We have recently developed sensitive and specific radioimmunoassays (RIAs) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II) and have measured the contents of both GnRHs in the rainbow trout brain. Our results showed that contents of the two GnRHs are variable among different brain regions. Therefore, in order to confirm the differential distribution of the two GnRHs by a different technique, we examined the distribution of immunoreactive sGnRH and cGnRH-II in the brain of masu salmon by using immunocytochemical techniques. sGnRH immunoreactive (ir) cell bodies were scattered in the transitional areas between the olfactory nerve and the olfactory bulb, the ventral olfactory bulb, between the olfactory bulb and the telencephalon, the ventral telencephalon, and the preoptic area. These sGnRH-ir cell bodies were dispersed in a strip-like region running rostrocaudally in the most ventral part of the ventral telencephalon. sGnRH-ir fibers were distributed in the various brain regions from the olfactory bulb to the spinal cord. They were especially abundant in the olfactory bulb, ventral telencephalon, preoptic area, hypothalamus, deep layers of the optic tectum, and thalamus. sGnRH-ir fibers also innervated the pituitary directly. cGnRH-II-ir cell bodies were found in the nucleus of the medial longitudinal fasciculus (nMLF). The distribution of cGnRH-II-ir fibers was similar to that of sGnRH-ir fibers, except that cGnRH-II-ir fibers were absent in the pituitary. The number of cGnRH-II-ir fibers was much fewer than that of sGnRH-ir fibers. The results of the present immunocytochemical study are in basic agreement with those of our previous RIA study. Thus, we suggest that in masu salmon, sGnRH not only regulates gonadotropin (GTH) release from the pituitary but also functions as a neuromodulator in the brain, whereas cGnRH-II functions only as a neuromodulator.

Although reduced fetal growth in response to hypoxia has been appreciated for decades, we have a poor understanding of the effects of hypoxia on embryonic development and the underlying cellular and molecular mechanisms. Here we show that hypoxia treatment not only resulted in embryonic growth retardation but also caused significant delay in developmental zebrafish ͉ heart development ͉ craniofacial skeleton ͉ morphogenesis ͉ oxygen H ypoxic stress causes major metabolic changes in all organisms requiring oxygen. Hypoxic stress also influences fetal growth and development and the pathogenesis of several human diseases, including intrauterine growth restriction (IUGR) (1-3). Reduced birth weight is also observed at high altitudes (4). IUGR not only increases fetal and neonatal morbidity and mortality, but also increases the risk of having adult diseases, such as cardiovascular disease, type-2 diabetes, obesity, and hypertension (5).Recent evidence suggests that hypoxia may influence fetal growth through its connection to the insulin-like growth factor (IGF) signaling system. IGFs are well known fetal growth factors (6-9). Several groups have reported that the circulating level of IGF-binding protein (IGFBP)-1, a secreted protein that binds to IGF in extracellular environments, is elevated in IUGR fetuses (10-12) and that there is a striking inverse correlation between IGFBP-1 levels and fetal size (13). In addition, higher maternal serum IGFBP-1 levels are found at higher altitude (14). In vitro studies with cultured human cells and in vivo studies with mammalian animal models suggest that IGFBP-1 gene expression is elevated in hypoxic conditions (15)(16)(17) and that this up-regulation is mediated through the hypoxia-inducible factor (HIF)-1 pathway (16). Because IGFBP-1 binds IGFs with high affinity and inhibits IGF activities on cell growth in vitro (18,19) and because IGFBP-1-overexpressing transgenic mice had reduced birth weight (20-22), it was postulated that the elevated IGFBP-1 plays a major role in hypoxia-caused IUGR by binding fetal IGFs and inhibiting their growth-promoting activities (3,16,17). This appealing model, however, has not been directly tested in vivo, and a causative relationship between the elevated IGFBP-1 expression and IUGR has not been established. Moreover, the impact of hypoxia on early developmental processes, such as morphogenesis, is poorly understood, and the role of IGFBP-1, if any, in mediating the hypoxic effects on embryonic development is unknown.The zebrafish has now become an informative vertebrate model organism for the study of IGF signaling in early development (23). Zebrafish embryos develop externally, eliminating the complication of maternal compensation. Fast developing and transparent zebrafish embryos make it possible to manipulate environmental factors and observe the phenotypic changes in organ formation in real time. Furthermore, major components of the zebrafish IGFsignaling pathway, including IGF ligands, receptors, IGFBPs, and intracellular signal transdu...

Follicle-stimulating hormone (FSH) plays important roles in spermatogenesis. However, the biologic activity of FSH can vary in different vertebrate classes, and the definitive function of FSH has not been established. In this study, we investigated the functions of FSH on spermatogenesis using an in vitro culture system for Japanese eel testis. The eel Fsh receptor was expressed in testis tissue during the whole process of spermatogenesis, mainly by Leydig cells that produce steroid hormones and by Sertoli cells surrounding type A spermatogonia and early type B spermatogonia. In an in vitro organ culture, recombinant eel Fsh (r-eFsh) induced complete spermatogenesis from the proliferation of spermatogonia to spermiogenesis during 36 days of culture; also, spermatozoa were observed in the testicular fragments. Spermatogenesis induced by r-eFsh was inhibited by trilostane, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. However, trilostane did not inhibit spermatogenesis induced by 11-ketotestosterone. These results clearly show that the main function of FSH in eel is to induce spermatogenesis via stimulating androgen production.

The present study characterizes gonadotropin-releasing hormone (GnRH) neuronal groups that are located in several different brain regions by investigating GnRH molecular species and projection patterns in an anabantid fish, Colisa lalia. First, we examined the molecular species of GnRHs in extracts of the brain and the pituitary by reverse-phase high-performance liquid chromatography followed by radioimmunoassays. We found salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and an unfamiliar GnRH-like substance. Next, to examine the distribution of each GnRH molecule in different GnRH neuronal groups, we performed immunohistochemistry using four kinds of antisera and an antibody. Furthermore, we performed brain lesioning experiments of terminal nerve (TN) cells, the most conspicuous GnRH-immunoreactive cells in Colisa lalia. Comparisons of immunoreactive structures between TN-lesioned fish and untreated fish elucidated the projection area of each neuronal group. Three major neuronal groups were observed. TN-GnRH cells, which are located in the transitional area between the olfactory bulb and the telencephalon, showed strong sGnRH and weaker cGnRH-II immunoreactivity. TN-GnRH cells projected to wide areas of the central nervous system from the olfactory bulb to the spinal cord. The second group, located in the preoptic area, showed only sGnRH immunoreactivity and projected only to the pituitary. The third one, located in the midbrain tegmentum, exhibited strong cGnRH-II and weaker sGnRH immunoreactivity. This cell group projected mainly to brain regions posterior to the hypothalamus and the spinal cord. These different projection patterns suggest functional differentiation of each GnRH neuronal group.

SUMMARYTo elucidate the mechanisms associated with water absorption in the intestine, we compared drinking and intestinal water absorption in freshwater-and seawater-adapted Japanese eels, and investigated a possible involvement of aquaporin (AQP) in the absorption of water in the intestine. Seawater eels ingested more water than freshwater eels, the drinking rate being 0.02 ml kg-1 h-1 in fresh water and 0.82 ml kg-1h-1 in sea water. In intestinal sacs prepared from freshwater and seawater eels, water absorption increased in time- and hydrostatic pressure-dependent manners. The water absorption rates were greater in seawater sacs than in freshwater sacs, and also greater in the posterior intestine than in the anterior. In view of the enhanced water permeability in the intestine of seawater eel, we cloned two cDNAs encoding AQP from the seawater eel intestine, and identified two eel homologues (S-AQP and L-AQP) of mammalian AQP1. S-AQP and L-AQP possessed the same amino acid sequence, except that one amino acid was lacking in S-AQP and two amino acids were substituted. Eel AQP1 was expressed predominantly in the intestine, and the expression levels were higher in seawater eel than in freshwater eel. Immunocytochemical studies revealed intense AQP1 immunoreaction in the apical surface of columnar epithelial cells in seawater eel, in which the immunoreaction was stronger in the posterior intestine than in the anterior. In contrast, the immunoreaction was faint in the freshwater eel intestine. Preferential localization of AQP1 in the apical membrane of epithelial cells in the posterior intestine of seawater eel indicates that this region of the intestine is responsible for water absorption, and that AQP1 may act as a water entry site in the epithelial cells.

Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.

Hypoxia triggers the transcription of regulatory genes that promote O 2 delivery and anaerobic metabolism, suppress major energy-requiring processes, and inhibit growth and development in animals ranging from invertebrates to mammals (14,15,40). Hypoxia also influences several human pathological processes, such as tumorigenesis and intrauterine growth restriction (IUGR). The majority of these transcriptional responses to hypoxia are mediated by the hypoxia-inducible factor 1 (HIF-1) complex. HIF-1 is a heterodimeric complex composed of 45). HIF-1␤, also known as aryl hydrocarbon receptor nuclear translocator, is constitutively expressed and insensitive to O 2 availability. When oxygen levels are high, HIF-1␣ is bound to the von Hippel-Lindau tumor suppressor (pVHL) and targeted for ubiquitination and proteosomal degradation (30, 34). Hypoxic conditions inhibit this degradation, which allows HIF-1␣ to accumulate in the cell (16,18,27). HIF-1␣ is then translocated to the nucleus, dimerizes with HIF-1␤, binds to DNA, and activates target gene expression (3). Hypoxia response elements (HREs) are cis-regulatory DNA sequences that specifically bind to HIF-1 and are required for transcriptional induction upon hypoxia exposure.Insulin-like growth factor binding protein 1 (IGFBP-1) is a hypoxia-inducible gene. Earlier studies have shown that circulating levels of IGFBP-1 are elevated in IUGR fetuses (7,12,42,43). In vitro studies using cultured human cells and in vivo studies using rodent and fish models suggest that IGFBP-1 gene expression is elevated under hypoxic conditions (13,29,31,35,41). Since IGFBP-1 binds IGFs and inhibits IGF action on cell growth in vitro (4, 9) and because IGFBP-1 overexpression reduced birth weights in mice (5,11,36), it was postulated that elevated IGFBP-1 plays a major role in hypoxia-induced IUGR by binding IGFs and inhibiting their growth-promoting activities (41). Using the transparent and free-living zebra fish embryo as a model system, we have recently shown that (i) hypoxia strongly up-regulates IGFBP-1 expression and delays growth and developmental rate in zebra fish embryos; (ii) IGFBP-1 knockdown partially abrogates these hypoxic effects, whereas IGFBP-1 overexpression decreases growth and developmental rates under normoxia; and (iii) reintroduction of IGFBP-1 to the knocked down embryos restores the hypoxic effects (19). These findings provide strong evidence arguing that up-regulation of IGFBP-1 by hypoxia plays a key role in coordinating embryonic growth rate and developmental timing in response to environmental oxygen availability. Although there is in vitro evidence that overexpression of HIF-1␣ in cultured human hepatoma (HepG2) cells increases human IGFBP-1 promoter activity (41), how hypoxia triggers IGFBP-1 gene expression in vivo is not clear, and the cis-regulatory elements responsible for hypoxia-induced IGFBP-1 transcription in vivo are not well defined.The objectives of this study are (i) to determine when the HIF-1 pathway becomes operational in early development and

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