The central roles of luteinizing hormone (LH), progestin and their receptors for initiating ovulation have been well established. However, signaling pathways and downstream targets such as proteases that are essential for the rupture of follicular cells are still unclear. Recently, we found anovulation in nuclear progestin receptor (Pgr) knockout (Pgr-KO) zebrafish, which offers a new model for examining genes and pathways that are important for ovulation and fertility. In this study, we examined expression of all transcripts using RNA-Seq in preovulatory follicular cells collected following the final oocyte maturation, but prior to ovulation, from wild-type (WT) or Pgr-KO fish. Differential expression analysis revealed 3567 genes significantly differentially expressed between WT and Pgr-KO fish (fold change⩾2, p<0.05). Among those, 1543 gene transcripts were significantly more expressed, while 2024 genes were significantly less expressed, in WT than those in Pgr-KO. We then retrieved and compared transcriptional data from online databases and further identified 661 conserved genes in fish, mice, and humans that showed similar levels of high (283 genes) or low (387) expression in animals that were ovulating compared to those with no ovulation. For the first time, ovulatory genes and their involved biological processes and pathways were also visualized using Enrichment Map and Cytoscape. Intriguingly, enrichment analysis indicated that the genes with higher expression were involved in multiple ovulatory pathways and processes such as inflammatory response, angiogenesis, cytokine production, cell migration, chemotaxis, MAPK, focal adhesion, and cytoskeleton reorganization. In contrast, the genes with lower expression were mainly involved in DNA replication, DNA repair, DNA methylation, RNA processing, telomere maintenance, spindle assembling, nuclear acid transport, catabolic processes, and nuclear and cell division. Our results indicate that a large set of genes (>3000) is differentially regulated in the follicular cells in zebrafish prior to ovulation, terminating programs such as growth and proliferation, and beginning processes including the inflammatory response and apoptosis. Further studies are required to establish relationships among these genes and an ovulatory circuit in the zebrafish model.

The study of the evolutionary history of genes related to human disease lies at the interface of evolution and medicine. These studies provide the evolutionary context on which medical researchers should work, and are also useful in providing information to suggest further genetic experiments, especially in model species where genetic manipulations can be made. Here we studied the evolution of the β-adrenoreceptor gene family in vertebrates with the aim of adding an evolutionary framework to the already abundant physiological information. Our results show that in addition to the three already described vertebrate β-adrenoreceptor genes there is an additional group containing cyclostome sequences. We suggest that β-adrenoreceptors diversified as a product of the two whole genome duplications that occurred in the ancestor of vertebrates. Gene expression patterns are in general consistent across species, suggesting that expression dynamics were established early in the evolutionary history of vertebrates, and have been maintained since then. Finally, amino acid polymorphisms that are associated to pathological conditions in humans appear to be common in non-human mammals, suggesting that the phenotypic effects of these mutations depend on epistatic interaction with other positions. The evolutionary analysis of the β-adrenoreceptors delivers new insights about the diversity of these receptors in vertebrates, the evolution of the expression patterns and a comparative perspective regarding the polymorphisms that in humans are linked to pathological conditions.

G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent productive drug targets. Orphan GPCRs, which have unknown endogenous ligands, are considered drug targets and consequently have attracted great interest in identifying their endogenous cognate ligands for deorphanization. However, additional studies have shown that GPCRs, including many orphan GPCRs, can constitutively activate G protein signaling in a ligand-independent manner. GPR39 is such an orphan GPCR with constitutive activity. Here, we performed a phylogenetic and selection analysis of GPR39 in vertebrates, and we found that GPR39 underwent positive selection in different branches of vertebrates. Using luciferase reporter assays, we demonstrated that human, frog and chicken GPR39 can constitutively activate Gq and G12 signaling pathways in a ligand-independent manner. Zebrafish GPR39 can constitutively activate Gs, Gq and G12 signaling pathways in a ligand-independent manner. We further found that the zebrafish-H2967.35 site is crucial for the activity of the Gs signaling pathway. In addition, our mutagenesis studies indicated that the positive selection sites of GPR39 from different species had important effects on the constitutive activity of the receptor. Our results revealed the adaptive evolution of GPR39 in diverse directions, which led to differences in constitutive activity.

This article will review current knowledge on CXCR in fish, that represent three distinct vertebrate groups: Agnatha (jawless fishes), Chondrichthyes (cartilaginous fishes) and Osteichthyes (bony fishes). With the sequencing of many fish genomes, information on CXCR in these species in particular has expanded considerably. In mammals, 6 CXCRs have been described, and their homologues will be initially reviewed before considering a number of atypical CXCRs and a discussion of CXCR evolution.

Vasoinhibin is a pleiotropic protein hormone with endocrine, autocrine, and paracrine effects on blood vessel growth, permeability, and dilation, and a role in several human diseases. It is generated by proteolytic cleavage of the pituitary hormone prolactin by cathepsin D. Several isoforms with a variation in the number of amino acids and corresponding molecular mass exist. This in silico study investigated the cathepsin D cleavage sites in prolactin responsible for the generation of vasoinhibin in vertebrate species. Ninety-one prolactin protein sequences from species of the taxa primates, rodents, laurasiatheria, mammals, sauropsida, and fish were retrieved, and a multiple sequence alignment was performed. Each sequence was investigated for the presence of a vasoinhibin-generating cathepsin D cleavage site and its corresponding substrate affinity using a scoring system. Primates demonstrated the highest substrate affinity for the generation of the 15 kDa vasoinhibin isoform, and fish the highest affinity for the 16.8 kDa isoform. In both cases, this associates to the presence of leucine in the cleavage site, which is not present in species of the other taxa. In primate evolution, the presence of leucine in the cleavage site occurs with the emergence of simiiformes 42 million years ago and is conserved in higher primates across all subsequent speciation nodes. The 17.2 kDa vasoinhibin isoform has a constant substrate affinity in all taxa. The presence of leucine in vasoinhibin generating cleavage sites appears as an important feature of the molecular evolution of vasoinhibin.

Evolutionary studies of genes that have been functionally characterized and whose variation has been associated with pathological conditions represent an opportunity to understand the genetic basis of pathologies. α2-Adrenoreceptors (ADRA2) are a class of G protein-coupled receptors that regulate several physiological processes including blood pressure, platelet aggregation, insulin secretion, lipolysis, and neurotransmitter release. This gene family has been extensively studied from a molecular/physiological perspective, yet much less is known about its evolutionary history. Accordingly, the goal of this study was to investigate the evolutionary history of α2-adrenoreceptors (ADRA2) in vertebrates. Our results show that in addition to the three well-recognized α2-adrenoreceptor genes (ADRA2A, ADRA2B and ADRA2C), we recovered a clade that corresponds to the fourth member of the α2-adrenoreceptor gene family (ADRA2D). We also recovered a clade that possesses two ADRA2 sequences found in two lamprey species. Furthermore, our results show that mammals and crocodiles are characterized by possessing three α2-adrenoreceptor genes, whereas all other vertebrate groups possess the full repertoire of α2-adrenoreceptor genes. Among vertebrates ADRA2D seems to be a dispensable gene, as it was lost two independent times during the evolutionary history of the group. Additionally, we found that most examined species possess the most common alleles described for humans; however, there are cases in which non-human mammals possess the alternative variant. Finally, transcript abundance profiles revealed that during the early evolutionary history of gnathostomes, the expression of ADRA2D in different taxonomic groups became specialized to different tissues, but in the ancestor of sarcopterygians this specialization would have been lost.

Sex hormone-binding globulin (Shbg) is an important vertebrate blood carrier protein synthetized in the liver and involved in the transport and local regulation of sex steroids in target tissues. A novel shbg gene (shbgb) with a predominant ovarian expression was recently characterized. Being initially found only in salmonids, this shbgb was originally thought to result from the Salmonid-specific whole genome duplication. Using updated transcriptomic and genomic resources we identified Shbgb orthologs in non-salmonid teleosts (European eel, arowana), holosteans (spotted gar, bowfin), polypteriformes (reedfish), agnatha (sea lamprey) and in amphibians, and found that the classical Shbg gene (Shbga) displays a predominant hepatic expression whereas Shbgb has a predominant gonadal expression. Together, these results indicate that these two Shgb genes most likely originate from a whole genome duplication event at the root of vertebrate evolution, followed by numerous and independent losses and by tissue expression specialization of Shbga and Shbgb paralogs.

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-alpha and PdgfR-beta in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-alpha mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-beta mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-alpha and PdgfR-beta in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle.

In the present study the calcitonin receptor (CTR) sub-family of family B G-protein coupled receptors (GPCRs) in teleosts is evaluated and put in the context of the families overall evolution from echinodermates to vertebrates. Echinodermates, hemichordates, cephalochordates and tunicates have a single gene that encodes a receptor that bears similarity to the vertebrate calcitonin receptor (CTR) and calcitonin-like receptor (CTR/CLR). In tetrapods one gene encodes the calcitonin receptor (CALCR) and another gene the calcitonin receptor-like receptor (CALCRL). The evolution of CALCR has been under strong conservative pressure and a single copy is also found in fishes and high conservation of gene organisation and synteny exits from teleosts to human. A teleost specific CTR innovation that occurred after their divergence from holostei is the presence of several HBDs in the N-terminus. CALCRL had a different evolutionary trajectory from CALCR and although a single gene copy is present in tetrapods the sarcopterygii fish, the coelacanth, has 1 copy of CALCRL but also a fish specific form CALCRL3. The ray-finned fish, the spotted gar, has 1 copy of CALCRL and 1 of CALCRL3 but the teleost specific whole genome duplication has resulted in a CALCRL1 and CALCRL2 in addition to the fish specific CALCRL3. Strong conservation of CALCRL gene structure exists from human to fish. Promoter analysis in silico reveals that the duplicated CALCRL genes in the teleosts, zebrafish, takifugu, tetraodon and medaka, have divergent promoters and different putative co-regulated gene partners suggesting their function is different.

The golden rabbitfish Siganus guttatus is a reef fish with a restricted lunar-synchronized spawning rhythmicity and releases gametes simultaneously around the first quarter moon period during the spawning season. In order to understand the molecular aspects of the "circa" rhythms in this species, the full-length melatonin receptor (MT1) cDNA was cloned, and its diurnal/circadian regulation was examined. The full-length MT1 cDNA (1257 bp) contained an open reading frame that encodes a protein of 350 amino acids; this protein is highly homologous to MT1 of nonmammalian species. A high expression of MT1 mRNA with a day-night difference was observed in the whole brain, retina, liver, and kidney. When diurnal variations in MT1 mRNA expression in the retina and whole brain were examined using real-time quantitative RT-PCR, an increase in the mRNA expression was observed during nighttime in both tissues under conditions of light/dark, constant darkness, and constant light. This suggests that MT1 mRNA expression is under circadian regulation. The expression of MT1 mRNA in the cultured pineal gland also showed diurnal variations with high expression levels during nighttime; this suggests that the increased expression level observed in the whole brain is partially of pineal origin. Alternation of light conditions in the pineal gland cultures resulted in the changes in melatonin release into the culture medium as well as MT1 mRNA expression in the pineal gland. The present results suggest that melatonin and its receptors play an important role in the exertion of daily and circadian variations in the neural tissues.

Involvement of cyclooxygenase (COX) and prostaglandin (PG) synthesis during ovulation in Xenopus laevis ovarian follicles was investigated. X. laevis COX cDNAs were isolated from ovarian tissues by using reverse transcription-polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). In X. laevis ovary, expression of COX-2 but not COX-1 mRNA was up-regulated during gonadotropin-induced oocyte maturation and ovulation in vitro. Elevation of PGF2* synthesis was directly correlated with the up-regulation of COX-2 mRNA. Synthesis of PGE(2) also increased during periovulatory period, however, the concentrations were much lower than those of PGF2*Progesterone (P4) also induced up-regulation of COX-2 mRNA as well as ovulation. Actinomycin D (ActD) blocked P4-induced ovulation. The inhibition of ovulation by Act D was rescued by co-treatment with exogenous PGF2* in a dose dependent manner. A non-selective COX inhibitor (indomethacin) and selective COX-2 inhibitor (NS398) strongly inhibited the hCG- and P4-dependent production of PGF2*. Inhibitory effects of selective COX-1 inhibitor (SC560) on PGF2* production were lower than that of other inhibitors. Indomethacin and NS398 blocked P4-induced ovulation. NS398 did not block hCG-induced ovulation although it strongly suppressed PGF2* production. These results suggest that (1) in Xenopus ovarian follicles, PGF2* is synthesized during periovulatory period, similar to that in mammals, (2) PGF2* synthesis is regulated by de novo transcription of COX-2 but not COX-1, (3) PGF2* is necessary for P4-induced ovulation but may not be essential for hCG-induced ovulation, and other factor(s) may be involved in the hCG-induced ovulation.

Sex determination is controlled either by genetic or environmental factors. In mammals Sry initiates determination but no homologue of this gene exists in non-mammalian species. Other genes of the mammalian sex-determining pathway have been identified in gonads of different vertebrates. Sox9, Dax1, and Dmrt1 are expressed at the onset of gonadal development in birds and reptiles. In the sea turtle Lepidochelys olivacea, a species with temperature sex determination (TSD), Sox9 is expressed in undifferentiated gonads at male- (MPT) or female-promoting temperatures (FPT). At MPT, Sox9 remains expressed in male gonads, but at FPT it is downregulated coinciding with the onset of the ovarian morphologic differentiation and female sex determination. At MPT however, male sex is determined early than at FPT in still undifferentiated gonads suggesting that other genes maintain Sox9 expression in testis. Here we used RT-PCR to study the expression profiles of Dax1, Dmrt1, and Sox9 in gonads of embryos of L. olivacea incubated at MPT or at FPT. The profiles were correlated with sex determination during and after the temperature-sensitive period (TSP). Dax1 maintained similar levels at both temperatures during the TSP. The Dax1 expression level increased significantly in ovaries compared to testes at stage 27, once they were morphologically distinct. The expression levels of Dmrt1 were higher at MPT than at FPT at all stages, in contrast with Sox9 levels which were similar at both temperatures at stages 23-25. Together, current results suggest that, whereas Dax1 is not involved in TSD in L. olivacea, upregulation of Dmrt1 and downregulation of Sox9 may play a role in male and female sex determination, respectively.

Melanocortin (MC) systems are composed of MC peptides such as adrenocorticotropic hormone (ACTH), several molecular forms of melanocyte-stimulating hormones (MSHs) and MC receptors (MCRs). Here we demonstrated that the cartilaginous fish, Dasyatis akajei (stingray) expresses five subtypes of MCR genes-mc1r to mc5r-as in the case of teleost and tetrapod species. This is the first evidence showing the presence of the full repertoire of melanocortin receptors in a single of cartilaginous fish. Expression of respective stingray mcr cDNAs in Chinese hamster ovary cells revealed that Des-acetyl-α-MSH exhibited cAMP-producing activity indistinguishable to ACTH(1-24) on MC1R and MC2R, while the activity of Des-acetyl-α-MSH on MC3R, MC4R, and MC5R were similar to or slightly greater than that of ACTH(1-24). Notably, in contrast to the other vertebrates, MC2R did not require coexpression with a melanocortin receptor-2 accessory protein 1 (mrap1) cDNA for functional expression. One of the roles of MC system resides in regulation of the pituitary-interrenal (PI) axis-a homologue of tetrapod pituitary-adrenal axis. In stingray, interrenal tissues were shown to express mc2r and mc5r as major MCR genes. These results established the presence of functional PI axis in stingray at the level of receptor molecule. While MC2R participates in adrenal functions together with MRAP1 in tetrapod species, the fact that sensitivity of MC5R to Des-acetyl-α-MSH and ACTH(1-24) were two order of magnitude higher than MC2R without coexpression with MRAP1 suggested that MC5R could play a more important role than MC2R to transmit signals conveyed by ACTH and MSHs if MRAP1 is really absent in the stingray.

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.

Existing studies demonstrated that retinoic acid (RA) regulates meiotic initiation via stra8-independent pathway in teleosts which lack stra8 in their genomes. However, stra8 was recently identified from several fish species including Southern catfish (Silurus meridionalis). To explore the existence of stra8-dependent pathway in RA mediated meiotic initiation in fishes, in the present study, the genes encoding RA synthase aldh1a2 and catabolic enzyme cyp26a1 and cyp26b1 were cloned from the Southern catfish. By immunohistochemistry, Aldh1a2 signal was observed in gonads of both sexes during the meiotic initiation period. By real-time PCR, differentially expressed gene was observed for cyp26a1, but not for cyp26b1, in gonads during the meiotic initiation. Administration of exogenous RA or inhibition of endogenous RA degradation by either KET (RA catabolic enzyme inhibitor) or cyp26a1 knockdown using CRISPR/Cas9 induced advanced meiotic initiation in the ovaries as demonstrated by increased Stra8/stra8 expression and appearance of oocytes. In contrast, treatment with RA synthase inhibitor DEAB resulted in delayed meiotic initiation and Stra8/stra8 expression in the ovaries, which was rescued by exogenous RA administration. These results indicated that (1) RA triggers the onset of meiosis via stra8-dependent pathway in stra8 existing teleosts, as it does in tetrapods; (2) exogenous RA can rescue the endogenous RA deficiency; (3) Cyp26a1, instead of Cyp26b1, is the key catabolic enzyme involved in meiosis initiation in teleosts. Taken together, RA might trigger meiotic initiation via stra8-dependent and -independent pathway in different teleosts.

Gonadotropin-releasing hormone (GnRH) and its receptor are essential for reproduction in vertebrates. Although there are three major types of GnRH peptides and two major types of receptors in vertebrates, the pattern of distribution is unusual. Evidence is presented from genome mining that type I GnRHRs are not restricted to mammals, but can be found in the lobe-finned and cartilaginous fishes. This implies that this tail-less GnRH receptor emerged early in vertebrate evolution, followed by several independent losses in different lineages. Also, we have identified representatives from the three major GnRH peptide types (mammalian GnRH1, vertebrate GnRH2 and dogfish GnRH3) in a single cartilaginous fish, the little skate. Skate and coelacanth are the only examples of animals with both type I and II GnRH receptors and all three peptide types, suggesting this was the ancestral condition in vertebrates. Our analysis of receptor synteny in combination with phylogeny suggests that there were three GnRH receptor types present before the two rounds of whole genome duplication in early vertebrates. To further understand the origin of the GnRH peptide-receptor system, the relationship of vertebrate and invertebrate homologs was examined. Our evidence supports the hypothesis of a GnRH superfamily with a common ancestor for the vertebrate GnRHs, invertebrate (inv)GnRHs, corazonins and adipokinetic hormones. The invertebrate deuterostomes (echinoderms, hemichordates and amphioxus) have derived GnRH-like peptides, although one amphioxus GnRH with a syntenic relationship to human GnRHs has been shown to be functional. Phylogenetic analysis suggests that gene duplications in the ancestral bilaterian produced two receptor types, one of which became adipokinetic hormone receptor/GnRHR and the other corazonin receptor/invGnRHR. It appears that the ancestral deuterostome had both a GnRHR and invGnRHR, and this is still the case in amphioxus. During the transition to vertebrates both the invertebrate-type peptide and receptor were lost, leaving only the vertebrate-type system that presently exists.

A cDNA encoding thyroid stimulating hormone beta-subunit (TSHbeta) was cloned from pituitary of the Chinese soft-shell turtle, Pelodiscus sinensis, and its regulation of mRNA expression was investigated for the first time in reptile. The Chinese soft-shell turtle TSHbeta cDNA was cloned from pituitary RNA by reverse transcription and polymerase chain reaction (RT-PCR), and rapid amplification cDNA end (RACE) methods. The Chinese soft-shell turtle TSHbeta cDNA consists of 580-bp nucleotides, including 67-bp nucleotides of 5'-untranslated region (UTR), 402-bp of the open reading frame, and 97-bp of 3'-UTR followed by a 14 poly (A) trait. It encodes a precursor protein molecule of 133 amino acids with a putative signal peptide of 19 amino acids and a putative mature protein of 114 amino acids. The number and position of 12 cysteine residues, presumably forming six disulfide bonds, one putative asparagine-linked glycosylation site, and six proline residues that are found at positions for changing the backbone direction of the protein have been conserved in the turtle as in other vertebrate groups. The deduced amino acid sequence of the Chinese soft-shell turtle TSHbeta mature protein shares identities of 82-83% with birds, 71-72% with mammals, 49-57% with amphibians, and 44-61% with fish. The Chinese soft-shell turtle pituitaries were incubated in vitro with synthetic TRH (TSH-releasing hormone), thyroxine and triiodothyronine at doses of 10(-10) and 10(-8)M. TRH stimulated, while thyroid hormones suppressed, TSHbeta mRNA levels in dose-related manner. The sequences of cDNA and its deduced peptide of TSHbeta as well as the regulation of its mRNA level were reported for the first time in reptile.

The three-spot wrasse, Halichoeres trimaculatus, can change sex from female to male (i.e. protogyny) due to sharp decrease in endogenous estrogen. During the sex change, ovarian tissue degenerates and testicular tissue arises newly. Finally, ovarian tissue disappears completely and replaces into mature testis. In order to predict the molecular mechanisms controlling the processes of sex change, we investigated the expression patterns of four genes (rspo1, figla, sox9b and amh), which have been thought to be associated with ovarian/testicular differentiation in vertebrates. Expression levels of rspo1 and figla, which play important roles for ovarian differentiation in vertebrates, were stable until the middle stage of the sex change, and subsequently down-regulated. Therefore, it was indicated that decrease in rspo1 and figla could result from ovarian degeneration. On the other hand, basis on the expression pattern, it was indicated that sox9b and amh, which are involved in testicular differentiation in vertebrates, were implicated in testicular formation and spermatogenesis during the sex change as well. The present results could be fundamental information for investigating the relationship between these factors and E2 depletion, which is crucial trigger for sex change.

Spadefoot toad species display extreme variation in larval period duration, due in part to evolution of thyroid hormone (TH) physiology. Specifically, desert species with short larval periods have higher tail tissue content of TH and exhibit increased responsiveness to TH. To address the molecular basis of larval period differences, we examined TH receptor (TR) expression across species. Based on the dual function model for the role of TR in development, we hypothesized that desert spadefoot species with short larval periods would have (1) late onset of TR expression prior to the production of endogenous TH and (2) higher TR levels when endogenous TH becomes available. To test these hypotheses, we cloned fragments of TRα and TRβ genes from the desert spadefoot toads Scaphiopus couchii and Spea multiplicata and their non-desert relative Pelobates cultripes and measured their mRNA levels in tails using quantitative PCR in the absence (premetamorphosis) or presence (natural metamorphosis) of TH. All species express TRα and TRβ from the earliest stages measured (from just after hatching), but S. couchii, which has the shortest larval period, had more TRα throughout development compared to P. cultripes, which has the longest larval period. TRβ mRNA levels were similar across species. Exogenous T3 treatment induced faster TH-response gene expression kinetics in S. couchii compared to the other species, consistent with its higher TRα mRNA expression and indicative of a functional consequence of more TRα activity at the molecular level. To directly test whether higher TRα expression may contribute to shorter larval periods, we overexpressed TRα via plasmid injection into tail muscle cells of the model frog Xenopus laevis and found an increased rate of muscle cell death in response to TH. These results suggest that increased TRα expression evolved in S. couchii and contribute to its higher metamorphic rates.

Steroid hormones are a key regulator of reproductive biology in vertebrates, and are largely regulated via nuclear receptor families. Estrogen signaling is regulated by two estrogen receptor (ER) subtypes alpha and beta in the nucleus. In order to understand the role of estrogen in vertebrates, these ER from various species have been isolated and were functionally analyzed using luciferase reporter gene assays. Interestingly, species difference in estrogen sensitivity has been noted in the past, and it was reported that snake ER displayed highest estrogen sensitivity. Here, we isolated additional ER from three lizards: chameleon (Bradypodion pumilum), skink (Plestiodon finitimus), and gecko (Gekko japonicus). We have performed functional characterization of these ERs using reporter gene assay system, and found high estrogen sensitivity in all three species. Furthermore, comparison with results from other tetrapod ER revealed a seemingly uniform gradual pattern of ligand sensitivity evolution. In silico 3D homology modeling of the ligand-binding domain revealed structural variation at three sites, helix 2, and juncture between helices 8 and 9, and caudal region of helix 10/11. Docking simulations indicated that predicted ligand-receptor interaction also correlated with the reporter assay results, and overall squamates displayed highest stabilized interactions. The assay system and homology modeling system provides tool for in-depth comparative analysis of estrogen function, and provides insight toward the evolution of ER among vertebrates.