Neuroendocrinology of Reproduction and Social Behaviors in Reptiles: Advances Made in the Last Decade.

Among amniotes, reptiles are ectothermic and are clearly distinguished from mammals and birds. Reptiles show great diversity not only in species numbers, but also in ecological and physiological features. Although their physiological diversity is an interesting research topic, less effort has been made compared to that for mammals and birds, in part due to lack of established experimental models and techniques. However, progress, especially in the field of neuroendocrinology, has been steadily made. With this process, basic data on selected reptilian species have been collected. This review article presents the progress made in the last decade, which includes 1) behavioral regulation by sex steroid hormones, 2) regulation of seasonal reproduction by melatonin and GnRH, and 3) regulation of social interaction by arginine vasotocin. Through these research topics, we provide insights into the physiology of reptiles and the latest findings in the field of amniote neuroendocrinology.

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.

Characterization of G protein-coupled estrogen receptors in Japanese medaka, Oryzias latipes.

The G protein-coupled estrogen receptor 1 (Gper1) is a membrane-bound estrogen receptor that mediates non-genomic action of estrogens. A Gper1-mediating pathway has been implicated in reproductive activities in fish, including oocyte growth, but Gper1 has been characterized in only a very limited number of fish species. In this study, we cloned and characterized two genes encoding medaka (Oryzias latipes) Gper1s, namely, Gper1a and Gper1b, and phylogenic and synteny analyses suggest that these genes originate through a teleost-specific whole genome duplication event. We found that Gper1a induced phosphorylation of mitogen-activated protein kinase (MAPK) in 293T cells transfected with medaka Gper1s on exposure to the natural estrogen, 17ß-estradiol (E2) and a synthetic Gper1 agonist (G-1), and treatment with both E2 and G-1 also decreased the rate of spontaneous maturation in medaka oocytes. These findings show that the processes for oocyte growth and maturation are sensitive to estrogens and are possibly mediated through Gper1a in medaka. We also show that 17α-ethinylestradiol (EE2), one of the most potent estrogenic endocrine-disrupting chemicals, and bisphenol A (BPA, a weak environmental estrogen) augmented phosphorylation of MAPK through medaka Gper1s in 293T cells. Interestingly, however, treatment with EE2 or BPA did not attenuate maturation of medaka oocytes. Our findings support that Gper1-mediated effects on oocytes are conserved among fish species, but effects of estrogenic endocrine-disrupting chemicals on oocytes acting through Gper1 may be divergent among fish species.

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.

Chronology of embryonic and gonadal development in the Reeves' turtle, Mauremys reevesii.

Temperature-dependent sex determination (TSD) is a mechanism in which environmental temperature, rather than innate zygotic genotype, determines the fate of sexual differentiation during embryonic development. Reeves' turtle (also known as the Chinese three-keeled pond turtle, Mauremys reevesii) exhibits TSD and is the only species whose genome has been determined in Geoemydidae to date. Thus, M. reevesii occupy phylogenetically important position for the study of TSD and can be compared to other TSD species to elucidate the underlying molecular mechanism of this process. Nevertheless, neither embryogenesis nor gonadogenesis has been described in this species. Therefore, herein, we investigated the chronology of normal embryonic development and gonadal structures in M. reevesii under both female- and male-producing incubation temperatures (FPT 31 °C or MPT 26 °C, respectively). External morphology remains indistinct between the two temperature regimes throughout the studied embryonic stages. However, the gonadal ridges present on the mesonephros at stage 16 develop and sexually differentiate at FPT and MPT. Ovarian and testicular structures begin to develop at stages 18-19 at FPT and stages 20-21 at MPT, respectively, and thus, the sexual differentiation of gonadal structures began earlier in the embryos at FPT than at MPT. Our results suggest that temperature sensitive period, at which the gonadal structures remain sexually undifferentiated, spans from stage 16 (or earlier) to stages 18-19 at FPT and to stages 20-21 at MPT. Understanding the temperature-dependent differentiation in gonadal structures during embryonic development is a prerequisite for investigating molecular basis underlying TSD. Thus, the result of the present study will facilitate further developmental studies on TSD in M. reevesii.

Estrogen Receptor 1 (ESR1) Agonist Induces Ovarian Differentiation and Aberrant Müllerian Duct Development in the Chinese Soft-shelled Turtle, Pelodiscus sinensi.

Estrogens play critical roles in ovarian and reproductive organ development, but the molecular signaling pathways in non-mammalian vertebrates are not well understood. Studies of reptiles have indicated that administration of exogenous estrogens during embryonic development causes ovarian differentiation and presumptive male to female sex-reversal. The Chinese soft-shelled turtle, Pelodiscus sinensis, belongs to the family Trionychidae and exhibits genotypic sex determination system with ZZ/ZW sex chromosomes. In order to assess the role of estrogens and their signaling pathway on sex determination and differentiation, P. sinensis eggs were given a single administration of endogenous estrogen,17ß-estradiol (E2) or a synthetic estrogen receptor 1 (ESR1) agonist, 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) in ovo during gonadal differentiation, and the subsequent effects were examined during a final developmental stage prior to hatching. The administration of both E2 and PPT induced ovarian differentiation in genetic male embryos. Intriguingly, PPT but not E2 induced the Müllerian duct enlargement and aberrant glandular development. These data suggest that ovarian differentiation and reproductive tract anomalies induced by the exogenous estrogen exposure act through ESR1 in the Chinese soft-shelled turtles.

Controlled Cre/loxP site-specific recombination in the developing brain in medaka fish, Oryzias latipes.

BACKGROUND: Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain. METHODOLOGY/PRINCIPAL FINDINGS: To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0-1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2-3 dpf embyos compared with 0-1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish. CONCLUSIONS/SIGNIFICANCE: We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.