Tomoko Soga

Neurons synthesizing gonadotropin-releasing hormone mRNA subtypes have multiple developmental origins in the medaka

The origins of the different populations of gonadotropin‐releasing hormone (GnRH)‐containing neurons in the brains of two genotypes (HO4C; HNI‐II) of medaka Oryzias latipes were analyzed at different stages of development (day 1 after fertilization through adulthood), by using oligonucleotide probes specific to salmon‐, seabream‐, and chicken II‐GnRH mRNA and antisera against specific GnRH peptides. Between the two genotypes, there was no difference in the site and time of GnRH expression or the final pattern of GnRH neuronal organization. In the adult fish of both sexes, salmon GnRH mRNA and peptide‐containing neurons were seen in the terminal nerve ganglia (nucleus olfactoretinalis; NOR) and chicken II‐GnRH mRNA and peptide‐containing neurons in the midbrain tegmentum. GnRH cells at the base of the olfactory placode (1–2 cells) and in the midbrain tegmentum were first seen in 1‐day‐old fish of both genotypes. On day 15, lightly immunoreactive GnRH cells were seen in the NOR of only HNI genotype. By day 30, GnRH expression in the NOR and in the midbrain was prominent. GnRH cells along the basal olfactory bulb and basal telencephalon were occasionally seen in animals 30 days or older. This developmental study shows differential distribution of salmon and chicken II‐GnRH mRNA subtypes and emphasizes their separate embryonic origins from the olfactory apparatus (salmon‐GnRH) and the ependymal cells of the third ventricle (chicken II‐GnRH). The absence of preoptic GnRH hybridization signals, immunoreactivity and the lack of GnRH fibers in the pituitary suggests that the preoptic GnRH neurons are distinct from the olfactory derived‐terminal nerve GnRH neurons, and that the GnRH neurites reported in the pituitary of teleost must be of preoptic origin. J. Comp. Neurol. 401:217–226, 1998.

Effect of ER-β gene disruption on estrogenic regulation of anxiety in female mice

It has been shown that long-term estrogen treatment in gonadectomized female mice increases anxiety levels. On the other hand, a recent study has reported that estrogen may down-regulate the levels of anxiety by acting through estrogen receptor (ER) beta. In the present study, we investigated the role of ER-beta in the regulation of anxiety levels in female mice after long-term estrogen treatment. Gonadectomized ER-beta knockout (betaERKO) female mice and their wild type (betaWT) littermates were implanted several different doses (experiment 1: 2.0 microg/day, experiment 2: 1.0, 0.4, 0.2 or 0.1 microg/day) of an estradiol benzoate (EB) or placebo pellet. Ten days after pellet implant, behavioral tests commenced to measure the anxiety levels (experiment 1: light-dark transition test (LDT), experiment 2: LDT, elevated plus maze test (EPM) and social investigation test (SIT)). We found that, at higher-doses, long-term treatment of EB had anxiogenic effects in both betaWT and betaERKO mice as indicated by a decrease of the time spent in the light side and the number of transitions between two sides during LDT. In contrast, several behavioral measurements indicated that the lower-doses treatment of EB might reduce the anxiety levels possibly through ER-beta. Particularly, the anxiolytic effects of EB in the SIT were more pronounced in betaWT mice than betaERKO mice. Together, the findings in the present study suggest that estrogen may have both anxiolytic and anxiogenic effects in female mice, and that ER-beta gene disruption did not affect anxiogenic regulation by estrogen in female mice, but partially affected anxiolytic regulation.

Spatio‐Temporal Expression of Gonadotropin‐Releasing Hormone Receptor Subtypes in Gonadotropes, Somatotropes and Lactotropes in the Cichlid Fish

The description of two or more forms of gonadotropin‐releasing hormone (GnRH) in most vertebrates suggests multiple roles for this family of peptide hormones. In order to verify these functions, we analysed the anatomical location, time of initial expression and ontogenic changes in three distinct GnRH receptors (GnRH‐Rs) in developing and sexually mature tilapia, using antisera raised against the extracellular loop three of the receptor, which is a determinant in ligand‐selectivity and receptor coupling to signalling pathways. In all age groups, including males and females, using in situ hybridization and double‐label immunological methods, GnRH‐R type IA was colocalized in cells containing luteinizing hormone (LH) β‐subunit in the pituitary. GnRH‐R type IB was visualized in prolactin cells and LH cells. The type III GnRH‐R was expressed in growth hormone cells. On day 8 after fertilization, GnRH‐R type III was first seen in growth hormone cells and, subsequently, on day 15, GnRH‐Rs type IA and type IB were first seen in LH and prolactin cells, respectively. On day 25, the receptor occupied area per pituitary and the staining intensity of GnRH‐R type IA increased significantly, consistent with the hypothesis that differentiation of GnRH neurones and their inputs to the pituitary coincide precisely with gonadal sex differentiation and steroidogenesis in tilapia. The differential distribution of GnRH‐Rs in the pituitary provides the first clear evidence that the three native GnRH variants in tilapia have cognate receptors, each capable of regulating different pituitary endocrine cells.

Localization of the three GnRH types and GnRH receptors in the brain of a cichlid fish: Insights into their neuroendocrine and neuromodulator functions.

The cognate receptor for any of the known gonadotropin‐releasing hormones (GnRHs) has not been directly demonstrated. In order to establish this and shed light on the functions of GnRH types, we analyzed the neuroanatomical location and time of initial expression of three distinct GnRH receptors (GnRH‐Rs) and the three endogenous GnRHs in the brain of developing and sexually mature tilapia Oreochromis niloticus using immunocytochemistry. In all age groups, including males and females, GnRH‐RIA was seen specifically in gonadotropes (Parhar et al. [2002] J Neuroendocrinol 14:657–665) but was undetectable in the brain. On day 8 after fertilization, GnRH‐RIB was first seen in the periventricular hypothalamus (lateral recess nucleus, posterior recess nucleus, posterior tuberal nucleus) and GnRH‐RIII in the olfactory epithelium, olfactory bulb, telencephalon, preoptic region, mediobasal hypothalamus, thalamus, mesencephalon, and in the hindbrain. Double‐label immunocytochemistry showed GnRH1 (Ser8 GnRH)‐immunoreactive neuronal processes projecting mainly to the proximal pars distalis of the pituitary, while GnRH2 (His5, Trp7, Tyr8 GnRH) and GnRH3 (Trp7, Leu8 GnRH) fibers were observed in close association with cells containing GnRH‐RIB and GnRH‐RIII in the brain. These results suggest that GnRH‐RIA might be hypophysiotropic in nature, whereas GnRH‐RIB and GnRH‐RIII could have additional neuromodulatory functions. Further, evidence of close proximity of GnRH‐R‐containing cells and neuronal processes of multiple GnRH types suggests complex cross‐talk between several GnRH ligands and GnRH‐Rs. J. Comp. Neurol. 487:28–41, 2005.

Immunoneutralization of gonadotropin-releasing hormone type-III suppresses male reproductive behavior of cichlids

To investigate the roles of gonadotropin-releasing hormone (GnRH) types in reproductive behaviors, antisera against GnRH1, GnRH2 and GnRH3 were stereotaxically administered into the intracerebroventricular region to neutralize the three native GnRH types in the brain of male tilapia Oreochromis niloticus. Reproductive behaviors (nest-building and aggressive behaviors), and morphological changes of the three GnRH systems were investigated by immunocytochemistry. GnRH1, GnRH2 and GnRH3 immunoreactive fibers were significantly decreased following injections of GnRH antisera indicating successful neutralization of their respective endogenous GnRH peptides. GnRH1- and GnRH2-immunoneutralization did not inhibit reproductive behaviors but GnRH3-immunoneutralization significantly decreased nest-building ability (Saline: 26.5 +/- 3.7%/day versus GnRH3: 6.1 +/- 2.9%/day, P < 0.001), nest size (Saline: 0.67 +/- 0.09 points versus GnRH3: 0.10 +/- 0.05 points, P < 0.0002) and aggressive behavior (Saline: 2.34 +/- 0.19 points versus GnRH3 1.06 +/- 0.12 points, P < 0.0001). These observations provide evidence that GnRH3 is a potent neuromodulator of reproductive behaviors in male tilapia.

FSH and LH-β subunits in the preoptic nucleus: ontogenic expression in teleost

In the present study we cloned, sequenced, and confirmed the presence of mRNAs of gonadotropins (FSH-β, LH-β subunits) from the brain and pituitary of tilapia, Oreochromis niloticus. Further, we examined the spatio-temporal expression pattern of FSH-β and LH-β in the brain and pituitary of two species of teleost (tilapia, O. niloticus; sockeye salmon, Oncorhynchus nerka), using in situ hybridization and immunological methods. The expression of FSH and LH immunoreactivity appeared simultaneously in the brain and pituitary (tilapia, 14 days; sockeye, 51 days after fertilization). In the pituitary, FSH mRNA and peptide expressing cells were distinct from LH expressing cells located in the ventral proximal pars distalis. In the brain, FSH and LH immunoreactivity was co-localized in cells of the preoptic nucleus parvocellularis, magnocellularis, and gigantocellularis. Fibers immunoreactive to FSH and LH antisera were seen along the forebrain-hypothalamus and in the neurohypophysis of the pituitary. Double-label immunofluorescence revealed FSH and LH immunoreactivity co-localized in arginine vasotocin synthesizing preoptic neurons. Our results show that FSH and LH-producing cells have developmental origins in the brain as well as in the pituitary. In addition, we propose that the brain-derived gonadotropins may function as hypophysiotropic hormones that regulate pituitary cells and along with arginine vasotocin could act as neuromodulators of reproductive behaviors.

Testosterone differentially regulates expression of GnRH messenger RNAs in the terminal nerve, preoptic and midbrain of male tilapia

The purpose of the present study was to examine the regulation of three molecular variants of gonadotropin-releasing hormone (GnRH)-encoding mRNAs by testosterone in the male tilapia Oreochromis niloticus. Tilapias castrated for two weeks were injected intraperitoneally with sesame oil or 5 microgram/g testosterone for 7 days. In situ hybridization histochemistry was performed using 35S-labelled 30-mer antisense oligonucleotide probes complementary to exon two (bases 1-30) of salmon-, seabream-, and chicken II-GnRH. Computerized image analysis was performed to quantify GnRH mRNA expression in the terminal nerve ganglia (nucleus olfactoretinalis) and in individual cells of the preoptic area and the midbrain tegmentum. Testosterone treatment significantly elevated terminal nerve salmon-GnRH mRNA, reduced preoptic seabream-GnRH mRNA but had no effect on midbrain chicken II-GnRH mRNA levels. The total number and size of preoptic and midbrain GnRH mRNA-containing neurons or the total volume of the terminal nerve ganglia in testosterone-treated animals did not differ significantly from oil-treated animals. The midbrain chicken II-GnRH neurons are not targets of testosterone. These results demonstrate for the first time differential regulation of subpopulations of GnRH neurons with molecular diversity and different topography.

Neonatal dexamethasone exposure down-regulates GnRH expression through the GnIH pathway in female mice.

Synthetic glucocorticoid (dexamethasone; DEX) treatment during the neonatal stage is known to affect reproductive activity. However, it is still unknown whether neonatal stress activates gonadotropin-inhibitory hormone (GnIH) synthesizing cells in the dorsomedial hypothalamus (DMH), which could have pronounced suppressive action on gonadotropin-releasing hormone (GnRH) neurons, leading to delayed pubertal onset. This study was designed to determine the effect of neonatal DEX (1.0mg/kg) exposure on reproductive maturation. Therefore, GnRH, GnIH and GnIH receptors, G-protein coupled receptors (GPR) 147 and GPR74 mRNA levels were measured using quantitative real-time PCR in female mice at postnatal (P) days 21, 30 and in estrus stage mice, aged between P45-50. DEX-treated females of P45-50 had delayed vaginal opening, and irregular estrus cycles and lower GnRH expression in the preoptic area (POA) when compared with age-matched controls. The expression levels of GPR147 and GPR74 mRNA in the POA increased significantly in DEX-treated female mice of P21 and P45-50 compared to controls. In addition, GPR147 and GPR74 mRNA expression was observed in laser captured single GnRH neurons in the POA. Although there was no difference in GnIH mRNA expression in the DMH, immunostained GnIH cell numbers in the DMH increased in DEX-treated females of P45-50 compared to controls. Taken together, the results show that the delayed pubertal onset could be due to the inhibition of GnRH gene expression after neonatal DEX treatment, which may be accounted for in part by the inhibitory signals from the up-regulated GnIH-GnIH receptor pathway to the POA.

Citalopram (antidepressant) administration causes sexual dysfunction in male mice through RF-amide related peptide in the dorsomedial hypothalamus

Citalopram is the most potent selective serotonin reuptake inhibitor (SSRI) which is used as an antidepressant but causes sexual dysfunction. Whether citalopram induced sexual dysfunction is a result of gonadotropin-releasing hormone (GnRH), kisspeptin or RF-amide related peptide (RFRP) alteration is unknown. In this study, we tested mice for sexual behavior after vehicle (0.9% NaCl) and citalopram treatment (5 mg/kg) daily for 1 day (acute) and 21 or 28 days (chronic). Effects of acute and chronic treatments on neuronal numbers and mRNA expression of GnRH, kisspeptin and RFRP were measured. In addition, RFRP fiber projections to preoptic (POA)-GnRH neurons were analyzed using double-label immunohistochemistry. The expression of 14 different serotonin receptor types mRNA was examined in immunostained laser dissected single RFRP neurons in the dorsomedial hypothalamus (DMH), however only 11 receptors types were identified. Acute citalopram treatment did not affect sexual behavior, whereas, the total duration of intromission was reduced with chronic treatment. There was no effect in the expression of kisspeptin (neuronal numbers and mRNA) in the anteroventral periventricular nucleus and the arcuate nucleus and expression of GnRH (neuronal numbers and mRNA) in the POA after citalopram treatment. However, RFRP neuronal numbers in the DMH and fiber projections to the POA were significantly increased after chronic citalopram treatment, which suggests citalopram induced inhibition of sexual behavior involves the modulation of RFRP through serotonin receptors in the DMH.

Caffeine neuroprotects against dexamethasone-induced anxiety-like behaviour in the Zebrafish (Danio rerio).

The early-life stress has critical impact on brain development which can lead to long-term effects on brain functions during adulthood. It has been reported that caffeine possesses a protective effect in neurodegenerative diseases. Thus, this study investigates the potential of caffeine to protect brain functions from adverse effects due to stress exposure during early-life development in the male zebrafish. In the first part of this study, synthetic glucocorticoid, dexamethasone (DEX) (2-200 mg/L for 24 h) was used to induce stress effects in the zebrafish larvae from 4 to 5 days post-fertilisation (dpf) and the effect of DEX administration on zebrafish larvae on anxiety-like behaviour during adulthood in novel tank test was investigated. Next, the possible protective effect of caffeine pre-treatment (5-50 mg/L for 24 h from 3 to 4dpf) before DEX administration was studied. DEX-treated adult male zebrafish showed higher anxiety levels in behavioural tests, as seen in longer latency to enter the top part of the tank, lower transition numbers between the top and bottom parts with more time spent at the bottom and lesser time spent at the top and lower distance travelled at top part. The effect of DEX on anxiety-like behaviour was dose-dependent. Importantly, adult male zebrafish pre-treated with caffeine before DEX treatment did not show any anxiety-like behaviour. These results show that exposure to stress during early-life leads to anxiety-like behaviour in the adult male zebrafish but pre-treatment with caffeine protects from stress-induced anxiety.