Phylogeny of g6pc1 Genes and Their Functional Divergence among Sarcopterygian Vertebrates: Implications for Thermoregulatory Strategies.

Glucose-6-phosphatase catalytic subunit 1 (G6PC1) catalyzes the final rate-limiting step in endogenous glucose production and is critically important for glucose homeostasis. Although a single g6pc1 gene is present in mammals, other vertebrates possess two to five paralogs. Functional divergence between paralogs has been reported in actinopterygians and has been implicated in the acquisition of adaptive characteristics. Such reports make sarcopterygian g6pc1 an interesting research topic because unlike the aquatic habitat of actinopterygians, sarcopterygians have successfully adapted to terrestrial environments. However, little is known about the evolution of sarcopterygian g6pc1. In the present study, the evolutionary history of sarcopterygian g6pc1 was investigated using molecular phylogeny, synteny analyses, and comparison of the genomic environment. Functional divergence between paralogs was also investigated in a reptilian species, the Japanese gecko, with a focus on gene expression in the liver. Evolutionary analyses suggested that amphibians and amniotes acquired duplicated genes independently. Among the amniotes, gene duplication occurred at the root of the reptilian-avian lineage, giving rise to g6pc1-1 and g6pc1-2 classes. While the avian lineage subsequently lost the g6pc1-1, the reptiles retained both classes. This co-occurrence of gene loss and endothermy acquisition, together with the observation that mammals possess only a single gene, suggests that the duplicated g6pc1 is dispensable for endotherms. Quantitative RT-PCR analyses revealed that the two gecko genes respond differently to E2 administration, as the expression of g6pc1-1 was downregulated by E2, whereas g6pc1-2 showed no significant response. Such paralog-specific responses suggest functional divergence between paralogs, which is possibly related to reproduction.

Molecular Evolution of Two Distinct dmrt1 Promoters for Germ and Somatic Cells in Vertebrate Gonads.

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.

Expressional changes of AMH signaling system in the quail testis induced by photoperiod.

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 promote AMH expression in mammals (SF1, SOX9, WT1 and GATA4) 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.

Molecular characterization of insulin from squamate reptiles reveals sequence diversity and possible adaptive evolution.

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.

Regulation of the cell proliferation and migration as extra-pituitary functions of GnRH.

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.

Neurotrophic effect of gonadotropin-releasing hormone on neurite extension and neuronal migration of embryonic gonadotropin-releasing hormone neurons in chick olfactory nerve bundle culture.

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.

Identification and expression analysis of peroxisome proliferator-activated receptors cDNA in a reptile, the leopard gecko (Eublepharis macularius).

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.

Molecular identification of single hormone-encoding proglucagon cDNA isoforms from squamates and their abundant expression.

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.

Comparative analysis of the pituitary and ovarian GnRH systems in the leopard gecko: signaling crosstalk between multiple receptor subtypes in ovarian follicles.

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.

Sex difference in Ad4BP/SF-1 mRNA expression in the chick-embryo brain before gonadal sexual differentiation.

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.

A system for receptor functional analysis based on c-fos mRNA expression: analysis of GnRH receptors as a test system.

This report describes the establishment of a system for assessing receptor activation by RT-PCR-based detection of c-fos mRNA induction. In this system, COS-7 cells were transiently transfected with GnRH receptor expression plasmid, and ligand-induced c-fos expression was quantified by the RT-competitive PCR method. The results were compared with those of a conventional inositol phosphate (IP) assay. Changes in c-fos expression levels were observed in a dose- and ligand-dependent manner. Similar tendencies were observed in ligand selectivity between c-fos expression and IP production. The novel system developed and established in the present study is sensitive by using RT-PCR and convenient because it requires only basic methods of cell culture and molecular biology. It also has the merit that it does not need any specific measuring devices or radioactive substances. Given the ability of c-fos to respond to diverse stimuli, the present system may be applicable for various receptors for bioactive substances in addition to GnRH receptor, and useful for various purposes including screening ligands for orphan receptors.

Gonadotropin-releasing hormone induces actin cytoskeleton remodeling and affects cell migration in a cell-type-specific manner in TSU-Pr1 and DU145 cells.

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.

Molecular and evolutionary characterization of the GnRH-II gene in the chicken: distinctive genomic organization, expression pattern, and precursor sequence.

Of all the structural variants of GnRH (gonadotropin-releasing hormone), GnRH-II has been found to be universally present in and uniquely conserved among jawed vertebrates without any sequence substitutions. Our previous study found that the GnRH-II precursor sequences have become divergent in the lineage of eutherian mammals, based on a comparison between reptilian and mammalian GnRH-II. To elucidate the molecular evolution of GnRH-II throughout amniotes, we have performed the first identification of the avian GnRH-II cDNA/gene from the chicken, the species used for the initial discovery of GnRH-II peptide. Gene arrangement around the GnRH-II in the chicken was similar to that in mammals; however, a gene MRPS26 was partly overlapped with the downstream part of the GnRH-II in the chicken. It was identified that the GnRH-II/MRPS26 locus generated at least five distinct types of transcripts with different expression patterns and three of them may produce functional GnRH-II decapeptide. Sequence comparison revealed that the prepro-GnRH-II polypeptide of the chicken was substantially different from those of other species regarding the length and similarity. The present results strongly indicated that considerable variations were generated in the precursor sequence of the evolutionarily conserved GnRH-II during amniote evolution. It was also suggested that the sequence divergence seen in the chicken may have occurred independently of that in the mammalian lineage.

Molecular characterization of thyroid hormone receptors from the leopard gecko, and their differential expression in the skin.

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.

Identification of the reptilian prolactin and its receptor cDNAs in the leopard gecko, Eublepharis macularius.

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.

The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella.

The gene product of EFHC1 recently implicated in juvenile myoclonic epilepsy (JME) was found to be a homolog of Chlamydomonas axonemal protein Rib72, whose homologs are present in a wide variety of organisms that have motile cilia and flagella. Western blot analyses and immunofluorescence localization of the mouse ortholog mRib72-1/Efhc1 indicated that it is indeed abundantly present in sperm flagella and tracheal cilia but only in a small amount in the brain. It is not present in immotile primary cilia. These observations raise the possibility that malfunction of motile cilia is involved in the development of JME.

Molecular cloning of P450 aromatase from the leopard gecko and its expression in the ovary.

In this study, we identified the cDNA of P450 aromatase in the leopard gecko, a lizard with temperature-dependent sex determination. The cDNA encodes a putative protein of 505 amino acids. The deduced amino acid sequence of leopard gecko aromatase cDNA showed 80% identity with that of turtles, 70% with humans and 77% with chickens. This is the first report of the identification of P450 aromatase cDNA in squamata species. It has been reported that this gene is expressed in different layers of cells in the ovary of mammalian species and avian species. Thus, we also investigated cells expressing the mRNA of this gene in the ovary of the leopard gecko by RT-PCR and in situ hybridization. The mRNA expression of leopard gecko P450 aromatase was localized in both the thecal and granulosa cell layers in the ovary. The expression in thecal and granulosa cell layers was examined in the largest follicle, second largest follicle and third largest follicle by RT-PCR. A higher level of mRNA expression was observed in the granulosa cell layer of the second largest follicle than in other cell layers. This result may reflect the characteristics of follicles in species with automonochronic ovulation.

Identification and characterization of the reptilian GnRH-II gene in the leopard gecko, Eublepharis macularius, and its evolutionary considerations.

To elucidate the molecular phylogeny and evolution of a particular peptide, one must analyze not the limited primary amino acid sequences of the low molecular weight mature polypeptide, but rather the sequences of the corresponding precursors from various species. Of all the structural variants of gonadotropin-releasing hormone (GnRH), GnRH-II (chicken GnRH-II, or cGnRH-II) is remarkably conserved without any sequence substitutions among vertebrates, but its precursor sequences vary considerably. We have identified and characterized the full-length complementary DNA (cDNA) encoding the GnRH-II precursor and determined its genomic structure, consisting of four exons and three introns, in a reptilian species, the leopard gecko Eublepharis macularius. This is the first report about the GnRH-II precursor cDNA/gene from reptiles. The deduced leopard gecko prepro-GnRH-II polypeptide had the highest identities with the corresponding polypeptides of amphibians. The GnRH-II precursor mRNA was detected in more than half of the tissues and organs examined. This widespread expression is consistent with the previous findings in several species, though the roles of GnRH outside the hypothalamus-pituitary-gonadal axis remain largely unknown. Molecular phylogenetic analysis combined with sequence comparison showed that the leopard gecko is more similar to fishes and amphibians than to eutherian mammals with respect to the GnRH-II precursor sequence. These results strongly suggest that the divergence of the GnRH-II precursor sequences seen in eutherian mammals may have occurred along with amniote evolution.

Proliferation of TSU-Pr1, a human prostatic carcinoma cell line is stimulated by gonadotropin-releasing hormone.

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.

GnRH as a cell proliferation regulator: mechanism of action and evolutionary implications.

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.