Functional Authentication of a Novel Gastropod Gonadotropin-Releasing Hormone Receptor Reveals Unusual Features and Evolutionary Insight.

A gonadotropin-releasing hormone (GnRH)-like molecule was previously identified in a gastropod, Aplysia californica, and named ap-GnRH. In this study, we cloned the full-length cDNA of a putative ap-GnRH receptor (ap-GnRHR) and functionally authenticated this receptor as a bona fide ap-GnRHR. This receptor contains two potential translation start sites, each accompanied by a Kozak sequence, suggesting the translation of a long and a short form of the receptor is possible. The putative ap-GnRHR maintains the conserved structural motifs of GnRHR-like receptors and shares 45% sequence identity with the octopus GnRHR. The expression of the putative ap-GnRHR short form is ubiquitous in all tissues examined, whereas the long form is only expressed in parts of the central nervous system, osphradium, small hermaphroditic duct, and ovotestis. The cDNA encoding the long or the short receptor was transfected into the Drosophila S2 cell line and subject to a radioreceptor assay using 125I-labeled ap-GnRH as the radioligand. Further, the transfected cells were treated with various concentrations of ap-GnRH and measured for the accumulation of cAMP and inositol monophosphate (IP1). Radioreceptor assay revealed that only the long receptor bound specifically to the radioligand. Further, only the long receptor responded to ap-GnRH with an increased accumulation of IP1, but not cAMP. Our studies show that despite the more prevalent expression of the short receptor, only the long receptor is the functional ap-GnRHR. Importantly, this is only the second report on the authentication of a protostome GnRHR, and based on the function and the phylogenetic grouping of ap-GnRHR, we suggest that this receptor is more similar to protostome corazonin receptors than chordate GnRHRs.

Opposite-sex housing reactivates the declining GnRH system in aged transgenic male mice with FGF signaling deficiency.

The continued presence of gonadotropin-releasing hormone (GnRH) neurons is required for a healthy reproductive lifespan, but factors that maintain postnatal GnRH neurons have not been identified. To begin to understand these factors, we investigated whether 1) fibroblast growth factor (FGF) signaling and 2) interactions with the opposite sex are involved in the maintenance of the postnatal GnRH system. A transgenic mouse model (dnFGFR mouse) with the targeted expression of a dominant-negative FGF receptor (dnFGFR) in GnRH neurons was used to examine the consequence of FGF signaling deficiency on postnatal GnRH neurons. Male dnFGFR mice suffered a significant loss of postnatal GnRH neurons within the first 100 days of life. Interestingly, this loss was reversed after cohabitation with female, but not male, mice for 300-550 days. Along with a rescue in GnRH neuron numbers, opposite-sex housing in dnFGFR males also increased hypothalamic GnRH peptide levels, promoted a more mature GnRH neuronal morphology, facilitated litter production, and enhanced testicular morphology. Last, mice hypomorphic for FGFR3 exhibited a similar pattern of postnatal GnRH neuronal loss as dnFGFR males, suggesting FGF signaling acts, in part, through FGFR3 to enhance the maintenance of the postnatal GnRH system. In summary, we have shown that FGF signaling is required for the continued presence of postnatal GnRH neurons. However, this requirement is not absolute, since sexual interactions can compensate for defects in FGFR signaling, thereby rescuing the declining GnRH system. This suggests the postnatal GnRH system is highly plastic and capable of responding to environmental stimuli throughout adult life.

Maternal behavior in transgenic mice with reduced fibroblast growth factor receptor function in gonadotropin-releasing hormone neurons.

BACKGROUND: Fibroblast growth factors (FGFs) and their receptors (FGFRs) are necessary for the proper development of gonadotropin-releasing hormone (GnRH) neurons, which are key activators of the hypothalamo-pituitary-gonadal axis. Transgenic mice that have the targeted expression of a dominant negative FGFR (dnFGFR) in GnRH neurons (dnFGFR mice) have a 30% decrease of GnRH neurons. Additionally, only 30-40% of the pups born to the transgenic dams survive to weaning age. These data raised the possibility that FGFR defects in GnRH neurons could adversely affect maternal behavior via novel mechanisms. METHODS: We first determined if defective maternal behavior in dnFGFR mothers may contribute to poor pup survival by measuring pup retrieval and a battery of maternal behaviors in primiparous control (n=10-12) and dnFGFR (n=13-14) mothers. Other endocrine correlates of maternal behaviors, including plasma estradiol levels and hypothalamic pro-oxyphysin and GnRH transcript levels were also determined using enzyme-linked immunoassay and quantitative reverse transcription polymerase chain reaction, respectively. RESULTS: Maternal behaviors (% time crouching with pups, time off pups but not feeding, time feeding, and total number of nesting bouts) were not significantly different in dnFGFR mice. However, dnFGFR dams were more likely to leave their pups scattered and took significantly longer to retrieve each pup compared to control dams. Further, dnFGFR mothers had significantly lower GnRH transcripts and circulating E2, but normal pro-oxyphysin transcript levels. CONCLUSIONS: Overall, this study suggests a complex scenario in which a GnRH system compromised by reduced FGF signaling leads to not only suboptimal reproductive physiology, but also suboptimal maternal behavior.

Fibroblast growth factor signaling in the developing neuroendocrine hypothalamus.

Fibroblast growth factor (FGF) signaling is pivotal to the formation of numerous central regions. Increasing evidence suggests FGF signaling also directs the development of the neuroendocrine hypothalamus, a collection of neuroendocrine neurons originating primarily within the nose and the ventricular zone of the diencephalon. This review outlines evidence for a role of FGF signaling in the prenatal and postnatal development of several hypothalamic neuroendocrine systems. The emphasis is placed on the nasally derived gonadotropin-releasing hormone neurons, which depend on neurotrophic cues from FGF signaling throughout the neurons' lifetime. Although less is known about neuroendocrine neurons derived from the diencephalon, recent studies suggest they also exhibit variable levels of dependence on FGF signaling. Overall, FGF signaling provides a broad spectrum of cues that ranges from genesis, cell survival/death, migration, morphological changes, to hormone synthesis in the neuroendocrine hypothalamus. Abnormal FGF signaling will deleteriously impact multiple hypothalamic neuroendocrine systems, resulting in the disruption of diverse physiological functions.

Differential fibroblast growth factor 8 (FGF8)-mediated autoregulation of its cognate receptors, Fgfr1 and Fgfr3, in neuronal cell lines.

Fibroblast growth factors (FGFs) mediate a vast range of CNS developmental processes including neural induction, proliferation, migration, and cell survival. Despite the critical role of FGF signaling for normal CNS development, few reports describe the mechanisms that regulate FGF receptor gene expression in the brain. We tested whether FGF8 could autoregulate two of its cognate receptors, Fgfr1 and Fgfr3, in three murine cell lines with different lineages: fibroblast-derived cells (3T3 cells), neuronal cells derived from hippocampus (HT-22 cells), and neuroendocrine cells derived from hypothalamic gonadotropin-releasing hormone (GnRH) neurons (GT1-7 cells). GnRH is produced by neurons in the hypothalamus and is absolutely required for reproductive competence in vertebrate animals. Several lines of evidence strongly suggest that Fgf8 is critical for normal development of the GnRH system, therefore, the GT1-7 cells provided us with an additional endpoint, Gnrh gene expression and promoter activity, to assess potential downstream consequences of FGF8-induced modulation of FGF receptor levels. Results from this study suggest that the autoregulation of its cognate receptor represents a common downstream effect of FGF8. Further, we show that Fgfr1 and Fgfr3 are differentially regulated within the same cell type, implicating these two receptors in different biological roles. Moreover, Fgfr1 and Fgfr3 are differentially regulated among different cell types, suggesting such autoregulation occurs in a cell type-specific fashion. Lastly, we demonstrate that FGF8b decreases Gnrh promoter activity and gene expression, possibly reflecting a downstream consequence of altered FGF receptor populations. Together, our data bring forth the possibility that, in addition to the FGF synexpression group, autoregulation of FGFR expression by FGF8 represents a mechanism by which FGF8 could fine-tune its regulatory actions.

Fibroblast growth factor 8 signaling through fibroblast growth factor receptor 1 is required for the emergence of gonadotropin-releasing hormone neurons.

GnRH neurons are essential for the onset and maintenance of reproduction. Mutations in both fibroblast growth factor receptor (Fgfr1) and Fgf8 have been shown to cause Kallmann syndrome, a disease characterized by hypogonadotropic hypogonadism and anosmia, indicating that FGF signaling is indispensable for the formation of a functional GnRH system. Presently it is unclear which stage of GnRH neuronal development is most impacted by FGF signaling deficiency. GnRH neurons express both FGFR1 and -3; thus, it is also unclear whether FGFR1 or FGFR3 contributes directly to GnRH system development. In this study, we examined the developing GnRH system in mice deficient in FGF8, FGFR1, or FGFR3 to elucidate the individual contribution of these FGF signaling components. Our results show that the early emergence of GnRH neurons from the embryonic olfactory placode requires FGF8 signaling, which is mediated through FGFR1, not FGFR3. These data provide compelling evidence that the developing GnRH system is exquisitely sensitive to reduced levels of FGF signaling. Furthermore, Kallmann syndrome stemming from FGF signaling deficiency may be due primarily to defects in early GnRH neuronal development prior to their migration into the forebrain.

Alteration of the hypothalamic-pituitary-gonadal axis in estrogen- and androgen-treated adult male leopard frog, Rana pipiens.

BACKGROUND: Gonadal steroids, in particular 5 alpha-dihydrotestosterone (DHT) and 17 beta-estradiol (E2), have been shown to feed back on the hypothalamic-pituitary-gonadal (HPG) axis of the ranid frog. However, questions still remain on how DHT and E2 impact two of the less-studied components of the ranid HPG axis, the hypothalamus and the gonad, and if the feedback effects are consistently negative. Thus, the goal of the study was to examine the effects of DHT and E2 upon the HPG axis of the gonadally-intact, sexually mature male leopard frogs, Rana pipiens. METHODS: R. pipiens were implanted with silastic capsules containing either cholesterol (Ch, a control), DHT, or E2 for 10 or 30 days. At each time point, steroid-induced changes in hypothalamic GnRH and pituitary LH concentrations, circulating luteinizing hormone (LH), and testicular histology were examined. RESULTS: Frogs implanted with DHT or E2 for 10 days did not show significant alterations in the HPG axis. In contrast, frogs implanted with hormones for 30 days had significantly lower circulating LH (for both DHT and E2), decreased pituitary LH concentration (for E2 only), and disrupted spermatogenesis (for both DHT and E2). The disruption of spermatogenesis was qualitatively similar between DHT and E2, although the effects of E2 were consistently more potent. In both DHT and E2-treated animals, a marked loss of all pre-meiotic germ cells was observed, although the loss of secondary spermatogonia appeared to be the primary cause of disrupted spermatogenesis. Unexpectedly, the presence of post-meiotic germ cells was either unaffected or enhanced by DHT or E2 treatment. CONCLUSIONS: Overall, these results showed that both DHT and E2 inhibited circulating LH and disrupted spermatogenesis progressively in a time-dependent manner, with the longer duration of treatment producing the more pronounced effects. Further, the feedback effects exerted by both steroid hormones upon the HPG axis were largely negative, although the possibility exists for a stimulatory effect upon the post-meiotic germ cells.

Effects of steroid hormones on spermatogenesis and GnRH release in male Leopard frogs, Rana pipiens.

Several lines of evidence suggest reproduction in the ranid frogs is potently regulated by the gonadal steroids, in particular 5alpha-dihydrotestosterone (DHT) and 17beta-estradiol (E(2)), and a non-gonadal steroid, the stress hormone corticosterone (Cort). Little is known about how these steroid hormones act upon the GnRH system to regulate the downstream reproductive events. We address these gaps in our knowledge by investigating the effects of Cort, E(2), and DHT administration on the in vitro release of GnRH and on the spermatogenesis of adult male leopard frog, Rana pipiens. R. pipiens were implanted for 20 days with silastic capsules containing cholesterol (Ch; control), Cort, E(2), or DHT. Upon sacrifice, acute hypothalamic explants were cultured and measured for GnRH release, and testes processed for histological analysis. Although only E(2) implant significantly reduced the gonadosomatic index, all three steroid hormones altered spermatogenesis. Cort modestly but significantly reduced the presence of spermatids. The effects of E(2) and DHT were both stimulatory and inhibitory, depending on the stage of spermatogenesis. None of the steroid hormones altered baseline GnRH release. Interestingly, only E(2) significantly stimulated veratridine-induced GnRH release, suggesting E(2) treatment increased the releasable pool of GnRH and/or enhance the excitability of GnRH neurons. In sum, this is the first study to report the direct measurement of GnRH secretion in a poikilothermic tetrapod. Our results revealed potent but sometimes paradoxical effects of steroid hormones, especially E(2), on the reproductive regulation of the male R. pipiens.

Development of a novel enzyme immunoassay for the measurement of in vitro GnRH release from rat and bullfrog hypothalamic explants.

Gonadotropin-releasing hormone (GnRH) is critical for the initiation and maintenance of reproduction in vertebrates. Information regarding GnRH release is abundant in mammals, but absent in poikilothermic tetrapods. In this study, we established a novel GnRH enzyme immunoassay (EIA) to measure GnRH release over time from hypothalamic explants isolated from mature field-caught and commercially-acquired male bullfrogs, Rana catesbeiana. Hypothalamic explants from rats were used as a positive control to test the sensitivity and accuracy of our EIA and to ensure our in vitro system could detect GnRH pulses. Prominent GnRH pulses were present in the majority (9/10) of rat hypothalamic explants, but absent in all (17/17) of the commercial bullfrogs and the majority (5/8) of field-caught bullfrogs. In three cases where GnRH pulses were observed in field-caught bullfrogs, there was only one pulse during the 2-h incubation period; high-frequency pulses similar to those observed in rats were not observed. Veratridine, which opens voltage-gated sodium channels, stimulated GnRH release in all explants cultured in the presence of Ca(2+), demonstrating explant viability. The levels of both spontaneous and veratridine-induced GnRH release were significantly higher in field-caught than commercial bullfrogs. This study demonstrated, for the first time, the temporal pattern of GnRH release in a poikilothermic tetrapod. Further, our results suggest the levels and patterns of GnRH output in bullfrogs are subject to the dynamic regulation by physiological and environmental cues.