Project description:In the present study, the eggs of Chinese pond turtles (Mauremys reevesii) were incubated at three temperatures (26℃, 29℃ and 32℃). During the thermosensitive period (TSP) of incubation, the adrenal-kidney-gonad (AKG) complexes were sampled, and the transcriptome differences of the AKG samples were investigated using next-generation sequencing, then candidate genes associated with temperature-dependent sex determination of Reeves' Turtle were identified.
Project description:In the present study, the eggs of Chinese pond turtles (Mauremys reevesii) were incubated at three temperatures (26℃, 29℃ and 32℃). During the thermosensitive period (TSP) of incubation, the adrenal-kidney-gonad (AKG) complexes were sampled, and a comprehensive investigation for miRNAs was performed using next-generation small RNA sequencing.
Project description:Some differentially expressed genes (DEGs) that encode key enzymes involved in steroidogenic biosynthesis (CYP19A1) and key molecules related to gonadal functions (DMRT1, SOX9, AMH, FOXL2, WNT4, RSPO2, and GDF9) have been identified in adult gonadal RNA-seq studies of Reeves' pond turtle (Mauremys reevesii) with temperature-dependent sex determination (TSD). Gonadal functional maintenance and gametogenesis comprises a highly regulated and coordinated biological process, and increasing evidence indicates that microRNAs (miRNAs) may be involved in this dynamic program. However, it is not clear how the regulatory network comprising miRNAs changes the expression levels of these genes. In this study, miRNA sequencing of adult testis and ovary tissues from M. reevesii detected 25 known and 379 novel miRNAs, where 60 miRNAs were differentially expressed in the testis and ovary. A total of 1,477 target genes based on the differentially expressed miRNAs were predicted, where 221 target genes also exhibited differential expression. To verify the accuracy of the sequencing data, 10 differentially expressed miRNAs were validated by quantitative reverse transcription real-time PCR, and were found to be consistent with the transcriptome sequencing results. Moreover, several miRNA/target gene pairs, i.e., mre-let-7a-5p/mre-let-7e-5p and CYP19A1, mre-miR-200a-3p and DMRT1, mre-miR-101-3p and SOX9, and mre-miR-138-5p and AMH were identified. To explore the regulatory role of miRNAs, we conducted target gene enrichment analysis of the miRNAs and 221 target genes in the regulatory network. The signaling pathways related to gonadal functional maintenance and gametogenesis based on the DEGs and target genes were then compared. Our findings provide crucial information to facilitate further research into the regulatory mechanisms involving miRNAs in turtle species with TSD.
Project description:Knowledge of sex determination has important implications in physiology, ecology and genetics, but the evolutionary mechanisms of sex determination systems in turtles have not been fully elucidated, due to a lack of reference genomes. Here, we generate a high-quality genome assembly of Asian yellow pond turtle (Mauremys mutica) using continuous long-read (PacBio platform), Illumina, and high-throughput chromatin conformation capture (Hi-C) technologies. The M. mutica haplotype has a genome size of 2.23 Gb with a contig N50 of 8.53 Mb and scaffold N50 of 141.98 Mb. 99.98% sequences of the total assembly are anchored to 26 pseudochromosomes. Comparative genomics analysis indicated that the lizard-snake-tuatara clade diverged from the bird-crocodilian-turtle clade at approximately 267.0-312.3 Mya. Intriguingly, positive selected genes are mostly enriched in the calcium signaling pathway and neuroactive ligand-receptor interaction, which are involved in the process of temperature-dependent sex determination. These findings provide important evolutionary insights into temperature-dependent sex determination system.
Project description: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.
Project description:Sex determination is the process by which and original bipotential gonad differentiate into either a testis or ovaries. While mammals and birds determine their sex solely by genetic clues (genetic sex determination, GSD), other vertebrates like the turtle Trachemys scripta are influenced by environmental factors, like temperature (environmental sex determination, ESD). In both cases an initially bipotential gonad develops into either testes or ovaries in response to GSD or ESD cues. In order to shed light into the differences and similarities between sex determination systems we performed single-cell RNA-seq on Trachemys scripta developing gonads during the sex determination window.
Project description:Mauremys reevesii is a classical organism with temperature-dependent sex determination (TSD). Gonad development in early life has recently received considerable attention but gonadal maintenance after sex differentiation in turtles with TSD remains a mystery. In this study, we sequenced the transcriptomes for the adult testis and ovary using RNA-seq, and 36,221 transcripts were identified. In total, 1,594 differentially expressed genes (DEGs) were identified where 756 DEGs were upregulated in the testis and 838 DEGs were upregulated in the ovary. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the TGF-beta signaling pathway and Hedgehog signaling pathway have important roles in testis maintenance and spermatogenesis, whereas the Hippo signaling pathway and Wnt signaling pathway are likely to participate in ovary maintenance. We determined the existence of antagonistic networks containing significant specific-expressed genes and pathways related to gonadal maintenance and gametogenesis in the adult gonads of M. reevesii. The candidate gene Fibronectin type 3 and ankyrin repeat domains 1 (FANK1) might be involved with the regulation of testis spermatogenesis.
Project description:BackgroundReeves' Turtles (Mauremys reevesii) are economically important in aquaculture in China. Understanding the effects of incubation temperature and substrate moisture on embryos and hatchlings is of great significance for improving the artificial culture of M. reevesii. However, available studies have not yet determined the thermal and hydric optima for M. reevesii eggs, and the potential interaction between the two factors.MethodsIn this study, eggs of M. reevesii were incubated at five temperature levels (23, 26, 29, 32 and 35 °C, fluctuation range ± 0.5 °C). In each temperature level, there were three substrate moisture levels (1:0.5, 1:0.9 and 1:1.2, weight ratio of vermiculite to water). Thus, a total of 15 combinations of temperature and moisture were used to examine the effects of incubation temperature and substrate moisture on incubation duration, hatching success, hatchling phenotypes, post-hatching growth and hatchling survival.ResultsSubstrate moisture did not significantly affect most development parameters (except incubation duration and carapace width of hatchlings). Eggs incubated at low moisture level (1:0.5) had a longer incubation duration and produced hatchlings with smaller carapace widths than those incubated at medium (1:0.9) or high (1:1.2) moisture levels. Incubation temperature had a significant effect on incubation duration, hatching success, hatchling phenotypes and hatchling survival. Incubation duration decreased as incubation temperature increased. Eggs incubated at 23, 26 and 29 °C showed higher hatching success than those incubated at 32 and 35 °C. Hatchlings incubated at 32 °C were smaller in body size and mass than those incubated at 23, 26 and 29 °C. At 12 months of age, incubation temperature had no long-lasting effect on body mass, but hatchlings incubated at 23 and 35 °C had lower survival rates than those incubated at 26, 29 and 32 °C. For the development of embryos and hatchlings, the interaction between incubation temperature and substrate moisture was not significant.ConclusionsOur results indicate that incubation temperature has a significant influence on the development of embryos and hatchlings of M. reevesii, while substrate moisture only significantly affects the incubation duration and carapace width of hatchlings. The combination of an incubation temperature of 29 ± 0.5 °C and a substrate moisture level of 1:1.2 represented optimal incubation conditions in this experiment. Such incubation conditions are helpful in obtaining higher hatching success, shorter incubation duration and higher survival rates for this aquaculture species.
Project description:The environment surrounding the embryos has a profound impact on the developmental process and phenotypic outcomes of the organism. In species with temperature-dependent sex determination, gonadal sex is determined by the incubation temperature of the eggs. A mechanistic link between temperature and transcriptional regulation of developmental genes, however, remains elusive. In this study, we examine the changes in DNA methylation and histone modification patterns of the aromatase (cyp19a1) gene in embryonic gonads of red-eared slider turtles (Trachemys scripta) subjected to a temperature shift during development. Shifting embryos from a male-producing temperature (MPT) to a female-producing temperature (FPT) at the beginning of the temperature-sensitive period (TSP) resulted in an increase in aromatase mRNA expression while a shift from FPT to MPT resulted in decreased expression. DNA methylation levels at CpG sites in the promoter of the aromatase gene were high (70-90%) at the beginning of TSP, but decreased in embryos that were incubated at constant FPT and those shifted from MPT to the FPT. This decrease in methylation in the promoter inversely correlated with the expected increase in aromatase expression at the FPT. The active demethylation under the FPT was especially prominent at the CpG site upstream of the gonad-specific TATA box at the beginning of TSP and spread downstream of the gene including exon1 as the gonad development progressed. In embryos incubated at FPT, the promoter region was also labeled by canonical transcriptional activation markers, H3K4me3 and RNA polymerase II. A transcriptional repression marker, H3K27me3, was observed in temperature-shifted gonads of both temperature groups, but was not maintained throughout the development in either group. Our findings suggest that DNA hypomethylation and H3K4me3 modification at the aromatase promoter may be a primary mechanism that releases a transcriptional block of aromatase to initiate a cascade of ovarian differentiation.
Project description:Mauremys reevesii (Geoemydidae) is one of the most common and widespread semi-aquatic turtles in East Asia. The unusually long lifespan of some individuals makes this turtle species a potentially useful model organism for studying the molecular basis of longevity. In this study, pooled total RNA extracted from liver, spleen and skeletal-muscle of three adult individuals were sequenced using Illumina Hiseq 2500 platform. A set of telomere-related genes were found in the transcriptome, including tert, tep1, and six shelterin complex proteins coding genes (trf1, trf2, tpp1, pot1, tin2 and rap1). These genes products protect chromosome ends from deterioration and therefore significantly contribute to turtle longevity. The transcriptome data generated in this study provides a comprehensive reference for future molecular studies in the turtle.