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Among ectothermic reptiles, the order Squamata has adapted most successfully to the terrestrial environment. However, the physiological background of this success remains unknown. Since the regulation of energy metabolism provides an important insight into terrestrial adaption by ectothermic animals, we focused on proglucagon-derived peptides (PGDPs). In the process of cloning proglucagon mRNA in geckos, we identified several novel proglucagon (PG) cDNA isoforms. They were tissue-specifically and strongly expressed in the pancreas and small intestine of the geckos, suggesting their biological relevance. Therefore, in order to clarify whether these novel cDNA isoforms are phylogenetically conserved, we performed the additional molecular characterization of proglucagon cDNAs from several representative species of the Squamata and Testudine clade and examined the expression of proglucagon mRNAs in the small intestine and pancreas. In the present study, a total of 7 proglucagon cDNA isoforms were identified and divided into two groups (Classes A and B) based on the 3′-UTR sequence of each isoform. The longest isoform of each group (named PG-A1 and PG-B1, respectively) had the same molecular characteristics as those previously reported from chickens and reptiles, namely, PG-A and PG-B. Other 5 isoforms were novel-type cDNAs, and were the products of exon skipping (named PG-A2, PG-A2s, PG-B2, PG-B2s, and PG-B3). Some of these isoforms coded for only one peptide hormone (GLP-1 or GLP-2). This is the first identification of single hormone-encoding proglucagon cDNAs in vertebrates. Moreover, an expression analysis of these isoforms revealed that single hormone-encoding proglucagon mRNAs were predominantly expressed with tissue and lineage specificities in the reptile clade. Collectively, the present results suggest an independent regulatory system for GLP-1 and GLP-2 secretion and indicate the plasticity of proglucagon genes in expressing different isoforms in different tissues in Squamata. These results also provide insights into the plastic energy metabolic system of Squamata in accordance with various habitats in the terrestrial environment, supporting their successful prosperity.

Gonadal sex differentiation proceeds by the interplay of various genes including the transcription factors and secretory factors in a complex network. The sex-differentiating genes are expressed not only during early sex differentiation but also throughout the gonadal development and even in the adult gonads. In addition, the evidence that they actually function in the adult gonads have been accumulated from the studies using the conditional knockout mice. However, many previous studies were focused on one single gene though those genes function in a network. In this study, the expressions of various sex-differentiating genes were analyzed simultaneously in the adult testis of the Japanese quail (Coturnix japonica), whose testicular functions are dramatically changed by altering the photoperiod, to elucidate the roles of them in the adult gonad. Anti-Müllerian hormone (AMH) was significantly upregulated in the regressed testis induced by the short-day condition. The expressions of the transcription factors that promoteAMHexpression in mammals (SF1,SOX9,WT1andGATA4) were also increased in the regressed testis. Moreover, AMH receptor (AMHR2) showed similar expression pattern to its ligand. We also analyzed the expressions of other transforming growth factor beta (TGFB) superfamily members and their receptors. The expressions of the ligands and receptors of TGFB family, and follistatin and betaglycan in addition to inhibin subunits were increased in the regressed testis. These results suggest that AMH is involved in the adult testicular functions of the Japanese quail together with other TGFB superfamily members.

The Squamata are the most adaptive and prosperous group among ectothermic amniotes, reptiles, due to their species-richness and geographically wide habitat. Although the molecular mechanisms underlying their prosperity remain largely unknown, unique features have been reported from hormones that regulate energy metabolism. Insulin, a central anabolic hormone, is one such hormone, as its roles and effectiveness in regulation of blood glucose levels remain to be examined in squamates. In the present study, cDNAs coding for insulin were isolated from multiple species that represent various groups of squamates. The deduced amino acid sequences showed a high degree of divergence, with four lineages showing obviously higher number of amino acid substitutions than most of vertebrates, from teleosts to mammals. Among 18 sites presented to comprise the two receptor binding surfaces (one with 12 sites and the other with 6 sites), substitutions were observed in 13 sites. Among them was the substitution of HisB10, which results in the loss of the ability to hexamerize. Furthermore, three of these substitutions were reported to increase mitogenicity in human analogues. These substitutions were also reported from insulin of hystricomorph rodents and agnathan fishes, whose mitogenic potency have been shown to be increased. The estimated value of the non-synonymous-to-synonymous substitution ratio (ω) for the Squamata clade was larger than those of the other reptiles and aves. Even higher values were estimated for several lineages among squamates. These results, together with the regulatory mechanisms of digestion and nutrient assimilation in squamates, suggested a possible adaptive process through the molecular evolution of squamate INS. Further studies on the roles of insulin, in relation to the physiological and ecological traits of squamate species, will provide an insight into the molecular mechanisms that have led to the adaptivity and prosperity of squamates.

Novel types of symmetric bis-7-methoxytacrines connected by oligoethyleneoxy chains 3–5 and nonsymmetric monomeric 7-methoxytacrines containing hydroxyl-terminated oligoethyleneoxy chains 6–8 were prepared, and their in vitro/in silico effects on human recombinant AChE (hAChE) and human plasmatic butyrylcholinesterase (hBChE) were compared, with 7-MEOTA (2) as the standard compound. The symmetric bis-7-MEOTA derivatives 3–5 showed hAChE inhibition similar to that of 2. On the other hand, their effects on hBChE revealed an increasing inhibition trend when the oligoethyleneoxy units between the two 7-MEOTA moieties became longer. Accordingly, compounds 4 and 5 showed better selectivity towards hBChE. The most effective in the inhibition hAChE and hBChE was compound 8 with the longest oligoethyleneglycol chain, whereas compounds 6 and 7 resulted in similar IC50 values. A molecular modeling study using substrates 5 and 8 showed a possible binding conformation and protein–ligand interaction between the substrates and AChE/BChE.

A green procedure is described for the preparation of a series of ionic liquids where two 3-methylimidazolium cations were bridged with ethylene glycol chain as linkers. Diethylene glycol-bis(3-methylimidazolium) ditosylate, triethylene glycol- bis(3-methylimidazolium) ditosylate, and tetraethylene glycol-bis(3-methylimidazolium) ditosylate were heated with 3-methyl- imidazole under solvent-free condition to give the corresponding bis(3-methylimidazolium) ditosylate ionic liquids efficiently in a short time.

Glucagon is a linear peptide hormone of 29 amino acids secreted from α cells of the pancreas. Glucagon shares the same precursor molecule, proglucagon, with GLP-1 and GLP-2. By tissue-specific posttranslational processing, glucagon is secreted from pancreatic α cells whereas GLP-1 and GLP-2 are secreted from intestinal L cells. All these peptides have considerable sequence homology and form a subfamily within the secretin family. Among the members of the glucagon subfamily, the glucagon sequence is most highly conserved throughout vertebrates. Glucagon is the principal hyperglycemic hormone, and acts as a counterbalancing hormone to insulin. Glucagon generally elevates the level of blood glucose by promoting gluconeogenesis and glycogenolysis. Glucagon has the greatest effect on the liver although it affects many different organs in the body, such as adipose tissue, pancreas, brain, and kidney.

GLP-2 was first identified as a novel peptide encoded within a proglucagon precursor that also encodes glucagon and GLP-1. In the intestinal L-cells, PC1/3 liberates GLP-1 and GLP-2 as bioactive hormones, and GLP-1 and GLP-2 are co-secreted in response to nutrient ingestion. The principal role of GLP-2 appears to be the maintenance of growth and absorptive function of the intestinal mucosal villus epithelium. The expression of GLP-2 receptor is highly restricted, predominantly to the gastrointestinal tract and central nervous system. In the gut, GLP-2 receptor is localized to scattered enteroendocrine cells, enteric neurons, and subepithelial myofibroblasts, but is not localized to the known target enterocytes of GLP-2 tropic action. This result suggests that intestinotropic action of GLP-2 is indirect, and it has been demonstrated that KGF and IGF-1, secreted from subepithelial myofibroblasts, act as essential mediators in responses to GLP-2.

GIP is a 42-amino acid polypeptide hormone secreted from endocrine K cells in the intestinal epithelium, and was originally isolated as a gastric inhibitory polypeptide. After finding its glucose-dependent insulinotropic activity, known as the incretin effect, GIP was renamed as glucose-dependent insulinotropic peptide. The incretin effect is shared with another hormone, GLP-1, and there are two main incretin hormones. Both hormones, along with glucagon and GLP-2, are highly related in their molecular structure and biological function, and are evolutionally related to the glucagon peptide family. GIP expression is also reported in the submandibular salivary gland, the stomach, and the brain. The GIP receptor is expressed in a wide variety of organs and tissues, and GIP exerts a number of additional non-incretin actions, including inhibition of glucagon secretion and stimulation of adipogenesis.

The glucagon family peptides include glucagon, the glucagon-like peptides (GLP-1 and GLP-2), and GIP. This family of peptides is a subfamily within the secretin family, and shares a highly related molecular structure. The peptides are expressed in endocrine cells of the pancreas and gastrointestinal epithelium, and in specialized neurons in the brain. They share multiple biological functions in the regulation of nutrient homeostasis and energy metabolism through their distinct specific receptors. Not only are the hormone peptides related, but also their receptors. These receptors exhibit considerable amino acid sequence identity, and share similar structural properties with respect to ligand binding and signal transduction. With these characteristics, they are grouped as the glucagon receptor family in the secretin receptor superfamily, like their ligand peptides.

GLP-1 is a 30-aa peptide hormone secreted by intestinal L cells during feeding. GLP-1 shares the same precursor molecule, proglucagon, with glucagon and GLP-2, and their structure and physiological function are highly related with one another. There are two natural bioactive GLP-1s, GLP-1(7–37) and GLP-1(7–36)-amide, and unqualified designation GLP-1 covers truncated peptide. The major physiological function of GLP-1 is stimulation of the pancreatic β cells to secrete insulin as an incretin hormone. The first incretin hormone to be identified was GIP, which is also structurally related to GLP-1 and a member of the glucagon family. GLP-1 also has additional non-incretin actions, including suppression of glucagon secretion, inhibition of gastric motility, and promotion of satiety. Secretion of GLP-1 from intestinal L cells is stimulated by nutrients, hormones, and neural inputs. Insulin also inhibits GLP-1 release, suggesting that a feedback loop mechanism regulates GLP-1 secretion.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in regulating the reproductive function of vertebrates. These neurons are known to originate in the olfactory placode and migrate along olfactory-related axons to reach the forebrain during embryonic development. Although GnRH is suggested to be secreted during such migration, its physiological significance is unknown. This point is difficult to explore in vivo because recent studies suggest that GnRH is an important factor for normal brain development and that modification of the embryonic GnRH system by exogenous GnRH analogue or genetic methods would result in dysgenesis of the brain. Therefore, to study the role of GnRH in the migratory process of GnRH neurons, we established an in vitro chick embryonic olfactory nerve bundle explant model. Embryonic day 7.5-8 olfactory nerve bundles were cultured in a mixture of Matrigel and collagen gel. At day 3 of culture, GnRH neurons extended their unbranched neurites and migrated out from both edges of the explant. The nature of neurite extension and migratory behavior of GnRH neurons was well maintained in the gel containing 25% Matrigel and 50% collagen. With this culture system, we examined the effect of GnRH on the migrating GnRH neurons. Cetrorelix, a GnRH antagonist, was found to inhibit significantly neurite growth and neuronal migration of GnRH neurons, the effects of which were repressed by the addition of chicken GnRH-I. These results suggest that GnRH functions as one of the regulating factors of GnRH neuronal development by promoting neurite extension and neuronal migration.

Despite the physiological and evolutionary significance of lipid metabolism in amniotes, the molecular mechanisms involved have been unclear in reptiles. To elucidate this, we investigated peroxisome proliferators-activated receptors (PPARs) in the leopard gecko (Eublepharis macularius). PPARs belong to a nuclear hormone-receptor family mainly involved in lipid metabolism. Although PPARs have been widely studied in mammals, little information about them is yet available from reptiles. We identified in the leopard gecko partial cDNA sequences of PPARalpha and beta, and full sequences of two isoforms of PPARgamma. This is the first report of reptilian PPARgamma mRNA isoforms. We also evaluated the organ distribution of expression of these genes by using RT-PCR and competitive PCR. The expression level of PPARalpha mRNA was highest in the large intestine, and moderate in the liver and kidney. The expression level of PPARbeta mRNA was highest in the kidney and large intestine, and moderate in the liver. Similarly to the expression of human PPARgamma isoforms, PPARgammaa was expressed ubiquitously, whereas the expression of PPARgammab was restricted. The highest levels of their expression, however, were observed in the large intestine, rather than in the adipose tissue as in mammals. Taken together, these results showed that the profile of PPARbeta mRNA expression in the leopard gecko is similar to that in mammals, and that those of PPAR alpha and gamma are species specific. This may reflect adaptation to annual changes in lipid storage due to seasonal food availability.

Sex steroid hormones are known to play a central role in vertebrate sex determination and differentiation. However, the tissues in which they are produced or received during development, especially around the period of sex determination of the gonads, have rarely been investigated. In this study, we identified the cDNA sequence, including the full-length of the coding region of cholesterol side-chain cleavage enzyme (P450scc), from the leopard gecko; a lizard with temperature-dependent sex determination. Embryonic expression analysis of two steroidogenic enzymes, P450scc and P450 aromatase (P450arom), and four sex steroid hormone receptors, androgen receptor, estrogen receptor alpha and beta, and progesterone receptor, was subsequently conducted. mRNA expression of both steroidogenic enzymes was observed in the brain and gonads prior to the temperature-sensitive period of sex determination. The mRNAs of the four sex steroid hormone receptors were also detected in the brain and gonads at all stages examined. These results suggest the existence of a gonad-independent sex steroid hormone signaling system in the developing leopard gecko brain.

Sexual differentiation in the amniote brain is believed to be regulated by gonadal sex steroid hormones. Recently, however, the possibility of brain-autonomous sexual differentiation in avian and reptilian species has been reported. We conducted here an expressional analysis of genes related to sex steroid hormones in the chick-embryo brain before gonadal sexual differentiation. Female-specific P450 aromatase expression in the gonad was observed at day 6.5 of incubation, as previously reported, whereas the mRNAs of cholesterol side-chain cleavage enzyme, androgen receptor, and estrogen receptors alpha and beta were clearly expressed in all brain samples of both male and female embryos from day 4.5 of incubation. P450 aromatase was expressed in some brain samples before day 5.5 of incubation and in all brain samples after day 6 of incubation. The mRNA of Ad4BP/SF-1, a transcription factor that regulates steroidogenic enzymes, showed higher expression levels in the male brain than in the female brain at day 5.5 of incubation. This gene was expressed in the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Embryonic Ad4BP/SF-1 expression is reported to play an important role in the formation of this region. These results therefore suggest the involvement of a sex steroid hormone signaling system in brain-autonomous sexual differentiation.

GnRH regulates reproductive functions through interaction with its pituitary receptor in vertebrates. The present study demonstrated that the leopard gecko possessed two and three genes for GnRH ligands and receptors, respectively, though one of the three receptor subtypes had long been thought not to exist in reptiles. Each receptor subtype showed a distinct pharmacology. All types of ligands and receptors showed different expression patterns, and were widely expressed both inside and outside the brain. This report also shows a comparison of the pituitary and ovarian GnRH systems in the leopard gecko during and after the egg-laying season. All three receptor subtypes were expressed in both the whole pituitary and ovary; however, only one receptor subtype could be detected in the anterior pituitary gland. In situ hybridization showed spatial expression patterns of ovarian receptors, and suggested co-expression of multiple receptor subtypes in granulosa cells of larger follicles. Co-transfection of receptor subtypes showed a distinct pharmacology in COS-7 cells compared with those of single transfections. These results suggest that distinct signaling mechanisms are involved in the pituitary and ovarian GnRH systems. Seasonal and developmental variations in receptor expression in the anterior pituitary gland and ovarian follicles may contribute to the seasonal breeding of this animal.

Thyroid hormones (THs) play crucial roles in various developmental and physiological processes in vertebrates, including squamate reptiles. The effect of THs on shedding frequency is interesting in Squamata, since the effects on lizards are quite the reverse of those in snakes: injection of thyroxine increases shedding frequency in lizards, but decreases it in snakes. However, the mechanism underlying this differential effect remains unclear. To facilitate the investigation of the molecular mechanism of the physiological functions of THs in Squamata, their two specific receptor (TRalpha and beta) cDNAs, which are members of the nuclear hormone receptor superfamily, were cloned from a lizard, the leopard gecko, Eublepharis macularius. This is the first molecular cloning of thyroid hormone receptors (TRs) from reptiles. The deduced amino acid sequences showed high identity with those of other species, especially in the C and E/F domains, which are characteristic domains in nuclear hormone receptors. Expression analysis revealed that TRs were widely expressed in many tissues and organs, as in other animals. To analyze their role in the skin, temporal expression analysis was performed by RT-PCR, revealing that the two TRs had opposing expression patterns: TRalpha was expressed more strongly after than before skin shedding, whereas TRbeta was expressed more strongly before than after skin shedding. This provides good evidence that THs play important roles in the skin, and that the roles of their two receptor isoforms are distinct from each other.

GnRH was first identified as the hypothalamic decapeptide that promotes gonadotropin release from pituitary gonadotropes. Thereafter, direct stimulatory and inhibitory effects of GnRH on cell proliferation were demonstrated in a number of types of primary cultured cells and established cell lines. Recently, the effects of GnRH on cell attachment, cytoskeleton remodeling, and cell migration have also been reported. Thus, the effects of GnRH on various cell activities are of great interest among researchers who study the actions of GnRH. In this study, we demonstrated that GnRH induces actin cytoskeleton remodeling and affects cell migration using two human prostatic carcinoma cell lines, TSU-Pr1 and DU145. In TSU-Pr1, GnRH-I and -II induced the filopodia formation of the cells and promoted cell migration, whereas in DU145, GnRH-I and -II induced the formation of the cells with stress fiber and inhibited cell migration. In our previous studies, we reported the stimulatory and inhibitory effects of GnRH on the cell proliferation of TSU-Pr1 and DU145 cells. This study provides the first evidence for the effects of GnRH on actin cytoskeleton remodeling and cell migration of cells in which cell proliferation was affected by GnRH at the same time. Moreover, we also demonstrated that the same human GnRH receptor subtype, human type I GnRH receptor, is essential for the effects of GnRH-I and -II on actin cytoskeleton remodeling and cell migration in both TSU-Pr1 and DU145 cells using the technique of gene knock-down by RNA interference.

Gonadotropin-releasing hormone (GnRH) is thought to have diverse physiological functions. Understanding regulatory mechanisms of GnRH functions requires detailed knowledge of gene expressions of both GnRH ligands and receptors in a single species. This report concerns identification and molecular characterization of GnRH ligands and receptors in the spotted green pufferfish Tetraodon nigroviridis. It was identified that the pufferfish possessed three types of GnRH ligands and five types of GnRH receptors. All types of ligands and receptors showed different expression patterns, and were widely expressed both inside and outside the brain. Gonads expressed all the ligand and receptor subtypes. Two of five receptor subtypes could not be detected in the pituitary gland of reproductively active individuals, suggesting the existence of novel GnRH systems independent of hypothalamic–pituitary–gonadal axis. Alternative splicing was also observed for some receptor subtypes. The present results indicate that diversified gene expressions combined with molecular diversity contribute to the functional diversity of GnRH.

In spite of their physiological significance, there is no available information about the nucleotide sequences of prolactin (PRL) and its receptor in reptilian species. In order to fill this gap, PRL and its receptor cDNAs were identified in a reptilian species, the leopard gecko Eublepharis macularius. The deduced leopard gecko PRL polypeptide showed high identities with the corresponding polypeptides of other reptiles. The leopard gecko PRL receptor (PRLR) was estimated to have tandem repeated regions in its extracellular domain, which had been originally found in avian PRLR. Molecular phylogenetic analysis suggests that these tandem repeated regions were generated by the duplication of the extracellular region in the latest common ancestor among reptiles and birds. In addition, tissue distributions of PRL and PRLR in the leopard gecko were examined by the reverse transcription-polymerase chain reaction (RT-PCR). PRLR mRNA was detected in all tissues examined and highly expressed in the whole brain, pituitary, intestine, kidney, ovary, oviduct and testis. Whereas, PRL mRNA was expressed in the whole brain, pituitary, ovary and testis. The co-expressions of PRL and its receptor in some extrapituitary organs suggest that PRL acts as an autocrine/paracrine factor in such organs of the leopard gecko.

Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Studies on GnRH have demonstrated that GnRH has both stimulatory and inhibitory effects on cell proliferation depending on the cell type; however, the mechanisms of these effects remain to be elucidated. Against this background we used four human cell lines, TSU-Pr1, Jurkat, HHUA and DU145, and newly found that GnRH increased or decreased the colony-formation depending on the cell line. Moreover, we demonstrated that the stimulatory and inhibitory effects of GnRH exhibit distinct ligand selectivities. In order to investigate the molecular basis of these phenomena, analyses of the expression of human GnRH receptors were performed and, moreover, the effects of GnRH were analyzed under conditions in which human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor, which had been suspected of being nonfunctional because of alterations in its sequence, is involved in the effects of GnRH on cell proliferation. In this article, the influence of the autocrine activities of the cells is also reviewed, focusing on the characteristics of substances secreted from the four cell lines. Based on recent studies of GnRH and its receptors and our up-to-date findings, the evolutionary implications of GnRH action are discussed.

GnRH (gonadotropin-releasing hormone) is well-known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Progress in studies on GnRH demonstrated that GnRH has both inhibitory and stimulatory effects on cell proliferation depending on the cell type, and the mechanisms of these effects have been intensively studied. However, even human GnRH receptors which mediate GnRH stimulation have not been completely identified. In the present study, we showed that the inhibitory and stimulatory effects of GnRH on colony-formation using four cell lines and have demonstrated that the inhibitory and stimulatory effects of GnRH exhibit distinctly different patterns of ligand sensitivity. This result strongly suggests that the two opposite effects of GnRH occur via different types of GnRH receptors, however expressional analyses of human GnRH receptors did not exhibit the significantly different pattern between negatively and positively responding cell lines. Then, in order to identify the GnRH receptors involved in the two opposite effects, effects of GnRH were analysed under the conditions that human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor mediates GnRH stimulation and its splice variant determines the direction of the response to GnRH. These results are the first clear evidence for the functionality of human type II GnRH receptor. Therefore our novel findings are quite noticeable and will greatly contribute to the studies on the mechanisms of the effects of GnRH on cell proliferation in the future.

There have been numerous reports of the inhibitory effects of gonadotropin-releasing hormone (GnRH) and its agonistic and antagonistic analogues on carcinomas derived from various organs, and in particular the direct inhibitory effects have been extensively studied. On the other hand, several studies have indicated that GnRH stimulates the proliferation of lymphoid tissues and cells, suggesting that GnRH is an immunomodulator. However, there have been few reports showing a stimulatory effect of GnRH on cell lines not derived from lymphoid tissues, and the mechanism of the stimulatory effect has not been investigated in detail. In this study, the stimulatory effect of GnRH (100 pM) on TSU-Pr1, a human prostatic carcinoma cell line, was demonstrated, and the dose-depedency of this effect of GnRH (3.125 fM approximately 20 nM) was observed by measuring colony-formation. RT-PCR analysis showed that both human GnRH receptor 1 and 2 are expressed in TSU-Pr1 cells, suggesting that this stimulatory effect of GnRH occurs through GnRH receptor(s). To our knowledge, this is the first report showing the stimulatory effect of GnRH on a prostatic carcinoma cell line. Moreover, we also examined the effect of conditioned medium of TSU-Pr1 cells and found that it inhibited the GnRH activity only on TSU-Pr1 cells. This characteristic of the conditioned medium of TSU-Pr1 cells is different from that of HHUA or Jurkat cells described in our previous study. TSU-Pr1 cells the proliferation of which is stimulated by GnRH can yield important clues about the mechanisms of the effects of GnRH on cell proliferation.

Guinea pig gonadotropin-releasing hormone (gpGnRH) is predicted to have a unique structure among all known forms of GnRH molecule [Endocrinology 138 (1997) 4123] and it is of great interest to determine whether the unique structure of gpGnRH is manifested in the characteristics of the guinea pig GnRH receptor. In the present study, we isolated a full-length cDNA for a GnRH receptor from the pituitary gland of the guinea pig. The putative guinea pig GnRH receptor protein has an amino acid identity of 79–87% with mammalian type I GnRH receptors. The amino acid residues which have been demonstrated to be important for ligand binding and signal transduction were conserved in the guinea pig GnRH receptor. However, there are several specific amino acid substitutions among mammalian type I GnRH receptors. Moreover, though the guinea pig has generally been classified as a rodent, the putative GnRH receptor protein did not have some rodent-specific characteristics. Total IP assays demonstrated that the cloned guinea pig GnRH receptor is a functional GnRH receptor and that it shows different preference of ligand sensitivities from the rat GnRH receptor.

Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones function through their specific receptors, so the expression levels of the receptors may reflect the responsibility of target organs. However, there was no effective method to quantify the expression levels of these receptors in reptilian species. In this study, we established the competitive-PCR assay systems for the quantification of the mRNA expression levels of three sex steroid hormone receptors in the leopard gecko. These assay systems were successfully able to detect the mRNA expression level of each receptor in various organs of male adult leopard geckoes. The expression levels of mRNA of these receptors were highly various depending on the organs assayed. This is the first report regarding the tissue distributions of sex steroid hormone receptor expressions in reptile. The effects of environmental conditions on these hormone receptor expressions were also examined. After the low temperature and short photoperiod treatment for 6 weeks, only the androgen receptor expression was significantly increased in the testes. The competitive-PCR assay systems established in this report should be applicable for various studies of the molecular mechanism underlying the reproductive activity of the leopard gecko.

A number of studies have demonstrated that GnRH has anti-proliferative effects on various carcinomas of breast, ovary, endometrium, prostate, pancreas, and liver origin. In contrast, GnRH increases the proliferative activity of lymphoid tissues and cells, which suggests that GnRH is also an important immunomodulator. In a previous study, we demonstrated that the colony-forming efficiencies of HHUA (derived from human endometrial carcinoma) and Jurkat (derived from human mature leukemia) cells are affected by the GnRH agonist Buserelin, and that the conditioned media of HHUA and Jurkat cells severely affect the Buserelin activity. The latter finding suggests that substances in the culture medium have some relation to the GnRH activity. Therefore, in the present study, to evaluate the effect of serum supplements on the colony-forming efficiency assay, the assay was performed using 3 lots of fetal bovine serum (FBS) and 2 lots of Nu-Serum I, a semi-synthetic serum supplement. The results showed that the colony-forming efficiencies of HHUA and Jurkat cells fluctuated greatly depending on the lot of FBS. In contrast, Buserelin significantly affected the colony-forming efficiency to similar extents in the media containing both the lots of Nu-Serum I. These results strongly suggest that the constituents of the serum supplements also influence the effect of GnRH on cell proliferation. For further studies about the relationship between substances in the culture medium and the GnRH effects on cell proliferation, it will be necessary to use a completely defined medium, and that a semi-synthetic serum supplement such as Nu-Serum I will also be useful.

The transcription factor DMRT1 has important functions in two distinct processes, somatic-cell masculinization and germ-cell development in mammals. However, it is unknown whether the functions are conserved during evolution, and what mechanism underlies its expression in the two cell lineages. Our analysis of the Xenopus laevis and Silurana tropicalis dmrt1 genes indicated the presence of two distinct promoters: one upstream of the noncoding first exon (ncEx1), and one within the first intron. In contrast, only the ncEx1-upstream promoter was detected in the dmrt1 gene of the agnathan sand lamprey, which expressed dmrt1 exclusively in the germ cells. In X. laevis, the ncEx1- and exon 2-upstream promoters were predominantly used for germ-cell and somatic-cell transcription, respectively. Importantly, knockdown of the ncEx1-containing transcript led to reduced germ-cell numbers in X. laevis gonads. Intriguingly, two genetically female individuals carrying the knockdown construct developed testicles. Analysis of the reptilian leopard gecko dmrt1 revealed the absence of ncEx1. We propose that dmrt1 regulated germ-cell development in the vertebrate ancestor, then acquired another promoter in its first intron to regulate somatic-cell masculinization during gnathostome evolution. In the common ancestor of reptiles and mammals, only one promoter got function for both the two cell lineages, accompanied with the loss of ncEx1. In addition, we found a conserved noncoding sequence (CNS) in the dmrt1 5'-flanking regions only among amniote species, and two CNSs in the introns among most vertebrates except for agnathans. Finally, we discuss relationships between these CNSs and the promoters of dmrt1 during vertebrate evolution.

Recent studies have shown that gonadotropin-releasing hormone (GnRH) can exert various effects on the rat ovary by acting through its specific receptors. To determine the cell types responsive to the action of GnRH under physiological conditions in the ovary, distribution of the GnRH receptor mRNA was studied histologically by in situ hybridization in neonatal and adult rats. Expression of the luteinizing hormone receptor mRNA was also examined to judge the growing state of follicles and the corpora lutea. In neonatal rat ovaries, no significant GnRH receptor mRNA signal was detected until 5 days after birth. The expression was first observed at 10 days in the interstitial cells. At 15 days of age, the receptor mRNA was expressed in the granulosa cells of most preantral and early antral follicles, while no hybridization signal was detected in oocyte and theca cells. In adult cycling rats, GnRH receptor mRNA was detected mainly in the granulosa cells of most follicles and luteal cells. The granulosa cells of atretic follicles showed a very high level of the mRNA expression throughout their degenerating process. A strong hybridization signal was also detected in the mural granulosa cells of mature follicles. Newly formed (developing) corpora lutea exhibited signals with moderate intensity in the luteal cells, and the older ones showed weaker signals. The finding that the initial expression of GnRH receptor mRNA was seen in the interstitial cells of neonatal ovaries implies an unknown function of the ovarian GnRH receptor in ovarian development. The high level expression of GnRH receptor mRNA in atretic and mature follicles supports the putative roles of GnRH in the induction of follicular atresia and ovulation in rat ovaries.

Previously we reported that the experimental unilateral cryptorchidism altered the expression pattern of LH receptor mRNA in abdominal testis. To clarify whether the change was due to exposure of Leydig cells to high temperature in the abdomen, we examined the effect of unilateral efferent duct ligation on the expression. Two weeks after the operation, a relatively decreased expression of 1.8 kb transcript, increased expression of other transcripts, and degeneration of germ cells were observed in the efferent duct-ligated testes. As these changes were similar to those seen in abdominal testes of cryptorchid rats, exposure of Leydig cells to high temperature was not responsible for the changes in expression pattern of LH receptor mRNA. To examine the correlation between the changes in the receptor mRNA expression and the degeneration of seminiferous epithelium, expression of the mRNA was analyzed in unilaterally cryptorchid rats for 28 days after the operation. In scrotal testes which showed no hi...

We found that neuroblastoma × glioma hybrid NG108-15 cells accumulated lipofuscin-like autofluorescent materials during neuronal differentiation in culture in a medium containing 1% fetal calf serum, 1 mM dibutyryl cyclic AMP and 1 mM theophylline. The emission maximum of the lipofuscin-like autofluorescent materials was between 500 and 550 nm. Granules positive to acid phosphatase and periodic-acid Schiff were increased, as were the autofluorescent granules in NG108-15 cells. Thiolprotease inhibitors, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-4-aminobutylamide (E-64) and acetyl-Leu-Leu-Arg (leupeptin), markedly accelerated the accumulation of the lipofuscin-like autofluorescent materials in NG108-15 cells. On the other hand, activities of lysosomal thiolproteases, cathepsin B, C and L, were increased during neuronal differentiation. Protein content in the cells was gradually increased with the neuronal differentiation, and the rise was significantly accelerated when proteolysis was inhibited by E-64. These results suggest that the lipofuscin-like autofluorescent materials contain peptidic substances as a component, and indicate that the increase in hydrolytic activities of thiolproteases during neuronal differentiation is not enough for the hydrolysis of peptidic substrates, resulting in the accumulation of autofluorescent materials in NG108-15 cells.

Ontogeny and cellular localization of the lutropin receptor (LH-R) mRNA were studied in fowl gonads between day 5 of incubation and day 1 of posthatch. In situ hybridization using an antisense cRNA probe demonstrated that LH-R gene expression began at embryonic day 7 in females and day 14 in males. In female embryos, the specific hybridization was detected in not only developing left ovaries but also regressing right ovaries. The LH-R mRNA expression in the right ovary was detectable until hatch. LH-R mRNA was localized in the medullary cords in ovaries and interstitial tissues in testes. These results are consistent with previous reports on ontogenic analysis of LH-dependent steroidogenesis in chicken embryo gonads.

Components of the uterine fluid in mice with experimentally-induced adenomyosis and in controls were examined by proton nuclear magnetic resonance spectroscopy. One of the components was markedly different in mice with adenomyosis. As this component was estimated to be citrate by comparison with authentic samples (standard spectrum), its levels in uterine fluid, uterine tissue and blood were determined by enzymatic analysis. The fluid obtained from the uterus with adenomyosis showed significantly lower concentration of citrate than that from normal uterus. However, the uterine tissue concentration was significantly higher in the experimental mice with adenomyosis. There was no difference in the blood citrate level between the experimental and the control groups. Since adenomyosis was induced by chronic hyperprolactinemia, the change of citrate level in the uterus with this lesion might imply some effects of prolactin (PRL) on metabolism and/or secretion of citrate. However, in normal mice, no significant change was demonstrated in uterine citrate concentration after short-term experimental modulation of the circulating PRL level. Thus, it is unlikely that PRL can regulate directly citrate metabolism in the uterus, indicating some other cause for changes in citrate level accompanying the development of adenomyosis.

Matsuda M, Mori T, Park MK, Kawashima S. Modification of pancreatic digestive function by pituitary grafting in mice. Eur J Endocrinol 1995;133:221–6. ISSN 0804–4643 The effect of prolactin on the digestive potency of the acinar pancreas was examined in pituitary-grafted hyperprolactinemic mice, because our previous experiment showed that a marked proliferation of pancreatic acinar cells was induced by pituitary grafting in mice. To know whether the digestive function is modified, the tissue contents of pancreatic digestive enzymes, such as chymotrypsin, lipase, α-amylase and ribonuclease, were measured in the hyperprolactinemic mice. Pituitary grafting significantly increased the contents of chymotrypsin and lipase in the pancreas on day 12 after the operation without affecting intake of food, when compared to those in the sham-operated controls. On day 30, however, the differences between pituitary-grafted and control mice were no more discernible. Thus, the digestive enzyme activities are easily modified soon after the increase of circulating prolactin level. This effect of prolactin on the function of the pancreas may be responsible for "homeorhetic" control of nutrients during lactation. In another set of experiments in adrenalectomized–castrated or castrated mice, pituitary grafting induced an increase in the weight of the pancreas. In addition, adrenalectomy in combination with castration did not alter the pancreatic contents of chymotrypsin and lipase but decreased the amylase content. These results taken together seem to indicate that the effect of prolactin on the exocrine pancreas is not mediated by gonadal and adrenal steroid hormones. Manabu Matsuda, Zoological Institute, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Lipofuscin is the autofluorescent material, which accumulates with aging in the cells of various tissues. However, its autofluorescence characteristics are different among tissues. In the present study, the autofluorescence features of lipofuscin in the brain and adrenal were compared. In 18-21-month-old rats, the brain lipofuscin was granular and its autofluorescence was bright whitish-yellow to bright orange. On the contrary, the adrenal lipofuscin was not demarkated as granules, and its autofluorescence was subdued orange. The emission maximum of the bright whitish-yellow brain lipofuscin was 540 nm to 570 nm and that of the adrenal lipofuscin was 640 nm to 660 nm, when excited at 330 nm to 380 nm. When the spectra were drawn after correcting the wavelength-dependent bias of microspectrofluorometer, the autofluorescence spectra were consistent with microscopically observable tint. To conclude, the present results showed that the autofluorescence features of the bright whitish-yellow brain lipofuscin and the adrenal lipofuscin were quite different.

To clarify the target cells of GnRH in the ovary, in vivo expression of ovarian GnRH receptor mRNA was examined histologically by in situ hybridization in immature rats treated with PMSG only or in combination with hCG. Strong hybridization signals were observed in the granulosa cells of atretic follicles. However, no significant signals were found in the granulosa cells of healthy small, preantral, or early antral follicles. Healthy Graafian and preovulatory follicles also showed intense signals in their mural granulosa cells, but no signals were detected in the cumulus oophorus cells. Corpora lutea showed only weak signals, but luteinizing follicles probably after atresia exhibited signals of moderate intensity in their luteinized and remaining granulosa cells. No signals were detected in the theca cells and oocytes in all the follicles. Interstitial cells sometimes exhibited hybridization signals of moderate intensity, when the cells were eosinophilic. Pretreatment with different combinations of gonadotropins yielded different ovarian histology, but this had no influence on the localization of hybridization signals. These results, showing that the authentic GnRH receptor mRNA was expressed in a certain cell population in the rat ovary, suggest that the receptor is involved in the control of various ovarian functions including follicular development, atresia, ovulation, and luteinization after ovulation and follicular atresia.

GnRH was originally identified as a hypothalamic factor which promotes gonadotropin release from the pituitary and was named gonadotropin-releasing hormone (GnRH). However, broad tissue distributions of GnRH and the GnRH receptor in various extrapituitary tissues and organs have been revealed and it has been suggested that GnRH has extrapituitary effects such as neuromodulation, immunomodulation, and regulation of follicular atresia and ovulation. Although a number of studies have been performed on these effects, little is known about the molecular mechanisms and physiological settings in which GnRH exerts its activities in extrapituitary organs or tissues. Our recent studies had demonstrated that GnRH is able to regulate both cell proliferation and cell migration at much lower concentration than that in the peripheral circulation by using human carcinoma cell lines. Moreover, stimulating activity of GnRH on the developing chick embryonic GnRH neurons was also demonstrated and strongly suggests possible involvement of GnRH in some of extrapituitary functions. This mini-review intends to provide solid evidence of GnRH activity in the regulation of cell proliferation and migration and its physiological relevance in extra-pituitary functions. Recent other research, including that in various invertebrates, provides new insight into the evolutionary scenarios of GnRH signaling systems, and GnRH functions. Both proliferating and migrating activities are important fundamental cellular activities and could provide an important clue into understanding what the driving force behind the evolution of the GnRH signaling system was.

Gonadotropin-releasing hormone (GnRH) is a neuroendocrine peptide that plays a central role in the vertebrate hypothalamo-pituitary axis. The roles of GnRH in the control of vertebrate reproductive functions have been established, while its non-reproductive function has been suggested but less well understood. Here we show that the tunicate Ciona intestinalis has in its non-reproductive larval stage a prominent GnRH system spanning the entire length of the nervous system. Tunicate GnRH receptors are phylogenetically closest to vertebrate GnRH receptors, yet functional analysis of the receptors revealed that these simple chordates have evolved a unique GnRH system with multiple ligands and receptor heterodimerization enabling complex regulation. One of the gnrh genes is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord of vertebrates. Correspondingly, GnRH receptor genes were found to be expressed in the tail muscle and notochord of embryos, both of which are phylotypic axial structures along the nerve cord. Our findings suggest a novel non-reproductive role of GnRH in tunicates. Furthermore, we present evidence that GnRH-producing cells are present in the hindbrain and spinal cord of the medaka, Oryzias latipes, thereby suggesting the deep evolutionary origin of a non-reproductive GnRH system in chordates.

Matsuda M, Mori T, Park MK, Yanaihara N, Kawashima S. Enhanced cell proliferation by hyperprolactinemia in both exocrine and endocrine pancreas in mice. Eur J Endocrinol 1994;130:187–94. ISSN 0804–4643 Effects of hyperprolactinemia induced by ectopic anterior pituitary grafting on the pancreas were studied in male SHN mice. After pituitary grafting, the weight of pancreas rapidly increased. A similar increase in pancreatic weight was observed during lactation, a condition associated with elevated prolactin levels. Results of DNA and protein assays revealed that the increase in pancreatic weight in both pituitary-grafted and lactating mice was mainly due to the increase in the cell number, because the total DNA content per pancreas was greater in these mice than the controls. An increase in fluid volume or hypertrophy of cells also contributes to the weight increase; in contrast, the DNA and protein contents per unit tissue weight decrease. The rate of DNA synthesis determined by 5-bromo-2′-deoxyuridine labeling was higher both in acinar cells and islet B cells in pituitary-grafted mice han in the controls. Thus, hyperprolactinemia stimulates cell proliferation in exocrine pancreas as well as endocrine islets. The effect of prolactin seems to be indirect on acinar cells, because only B cells showed prolactin immunoreactivity in the mouse pancreas. In addition, insulin might not be a mediator of the prolactin effect on acinar cells, because the serum insulin level in pituitary-grafted mice failed to show any change. Manabu Matsuda, Zoological Institute, School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Expression of rat GnRH receptor mRNA in female rat pituitary was examined by in situ hybridization using 35S-labeled antisense RNA probe. Specific signals were detected in the pars distalis. For detailed analysis of the cellular expression of GnRH receptor mRNA, the pituitaries were double-stained by in situ hybridization of GnRH receptor mRNA and antisera against some pituitary hormones (LH, FSH, TSH and prolactin). The expression of GnRH receptor mRNA was detected in all the immunoreactive gonadotropes, but was not detectable in TSH or prolactin cells. Cellular expression of GnRH receptor mRNA in immunoreactive gonadotropes was compared during the estrous cycle and after ovariectomy. In the pituitary of normal cycling rats, the mean value of the cellular expression of the mRNA in estrus was significantly smaller than those of other phases of the cycle. In ovariectomized rats, the cellular expression was significantly in-creased 4 days after the operation as compared with normal cycling rats, and the elevated level of cellular expression was maintained until 21 days after ovariectomy. These results suggest that GnRH receptor expression in the pituitary is preferentially localized in gonadotropes, and the extent of cellular expression varies during the estrous cycle and after ovariectomy.

Zing finger AN1-type domain 3 (ZFAND3), also known as testis expressed sequence 27 (Tex27), is a gene found in the mouse testis, but its physiological function is unknown. We identified the full-length sequences of two isoforms (short and long) of ZFAND3 cDNA from Japanese quail and leopard gecko. This is the first cloning of avian and reptilian ZFAND3 cDNA. The two isoforms are generated by alternative polyadenylation in the 3'UTR and have the same ORF sequences encoding identical proteins. There were highly conserved regions in the 3'UTR of the long form near the polyadenylation sites from mammals to amphibians, suggesting that the features for determining the stability of mRNA or translation efficiency differ between isoforms. The deduced amino acid sequence of ZFAND3 has two putative zinc finger domains, an A20-like zinc finger domain at the N-terminal and an AN1-like zinc finger domain at the C-terminal. Sequence analysis revealed an additional exon in the genomic structures of the avian and reptilian ZFAND3 genes which is not present in mammals, amphibians, or fish, and this exon produces additional amino acid residues in the A20-like zinc finger domain. Expression analysis in Japanese quail revealed that the expression level of ZFAND3 mRNA was high in not only the testis but also the ovary, and ZFAND3 mRNA was expressed in both spermatides of the testis and oocytes of the ovary. While the short form mRNA was mainly expressed in the testis, the expression level of the long form mRNA was high in the ovary. These results suggest that ZFAND3 has physiological functions related to germ cell maturation and regulatory mechanisms that differ between the testis and ovary.

Prolyl endopeptidase and dipeptidyl peptidase IV are proline-specific peptidases, and are ubiquitously distributed in various tissues in mammals. The specific activities of these peptidases in both uterus and ovary were examined in the SHN strain of mice at estrus or diestrus. A marked change in prolyl endopeptidase activity was found in the uterus and ovary in intact mice during the estrous cycle, the activity being high at estrus and low at diestrus. In ovariectomized mice, prolyl endopeptidase activity was significantly higher in the uterus treated with progesterone or estradiol than in the uterus treated with vehicle oil only or a dopamine antagonist (perphenazine) which stimulates prolactin secretion. On the other hand, notable change in dipeptidyl peptidase IV activity during the estrous cycle was found only in the uterus of intact mice. The activity was low at estrus and high at diestrus. In ovariectomized mice, the uterus exposed to estradiol showed a lower dipeptidyl peptidase IV activity than the uteri treated with progesterone, the dopamine antagonist or vehicle oil. These findings reveal that there is a close correlation between the circulating level of ovarian steroids and the activities of these prolinespecific enzymes.

Inhibitory effects of danazol, an isoxazol derivative of synthetic steroid 17 alpha-ethinyl-testosterone, on the development of uterine adenomyosis, a pathological disorder of endometrial tissue defined as the presence of endometrial glands and stroma in the myometrium, were investigated in mice of SHN strain. Mice treated with 0.5 microgram danazol for 5 weeks during 4-9 weeks of age and killed at 21 weeks of age showed significantly lower incidence of the spontaneous development of adenomyosis than the age-matched intact control mice. The inhibitory effects of danazol were also evident in mice bearing pituitary isografts which were effective in inducing an early and a high incidence of adenomyosis. Furthermore, the treatment with danazol resulted in the decrease of serum levels of luteinizing hormone (LH) and prolactin (PRL) associated with hypofunction of ovaries and persistent diestrus. These results support the usefulness of danazol for the clinical treatment of gynecological disorders except for hypofunction of ovaries.

The ontogeny of gonadotrophs and thyrotrophs in the bullfrog pituitary was examined immunohistochemically using monoclonal antibodies against bullfrog lutropin beta-subunit (LH beta), follitropin beta-subunit (FSH beta) and its alpha-subunit, and polyclonal anti-human thyrotropin beta-subunit (TSH beta) serum. Immunoreactive alpha-subunit- and TSH beta-, but not FSH beta- and LH beta-containing cells were observed at embryonic stage 24 (Shumway's classification). Immunoreactive FSH beta cells first appeared at Taylor-Korllos stage V, and immunoreactive LH beta cells at stage X. Throughout metamorphosis, several gonadotrophs containing both FSH and LH were found in the ventrocaudal region, but most gonadotrophs contained only FSH. Immunoreactive alpha-subunit cells were always more frequent than the sum of immunoreactive beta-subunit cells, which was confirmed by quantitative studies using immunohistochemical and RIA techniques.

Transport of large proteins into the nucleus requires both a nuclear localization signal (NLS) and exposure of that signal to components of the transport machinery. In this report, polyclonal and monoclonal antibodies were generated against the NLS of SV40 large T antigen. Several of these antibodies immunoprecipitated large T antigen produced by in vitro transcription-translation and recognized T antigen expressed in cultured cells. Binding of the antibodies to T antigen was quantified using an indirect radioimmunoassay and found to be specifically inhibited by peptides corresponding to the T antigen NLS. The ability of NLS-specific antibodies to recognize large T antigen suggests that the NLS is exposed on the surface of T antigen. When one of the NLS-specific monoclonal antibodies was introduced into the cytoplasm of cells expressing T antigen, the antibody remained cytoplasmic. These results suggested either that cytoplasmic components compete for binding to the NLS or that the antibody dissociates from T antigen during transport into the nucleus. When an antibody directed against an epitope distinct from the NLS was microinjected into the cytoplasm of cells expressing large T antigen, both the antibody and antigen were transported into the nucleus. The observed stability of the antigen-antibody complex strongly suggest protein unfolding is not required for nuclear protein transport.

The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey Kidney COS-1 cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-1 cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-1 cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to express p62 in tissue culture cells will facilitate analysis of the role of this pore protein in nuclear transport.

Using beta and alpha subunits of bullfrog follitropin (FSH) III (pI 6.2), which were highly purified by HPLC, we generated three monoclonal antibodies (MCAs) to FSH beta subunit (FSH beta) and six to FSH alpha subunit (FSH alpha). They were produced by hybridomas derived from the myeloma X63.Ag8.653 and spleen lymphocytes from mice immunized with each subunit. Non-competitive binding tests revealed that one of the MCAs against FSH beta (BF3B25) bound strongly to intact FSH and its beta subunit, but not FSH alpha, lutropin (LH), LH alpha, and LH beta. The immunoblotting results also showed a similar immunological specificity for BF3B25. Cross-reactivity of bullfrog FSH against BF3B25 was 19.4%, when compared with FSH beta in the competitive inhibition assay system. On the other hand, noncompetitive binding tests and immunoblotting results showed that one of the MCAs against FSH alpha (BF3A20) bound strongly to intact LH and FSH and their alpha subunits, but not their beta subunits. The inhibition curves obtained using the alpha subunits of LH and FSH were similar. In the sexually mature bullfrog pituitary, immunoreactive FSH cells stained with MCA BF3B25 were distributed throughout the pars distalis, except for the rostral region, and were polygonal in shape, with well-developed cytoplasm. With respect to distribution and histological characteristics, the immunoreactive LH cells were very similar to the immunoreactive FSH cells when consecutive sections were stained with LH beta-specific MCA (BL4B11). However, immunoreactive TSH cells, revealed by anti-human TSH beta serum, formed clusters in the ventrocentral region of the pars distalis. In young adult pituitary, almost all of the gonadotrophs showed the coexistence of FSH and LH, but some gonadotrophs contained only FSH. The number of immunoreactive alpha-subunit cells stained by BF3A20 was always higher than the sum of the numbers of cells stained by the three beta-subunit-specific antibodies.

Gonadotophin-releasing hormone (GnRH) peptide released from the terminal nerve (TN)-GnRH neurones of the dwarf gourami primarily modifies the electrical properties of various neurones, including the TN-GnRH neurones themselves. However, our knowledge on the expression of GnRH receptors (GnRHRs) in the TN-GnRH neurones is still limited. Here, we used the single-cell reverse transcriptase-polymerase chain reaction after whole-cell patch-clamp recording to study the distribution of various GnRHR types expressed in the individual TN-GnRH neurones. We found that TN-GnRH neurones express two of the three types of GnRHRs cloned in the dwarf gourami: GnRHR1-2 and -R2, but not -R1-1. Furthermore, in agreement with our previous findings, all TN-GnRH neurones contained mRNAs of salmon GnRH but not chicken GnRH-II.