency or hypoprolactinemia that is associated with irregular reproductive cyclicity, gonadotropin imbalance, and impaired reproductive capabilities. These results reveal a previously unknown role for the cation channel Trpc5 in prolactin homeostasis of female mice and provide strategies to explore the genetic basis of reproductive disorders and other malfunctions associated with defective prolactin regulation in humans.

Cortisol differentially affects cell viability and reproduction-related gene expression in Atlantic cod pituitary cultures dependent on stage of sexual maturation.

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.

Effects of high and low temperature on expression of GnIH, GnIH receptor, GH and PRL genes in the male grass puffer during breeding season.

Gonadotropin-inhibitory hormone (GnIH) is a multifunctional hypophysiotropic neurohormone and has a stimulatory role in the control of reproduction in the grass puffer. To clarify the neuroendocrine mechanisms underlying the effect of changes in water temperature on reproduction in fish, we previously revealed that, in parallel to gonadal regression, both low and high temperature significantly decreased the expressions of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r), gonadotropin-releasing hormone 1 (gnrh1) in the brain and gonadotropin (GTH) subunits (fshb and lhb) in the pituitary of sexually mature male grass puffer. In this study, we examined the changes in expression of gnih and GnIH receptor gene (gnihr) in the brain and pituitary along with the genes for growth hormone (gh) and prolactin (prl) in the pituitary of male grass puffer exposed to low temperature (14 °C), normal temperature (21 °C, as initial control) and high temperature (28 °C) conditions for 7 days. The levels of gnih and gnihr mRNAs were significantly decreased in both low and high temperature conditions compared to normal temperature in the brain and pituitary. Similarly, the gh mRNA levels were significantly decreased in both low and high temperature conditions. The prl mRNAs showed no significant changes at high temperature, whereas drastically decreased at low temperature possibly by dysfunctional cold stress. Taken together, the present results suggest that, in addition to the inhibitory effect of temperature changes on the Kiss2/GnRH1/GTH system, the suppression of GnIH/GH system may also be involved in the termination of reproduction by high temperature at the end of breeding season.