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.

Ancient origins of metazoan gonadotropin-releasing hormone and their receptors revealed by phylogenomic analyses.

The discovery of genes related to gonadotropin-releasing hormones (GnRH) and their receptors from diverse species has driven important advances in comparative endocrinology. However, our view of the evolutionary histories and nomenclature of these gene families has become inconsistent as several different iterations of GnRH and receptor relationships have been proposed. Whole genome sequence data are now available for most of the major lineages of animals, and an exhaustive view of the phylogenies of GnRH and their receptors is now possible. In this paper, we leverage data from publically available whole genome sequences to present a new phylogenomic analysis of GnRH and GnRH receptors and the distant relatives of each across metazoan phylogeny. Our approach utilizes a phylogenomics pipeline that searches data from 36 whole genome sequences and conducts phylogenetic analyses of gene trees. We provide a comprehensive analysis of the major groupings of GnRH peptides, related hormones and their receptors and provide some suggestions for a new nomenclature. Our study provides a framework for understanding the functional diversification of this family of neuromodulatory peptides and their receptors.

Disruption of the Suprachiasmatic Nucleus in Fibroblast Growth Factor Signaling-Deficient Mice.

Fibroblast growth factor (Fgf) 8 is essential for the development of multiple brain regions. Previous studies from our laboratory showed that reduced Fgf8 signaling led to the developmental alterations of neuroendocrine nuclei that originated within the diencephalon, including the paraventricular (PVN) and supraoptic (SON) nuclei. To further understand the role of Fgf8 in the development of other hypothalamic nuclei, we examined if Fgf8 and its cognate receptor, Fgfr1, also impact the integrity of the suprachiasmatic nuclei (SCN). The SCN control an organism's circadian rhythm and contain vasoactive intestinal peptide (VIP)-producing neurons as the main input neurons. Mice hypomorphic for Fgf8, Fgfr1, or both were examined for their SCN volume and the number of VIP neurons on postnatal day (PN) 0; adult hypomorphic mice were further examined for SCN function by quantifying SCN neuronal activation using cFos as a marker. On PN0, mice homozygous for Fgf8 hypomorphy displayed the most severe reduction of the SCN volume and VIP neurons. Those heterozygous for Fgf8 hypomorphy alone or Fgf8 combined with Fgfr1 hypomorphy, called double heterozygotes (DH), showed normal SCN volume but significantly reduced VIP neurons, albeit less severely than the homozygotes. Adult wild type, heterozygous Fgf8 hypomorphs (F8 Het), and DH mice were also examined for SCN cFos activation at three time points: 1 h (morning), 6 h (afternoon), and 11 h (evening) after light onset. In F8 Het mice, a significant change in the pattern of cFos immunostaining that may reflect delayed morning SCN activation was observed. Overall, our studies provide evidence supporting that deficiencies in Fgf8 not only impact the structural integrity of the SCN but also the pattern of SCN activation in response to light.

Localization and functional characterization of a novel adipokinetic hormone in the mollusk, Aplysia californica.

Increasing evidence suggests that gonadotropin-releasing hormone (GnRH), corazonin, adipokinetic hormone (AKH), and red pigment-concentrating hormone all share common ancestry to form a GnRH superfamily. Despite the wide presence of these peptides in protostomes, their biological effects remain poorly characterized in many taxa. This study had three goals. First, we cloned the full-length sequence of a novel AKH, termed Aplysia-AKH, and examined its distribution in an opisthobranch mollusk, Aplysia californica. Second, we investigated in vivo biological effects of Aplysia-AKH. Lastly, we compared the effects of Aplysia-AKH to a related A. californica peptide, Aplysia-GnRH. Results suggest that Aplysia-AKH mRNA and peptide are localized exclusively in central tissues, with abdominal, cerebral, and pleural ganglia being the primary sites of Aplysia-AKH production. However, Aplysia-AKH-positive fibers were found in all central ganglia, suggesting diverse neuromodulatory roles. Injections of A. californica with Aplysia-AKH significantly inhibited feeding, reduced body mass, increased excretion of feces, and reduced gonadal mass and oocyte diameter. The in vivo effects of Aplysia-AKH differed substantially from Aplysia-GnRH. Overall, the distribution and biological effects of Aplysia-AKH suggest it has diverged functionally from Aplysia-GnRH over the course of evolution. Further, that both Aplysia-AKH and Aplysia-GnRH failed to activate reproduction suggest the critical role of GnRH as a reproductive activator may be a phenomenon unique to vertebrates.

Localization of a molluscan gonadotropin-releasing hormone in Aplysia californica by in situ hybridization and immunocytochemistry.

Gonadotropin-releasing hormone (GnRH) plays important roles in vertebrate reproduction. Recently, molecules structurally similar to vertebrate GnRH were discovered in mollusks, including a gastropod, Aplysia californica. As an important step toward understanding the function of A. californica GnRH (ap-GnRH), the present study examined the localization of ap-GnRH peptide and transcript in the central and peripheral tissues. Reverse transcription polymerase chain reaction (RT-PCR) revealed wide expression of ap-GnRH in all ganglia (abdominal, buccal, cerebral, and pedal ganglia) of the central nervous system (CNS) and in multiple peripheral organs. However, in situ hybridization (ISH) revealed that cells positive for ap-GnRH are detectable only in the CNS, with the pedal ganglia containing the highest number of ap-GnRH-positive neurons, followed by the cerebral and abdominal ganglia. Most neurons positive for the transcript were simultaneously positive for the peptide, although some discrepancies were observed in cerebral and abdominal ganglia. Overall, our data suggest the de novo synthesis of ap-GnRH is restricted to the CNS, with the pedal ganglia being the primary source of ap-GnRH. Our results support the notion that ap-GnRH is a bona-fide neuropeptide that may assume diverse central functions, including those unrelated to reproduction.

Molecular cloning and pharmacological characterization of two novel GnRH receptors in the lamprey (Petromyzon marinus).

This paper reports the identification, expression, binding kinetics, and functional studies of two novel type III lamprey GnRH receptors (lGnRH-R-2 and lGnRH-R-3) in the sea lamprey, a basal vertebrate. These novel GnRH receptors share the structural features and amino acid motifs common to other known gnathostome GnRH receptors. The ligand specificity and activation of intracellular signaling studies showed ligands lGnRH-II and -III induced an inositol phosphate (IP) response at lGnRH-R-2 and lGnRH-R-3, whereas the ligand lGnRH-I did not stimulate an IP response. lGnRH-II was a more potent activator of lGnRH-R-3 than lGnRH-III. Stimulation of lGnRH-R-2 and lGnRH-R-3 testing all three lGnRH ligands did not elicit a cAMP response. lGnRH-R-2 has a higher binding affinity in response to lGnRH-III than lGnRH-II, whereas lGnRH-R-3 has a higher binding affinity in response to lGnRH-II than IGnRH-III. lGnRH-R-2 precursor transcript was detected in a wide variety of tissues including the pituitary whereas lGnRH-R-3 precursor transcript was not as widely expressed and primarily expressed in the brain and eye of male and female lampreys. From our phylogenetic analysis, we propose that lGnRH-R-1 evolved from a common ancestor of all vertebrate GnRH receptors and lGnRH-R-2 and lGnRH-R-3 likely occurred due to a gene duplication within the lamprey lineage. In summary, we propose from our findings of receptor subtypes in the sea lamprey that the evolutionary recruitment of specific pituitary GnRH receptor subtypes for particular physiological functions seen in later evolved vertebrates was an ancestral character that first arose in a basal vertebrate.

Fibroblast growth factor signaling deficiencies impact female reproduction and kisspeptin neurons in mice.

Fibroblast growth factor (FGF) signaling is essential for the development of the gonadotropin-releasing hormone (GnRH) system. Mice harboring deficiencies in Fgf8 or Fgf receptor 1 (Fgfr1) suffer a significant loss of GnRH neurons, but their reproductive phenotypes have not been examined. This study examined if female mice hypomorphic for Fgf8, Fgfr1, or both (compound hypomorphs) exhibited altered parameters of pubertal onset, estrous cyclicity, and fertility. Further, we examined the number of kisspeptin (KP)-immunoreactive (ir) neurons in the anteroventral periventricular/periventricular nuclei (AVPV/PeV) of these mice to assess if changes in the KP system, which stimulates the GnRH system, could contribute to the reproductive phenotypes. Single hypomorphs (Fgfr1(+/-) or Fgf8(+/-)) had normal timing for vaginal opening (VO) but delayed first estrus. However, after achieving the first estrus, they underwent normal expression of estrous cycles. In contrast, the compound hypomorphs underwent early VO and normal first estrus, but had disorganized estrous cycles that subsequently reduced their fertility. KP immunohistochemistry on Postnatal Day 15, 30, and 60 transgenic female mice revealed that female compound hypomorphs had significantly more KP-ir neurons in the AVPV/PeV compared to their wild-type littermates, suggesting increased KP-ir neurons may drive early VO but could not maintain the cyclic changes in GnRH neuronal activity required for female fertility. Overall, these data suggest that Fgf signaling deficiencies differentially alter the parameters of female pubertal onset and cyclicity. Further, these deficiencies led to changes in the AVPV/PeV KP-ir neurons that may have contributed to the accelerated VO in the compound hypomorphs.

Gonadotropin-releasing hormone in protostomes: insights from functional studies on Aplysia californica.

Several protostomian molecules that structurally resemble chordate gonadotropin-releasing hormone (GnRH) have been identified through cloning, biochemical purification or data mining. These molecules share considerable sequence and structural similarities with chordate GnRH, leading to the current belief that protostomian and chordate forms of GnRH share a common ancestor. However, the physiological significance of these protostomian GnRH-like molecules remains poorly understood. This knowledge gap hampers our understanding of how GnRH has evolved functionally over time. This review provides a summary of our recent functional characterization of a GnRH-like molecule (ap-GnRH) in a gastropod mollusk, Aplysia californica, and presents preliminary proof for a cognate ap-GnRH receptor (ap-GnRHR). Our data reveal that ap-GnRH is a general neural regulator capable of exerting diverse central and motor effects, but plays little or no role in reproductive activation. This notion is supported by the abundance of a putative ap-GnRHR transcript in the central nervous system and the foot. Comparing these results to the available functional data from a cephalopod mollusk, Octopus vulgaris, we surmise that protostomian GnRH-like molecules are likely to assume a wide range of physiological roles, and reproductive activation is not an evolutionarily conserved role of these molecules. Future functional studies using suitable protostomian models are required to identify functional changes in protostomian GnRH-like molecules that accompany major taxa-level transitions.

Novel FGF8 mutations associated with recessive holoprosencephaly, craniofacial defects, and hypothalamo-pituitary dysfunction.

CONTEXT: Fibroblast growth factor (FGF) 8 is important for GnRH neuronal development with human mutations resulting in Kallmann syndrome. Murine data suggest a role for Fgf8 in hypothalamo-pituitary development; however, its role in the etiology of wider hypothalamo-pituitary dysfunction in humans is unknown. OBJECTIVE: The objective of this study was to screen for FGF8 mutations in patients with septo-optic dysplasia (n = 374) or holoprosencephaly (HPE)/midline clefts (n = 47). METHODS: FGF8 was analyzed by PCR and direct sequencing. Ethnically matched controls were then screened for mutated alleles (n = 480-686). Localization of Fgf8/FGF8 expression was analyzed by in situ hybridization in developing murine and human embryos. Finally, Fgf8 hypomorphic mice (Fgf8(loxPNeo/-)) were analyzed for the presence of forebrain and hypothalamo-pituitary defects. RESULTS: A homozygous p.R189H mutation was identified in a female patient of consanguineous parentage with semilobar HPE, diabetes insipidus, and TSH and ACTH insufficiency. Second, a heterozygous p.Q216E mutation was identified in a female patient with an absent corpus callosum, hypoplastic optic nerves, and Moebius syndrome. FGF8 was expressed in the ventral diencephalon and anterior commissural plate but not in Rathke's pouch, strongly suggesting early onset hypothalamic and corpus callosal defects in these patients. This was consolidated by significantly reduced vasopressin and oxytocin staining neurons in the hypothalamus of Fgf8 hypomorphic mice compared with controls along with variable hypothalamo-pituitary defects and HPE. CONCLUSION: We implicate FGF8 in the etiology of recessive HPE and potentially septo-optic dysplasia/Moebius syndrome for the first time to our knowledge. Furthermore, FGF8 is important for the development of the ventral diencephalon, hypothalamus, and pituitary.

Seasonal changes of brain GnRH-I, -II, and -III during the final reproductive period in adult male and female sea lamprey.

Sea lampreys are anadromous and semelparous, i.e., they spawn only once in their lifetime, after which they die. Sexual maturation is thus a synchronized process coordinated with the life stages of the lamprey. Recently, a novel gonadotropin-releasing hormone, lamprey GnRH-II (lGnRH-II), was identified in lampreys and suggested to have a hypothalamic role in reproduction (Kavanaugh et al., 2008). To further understand the role of lGnRH-II, changes in ovarian morphology, brain gonadotropin-releasing hormone (lGnRH-I, -II, and -III), and plasma estradiol were examined during the final two months of the reproductive season of adult male and female sea lamprey. The results showed significant correlations between water temperature, fluctuation of brain GnRHs, plasma estradiol and reproductive stages during this time. In males, lGnRH-I concentration increased early in the season, peaked, then declined with a subsequent increase with the final maturational stages. In comparison, lGnRH-II and -III concentrations were also elevated early in the season in males, dropped and then peaked in mid-season with a subsequent decline of lGnRH-II or increase of lGnRH-III at spermiation. In females, lGnRH-III concentration peaked in mid-season with a drop at ovulation while lGnRH-I remained unchanged during the season. In contrast, lGnRH-II concentrations in females were elevated at the beginning of the season and then dropped and remained low during the rest of the season. In summary, these data provide evidence that there are seasonal and differential changes of the three GnRHs during this final reproductive period suggesting specific roles for each of the GnRHs in male and female reproduction.

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.

The origins of the vertebrate hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroid (HPT) endocrine systems: new insights from lampreys.

The acquisition of a hypothalamic-pituitary axis was a seminal event in vertebrate evolution leading to the neuroendocrine control of many complex functions including growth, reproduction, osmoregulation, stress and metabolism. Lampreys as basal vertebrates are the earliest evolved vertebrates for which there are demonstrated functional roles for two gonadotropin-releasing hormones (GnRHs) that act via the hypothalamic-pituitary-gonadal axis controlling reproductive processes. With the availability of the lamprey genome, we have identified a novel GnRH form (lamprey GnRH-II) and a novel glycoprotein hormone receptor, lGpH-R II (thyroid-stimulating hormone-like receptor). Based on functional studies, in situ hybridization and phylogenetic analysis, we hypothesize that the newly identified lamprey GnRH-II is an ancestral GnRH to the vertebrate GnRHs. This finding opens a new understanding of the GnRH family and can help to delineate the evolution of the complex neuro/endocrine axis of reproduction. A second glycoprotein hormone receptor (lGpH-R II) was also identified in the sea lamprey. The existing data suggest the existence of a primitive, overlapping yet functional HPG and HPT endocrine systems in this organism, involving one possibly two pituitary glycoprotein hormones and two glycoprotein hormone receptors as opposed to three or four glycoprotein hormones interacting specifically with three receptors in gnathostomes. We hypothesize that the glycoprotein hormone/glycoprotein hormone receptor systems emerged as a link between the neuro-hormonal and peripheral control levels during the early stages of gnathostome divergence. The significance of the results obtained by analysis of the HPG/T axes in sea lamprey may transcend the limited scope of the corresponding physiological compartments by providing important clues in respect to the interplay between genome-wide events (duplications), coding sequence (mutation) and expression control level evolutionary mechanisms in definition of the chemical control pathways in vertebrates.

Origins of gonadotropin-releasing hormone (GnRH) in vertebrates: identification of a novel GnRH in a basal vertebrate, the sea lamprey.

We cloned a cDNA encoding a novel (GnRH), named lamprey GnRH-II, from the sea lamprey, a basal vertebrate. The deduced amino acid sequence of the newly identified lamprey GnRH-II is QHWSHGWFPG. The architecture of the precursor is similar to that reported for other GnRH precursors consisting of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide; however, the gene for lamprey GnRH-II does not have introns in comparison with the gene organization for all other vertebrate GnRHs. Lamprey GnRH-II precursor transcript was widely expressed in a variety of tissues. In situ hybridization of the brain showed expression and localization of the transcript in the hypothalamus, medulla, and olfactory regions, whereas immunohistochemistry using a specific antiserum showed only GnRH-II cell bodies and processes in the preoptic nucleus/hypothalamus areas. Lamprey GnRH-II was shown to stimulate the hypothalamic-pituitary axis using in vivo and in vitro studies. Lamprey GnRH-II was also shown to activate the inositol phosphate signaling system in COS-7 cells transiently transfected with the lamprey GnRH receptor. These studies provide evidence for a novel lamprey GnRH that has a role as a third hypothalamic GnRH. In summary, the newly discovered lamprey GnRH-II offers a new paradigm of the origin of the vertebrate GnRH family. We hypothesize that due to a genome/gene duplication event, an ancestral gene gave rise to two lineages of GnRHs: the gnathostome GnRH and lamprey GnRH-II.

Distribution of gonadotropin-releasing hormone (GnRH) by in situ hybridization in the tunicate Ciona intestinalis.

Gonadotropin releasing hormone (GnRH) is the key hypothalamic neurohormone that is critical in its role of reproduction in all vertebrates. There are currently twenty-four known forms of GnRH that have been identified, 14 in vertebrates and 10 in invertebrates. In tunicates, the primary structure of nine forms have been identified, all of which have been shown to stimulate gamete release. However, the distribution and function of the various GnRH peptides in tunicates have not been fully examined. Therefore, the objective of this study was to determine tissue specific expression of Ci-gnrh-1 and Ci-gnrh-2 in an adult tunicate, Ciona intestinalis, using reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. To examine the expression of the two GnRH genes, total RNA and genomic DNA were isolated from whole animals. Total RNA from neural tissue (cerebral ganglion and neural gland), testis, ovary, heart, and hepatic organ were also isolated. Results from RT-PCR indicated both forms are only expressed in the neural tissue. We extended these studies using fluorescent dual label in situ hybridization. GnRH expression was confirmed to be in the cerebral ganglion bordering the neural gland. These current data along with previous studies suggest that GnRH may be involved in reproduction in the protochordate.

Current knowledge of hagfish reproduction: implications for fisheries management.

This review briefly summarizes the latest findings on reproductive endocrinology of Atlantic hagfish (Myxine glutinosa) and implications for fisheries management. In response to a major decline or collapse of the fisheries (groundfish and anadromous species) industry in the Northeast, species that were once considered alternative or underutilized have and are being identified that may be suitable for commercial harvest, one such example is the hagfish. Hagfish in recent years have been sought after as valuable fish not only for their flesh, but also their skin. Currently, there are no regulations governing the harvesting of hagfish along the East Coast. There has been little to no information of the life history of hagfish including growth rate, age determination, reproductive biology, life span, and larval size at hatching. Thus, the level at which a sustainable fisheries for this species can be maintained is unknown. In some parts of the world, hagfish stocks are being depleted due to overfishing. In order for fisheries management to manage its hagfish stocks and develop a sustainable commercial hagfish fishery, critical information is needed to assist in determining the optimal use of this valuable resource.Key elements of the reproductive system have not been elucidated in hagfish. However, there is new evidence from recent reproductive studies that Atlantic hagfish may have a seasonal reproductive cycle. These data include seasonal changes in gonadotropin-releasing hormone (GnRH), gonadal steroids, estradiol and progesterone, corresponding to gonadal reproductive stages along with the putative identity of a functional corpus luteum. This newly acquired data may provide important information to fisheries managers of the East Coast.

Seasonal changes of gonadotropin-releasing hormone in the Atlantic hagfish Myxine glutinosa.

To investigate seasonal reproduction in Myxine glutinosa, we measured total brain gonadotropin-releasing hormone (GnRH) and determined gonadal stages of hagfish collected from the Gulf of Maine once a month for 12 months. Thirty hagfish from each of three different size classes of small (20-35 cm), medium (35-45 cm), and large (50-60+ cm) were sampled for brains and gonads. In the medium and large class hagfish there was an increase in GnRH concentrations during April and May that correlated with male and female gonadal maturity. Also in these size classes of female hagfish, there was a similar rise in GnRH in November and then again in January that preceded the highest incidence of large eggs (stage 7). The elevated GnRH may be influencing the onset of ovarian recrudescence which has been shown in other vertebrates. These data suggest an association of the concentration of brain GnRH with gonadal maturity and provide supportive evidence of a possible seasonal reproductive cycle in M. glutinosa shown in recent studies of [J. Exp. Zool. 301A (2004) 352], correlating steroid production with gonadal maturation.

In vitro and in vivo effects of GABA, muscimol, and bicuculline on lamprey GnRH concentration in the brain of the sea lamprey (Petromyzon marinus).

gamma-Aminobutyric acid (GABA) is a neurotransmitter with a demonstrated neuroregulatory role in reproduction in most representative species of vertebrate classes via the hypothalamus. The role of GABA on the hypothalamus-pituitary axis in lampreys has not been fully elucidated. Recent immunocytochemical and in situ hybridization studies suggest that there may be a neuroregulatory role of GABA on the gonadotropin-releasing hormone (GnRH) system in lampreys. To assess possible GABA-GnRH interactions, the effects of GABA and its analogs on lamprey GnRH in vitro and in vivo were studied in adult female sea lampreys (Petromyzon marinus). In vitro perfusion of GABA and its analogs at increasing concentrations (0.1-100 microM) was performed over a 3-h time course. There was a substantial increase of GnRH-I and GnRH-III following treatment of muscimol at 100 microM. In in vivo studies, GABA or muscimol injected at 200 microg/kg significantly increased lamprey GnRH concentration in the brain 0.5 h after treatment compared to controls in female sea lampreys. No significant change in lamprey GnRH-I or GnRH-III was observed following treatment with bicuculline. These data provide novel physiological data supporting the hypothesis that GABA may influence GnRH in the brain of sea lamprey.

Seasonal concentrations of reproductive steroids in the gonads of the Atlantic hagfish, Myxine glutinosa.

Changes in gonadal morphology, gonadal estradiol, and progesterone were examined in Atlantic hagfish, Myxine glutinosa, during a period of 17 months, beginning in April, 2001. Atlantic hagfish were captured from the ocean on a monthly basis. A total of 60 hagfish were divided into three different size classes of twenty hagfish each (small 20-35 cm, medium 35-45 cm, large 45-55+cm) and transported to the University of New Hampshire for sampling. Overall, in the medium and large size hagfish, estradiol and progesterone had significantly elevated peaks in January, 2001. There were significant increases in estradiol concentrations in January, with relatively low fluctuations in levels for the rest of the sampling period. Progesterone concentrations increased significantly in January, 2002, in medium and large hagfish, and remained elevated until June and April, 2002, for the two size classes respectively. The majority of hagfish sampled were females or hermaphrodites; few true males were identified in any of the samples. The number of females with large eggs increased following the estradiol peak in January and hermaphrodites with mature sperm were identified in the July, 2002, sample. These data represent the first evidence for a seasonal reproductive cycle in M. glutinosa and only the second seasonal reproductive cycle identified in any hagfish species.