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Background: The Asian yellow pond turtle (Mauremys mutica) is an important commercial freshwater aquaculture species in China. This species is a highly sexually dimorphic species, with males growing at a faster rate than females and exhibits temperature-dependent sex determination (TSD), in which the incubation temperature during embryonic development determines the sexual fate. However, the mechanisms of the sex determination or sex differentiation in the Asian yellow pond turtle are remain a mystery. Results: Temperature-specific gonadal transcriptomics of the Asian yellow pond turtle were performed during the thermosensitive period (stage 15) using RNA-seq technology to identify candidate genes that initiate gonadal differentiation. We uncovered candidates that were the first to respond to temperature. These candidates were sexually dimorphic in expression, reflecting differences in gonadal (Cirbp, Runx1) and germline differentiation (Vasa, Nanos1, Piwil2), gametogenesis (Hmgb3, Zar1, Ovoinhibitor-like, Kif4), steroid hormone biosynthesis (Hsd17b5, Hsd17b6), heat shock (Dnajb6, Hsp90b1, Hsp90aa1) and transient receptor potential channel genes (Trpm1, Trpm4, Trpm6, Trpv1). Conclusions: Our work will provide important genetic information to elucidate the mechanisms of sex control in the Asian yellow pond turtles, and will contribute important genetic resources for further studies of temperature-dependent sex determination in turtles. [ABSTRACT FROM AUTHOR]

Simple Summary: The Asian yellow pond turtle (Mauremys mutica) is widely traded in China, and its artificial breeding has now become a major industry. However, the insufficient offspring supply and reproductive decline of farmed turtles make wild turtles more vulnerable. The present study was mainly designed to quantify the fecundity of M. mutica and attempt to screen for good reproductive performance in females. The parent–offspring relationships of all offspring in four consecutive years were confirmed using sixteen microsatellite loci. We also summarised the reproductive results of all females and counted the annual number of offspring and the variation in the number of offspring. The females were then divided into three types (stable, undulating and levelling off) according to the continuity. This study can provide the basis and materials for the creation of a good reproductive group and the study of the reproductive biology of turtles in M. mutica aquaculture. The Asian yellow pond turtle (Mauremys mutica) is widely traded in China, and its artificial breeding has now become a major industry. However, the insufficient offspring supply and reproductive decline of farmed turtles make the wild turtles more vulnerable. The present study was mainly designed to quantify the fecundity of M. mutica and attempt to screen for good reproductive performance in females. The genetic variability of the population and its genetic structure were also analysed. The parent–offspring relationships of all offspring in four consecutive years were confirmed using sixteen microsatellite loci. The genetic variability between the parents and offspring was low, and offspring of different years also showed little variability. We summarised the reproductive results of all females and counted the annual number of offspring and the variation in the number of offspring. The females were then divided into three types (stable, undulating and levelling off) according to the continuity. We selected seven females with good reproductive ability, which provided 16.94% of the annual contributions, while there were two females that had no offspring in four years. We also analysed the possible reasons for this difference and the importance of carrying out a family survey. This research can provide the basis and materials for the creation of a good reproductive group and the study of the reproductive biology of turtles in M. mutica aquaculture. [ABSTRACT FROM AUTHOR]

Simple Summary: We explored the effect of maternal size on offspring size and number using a 4-year dataset based on paternity analyses in Asian yellow pond turtle, Mauremys mutica, and found that the maternal size significantly affected the offspring number but not offspring size. The offspring size was significantly correlated with maternal age. Our results suggest that the offspring size-number trade-off theory does not apply to cultured M. mutica and also provides basic data for the efficient artificial breeding management of M. mutica in captivity. Offspring size-number trade-off is a critical component of life-history theory and is important for further understanding the reproductive strategies of animals. The relationship between this trade-off and maternal size has been explored in several turtle species, except for the Asian yellow pond turtle, Mauremys mutica. To investigate how the maternal condition affects offspring size and number, we explored the relationships among the maternal body size and the number and size of cultured M. mutica hatchlings using a 4-year dataset. Our results showed that different females not only produced different sizes of offspring but also produced different numbers of offspring. No trade-off in egg size number was detected. According to regression analysis, we did not find that the maternal body size significantly influenced the offspring mass; however, we detected that the offspring size was significantly correlated with the clutch size and maternal age. The mean body mass of offspring increased with maternal age, and the clutch size varied significantly over four years, which was correlated with offspring size, maternal body size and age. However, the number of offspring per female increased with the maternal plastron length rather than age. Our results were inconsistent with the optimal offspring size theory in that females did not increase their offspring size but rather increased the offspring number to increase their fitness, which will also provide a basis for the efficient cultivation management of turtles. [ABSTRACT FROM AUTHOR]

Simple Summary: Gonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes' functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells' culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we isolated an ovarian cell line (fGSCs), from the juvenile Asian yellow pond turtle in vitro. The cultured fGSCs were characterized with germline stem cell specific markers' expression using transcriptome sequencing on Illumina platform, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence (IF). Our study aims to provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle even in reptiles, and also, the established cell line has potential applications to enrich the conservation strategy of aquatic animal germplasm resources. Gonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes' functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells' culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we established a new ovarian cell line from the young Asian yellow pond turtle (Mauremys mutica), which exhibited a normal diploid karyotype with high alkaline phosphatase activity. The cell line, designated as YTO2, was then characterized through the analysis of gene expression profiles. The transcriptome analysis and the reverse transcription polymerase chain reaction (RT-PCR) showed that the cells expressed germline genes such as tdrd7, nanos1, klf5, igtb1, hsd17b4 and rad51. Moreover, the immunostaining showed that the germ cell markers, Tdrd7 and Rad51 proteins, were detected predominant in cytoplasm of perinuclear region, while proliferation marker, PCNA, was dominantly observed in the nuclei of cultured cells. Intriguingly, the cells could respond to the retinoic acid induction with significantly increasing the expression level of some meiosis genes, including vasa, dazl, figla, and dmc1. Furthermore, YTO2 cells could be efficiently transfected with the pHBAd-BHG-EGFP adenovirus and properly expressed the exogenous genes. To sum up, an ovarian cell line of the Asian yellow pond turtle had been established and could be stably propagated under in vitro culture condition, as well as being capable of efficiently expressing the exogenous gene tdrd7. This cell line would provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle, even in reptiles. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Vasa, one of the best‐studied germ cell markers plays a critical role in germ cell development and differentiation in animals. Vasa deficiency would lead to male‐specific sterility in most vertebrates, but female sterility in the fly. However, the role of the vasa gene involved in germ cell differentiation is largely elusive. Here, we first characterized the expression profile of vasa products in the Asian yellow pond turtle by quantitative reverse‐transcription polymerase chain reaction and fluorescence immunostaining. The results showed that vasa messenger RNA (mRNA) is initially detected in embryos at stage 16, and then dramatically increased in embryos at stage 19. In particular, like the sex‐related genes, vasa mRNA exhibited differential expression in embryos between the male‐producing temperature (MPT, 25°C) and the female‐producing temperature (FPT, 33°C), whereas there was no difference in methylation levels of vasa promoter detected between FPT and MPT. In contrast, in the adult Asian yellow pond, the level of vasa mRNA was much higher in the testis than ovary. Moreover, the immunostaining on testicular sections and cells showed that Vasa protein was exclusively expressed in germ cells: Weak but detectable in spermatogonia, highest in spermatocytes, moderate and concentrated in chromatid bodies in spermatids and spermatozoa, and bare in somatic cells. The expression profile of Vasa protein is similar in turtle species studied so far but distinct from those in fish species in this study. The findings of this study would provide new insights into our understanding of the conservation and divergence of the vasa gene, even other germ cell genes across phyla. Research Highlights: vasa mRNA expression was much higher in the Asian yellow pond turtle embryos/embryonic gonads hatched with the FPT than with the MPT while no difference in methylation levels of vasa promoter was detected between FPT and MPT.In the adult Asian yellow pond, the level of vasa mRNA was found to be much higher in the testis than ovary.The immunostaining on testicular sections and cells showed that Vasa protein was exclusively expressed in germ cells: Weak but detectable in spermatogonia, highest in spermatocytes, moderate and concentrated in chromatid bodies in spermatids and spermatozoa, and barely detectable in somatic cells. [ABSTRACT FROM AUTHOR]

Biotic factors (e.g., maternal age) and abiotic factors (e.g., weather) play vital roles in reproduction. However, there is little information about the combined effects of maternal age and weather on the reproductive output of cultured Asian yellow pond turtles, Mauremys mutica. To address this issue, we surveyed and compared the reproductive output of 13-year-old and 25-year-old female turtles on non-rainy and rainy days. The results showed that older females produced larger eggs than younger females regardless of the weather conditions. Females laid larger clutch sizes (number of eggs per nest) under non-rainy conditions compared with those under rainy conditions. However, the variation in clutch frequency (number of clutches per day) was independent of maternal age and weather. There was no effect of an interaction between maternal age and weather on egg mass, clutch size or clutch frequency. Our results suggest that maternal age and weather had significant effects on egg mass and clutch size, respectively. These results imply that maternal age, together with weather conditions (mainly conditions of rain), can affect the reproductive output of M. mutica. Our results provide useful information for the artificial breeding and stock management of M. mutica. [ABSTRACT FROM AUTHOR]

The crucial roles of miRNAs in regulating animal growth, development, and disease resistance have been extensively reported, but their roles in relation to the reproductive capacity of aquatic animals (numbers of eggs laid and hatchlings), especially reptiles, remain unclear. In this study, high-throughput sequencing technology was used to screen miRNAs related to reproductive capacity based on the construction of a cDNA library of ovaries from higher-fecundity (HF) and lower-fecundity (LF) M. mutica. The results showed that 15,767,494 (93.98%) and 14,137,621 (94.17%) high-quality reads were obtained from the HF and LF groups, respectively. We screened 131 miRNAs that were differentially expressed between the HF and LF groups, of which 78 were upregulated and 53 were downregulated compared with the M. mutica reference genome. GO and KEGG pathway enrichment analyses of the target genes of differentially expressed miRNAs revealed significant differences in the enrichment frequencies of genes associated with ATP binding and proteolysis between the HF and LF groups, while the tricarboxylic acid cycle, glucagon signaling pathway and vitamin B6 metabolic pathway were shown to potentially help determine reproductive capacity. Ten miRNAs were verified by qRT-PCR to confirm the reliability and accuracy of the sequencing results, and a miRNA-mRNA target gene interaction network was constructed. These results will further our understanding of the regulatory mechanism of miRNAs in regards to turtle reproductive capacity. [Display omitted] • 131 differentially expressed miRNAs were detected between higher fecundity group and lower fecundity group. • There were 10 differentially expressed miRNAs validated by using qRT-PCR. • A miRNA-mRNA target gene interaction network was constructed. [ABSTRACT FROM AUTHOR]

Mitochondria function as an energy transfer organelle for cell metabolism with the energy being used for processes such as reproduction. To investigate whether mutations and copy number variations in mitochondrial genes are related to the reproductive capacity of the Asian yellow pond turtle, Mauremys mutica , there was exploration of the distribution frequency of 129 Single Nucleotide Polymorphism loci of six mitochondrial genes, ND1 , ND2 , COX2 , ND4 , Cytb andD- loop , of turtles from a relatively greater and lesser fecundity group by direct sequencing. The validation results for five candidate SNP loci in 83 female turtles indicated that only three SNP loci (C119T, A320G and A417C) in ND1 were positively correlated with reproductive capacity in M. mutica (P < 0.05). In addition, by constructing linear regression equations of the copy numbers of ND1 , ND4 , Cytb , D-loop , COX3 , and ATP6 (log10 transformed) genes and the mean offspring number of different female turtles during a 4-year period, the copy numbers of ND4 and ATP6 (log10 transformed) genes were positively correlated (P < 0.05) with the fecundity of female turtles. Results from the present study may provide useful genetic markers for breeding M. mutica with greater reproductive capacity. [ABSTRACT FROM AUTHOR]

Abstract The reproductive capacity (egg size and egg number) of most of oviparous animals, including the Asian yellow pond turtle (Mauremys mutica), is constrained by the maternal age and body size, but the mechanism determining the maternal reproductive ability remains unclear. To disclose how maternal age and size affect reproductive ability of M. mutica , we first identified the full-length cDNAs from estrogen receptor 1 (ESR1), bone morphogenetic protein receptor 1B (BMPR1B), and forkhead box L2 (FOXL2). The ESR1 open reading frame (ORF) was 1, 767 bp encoding 588 amino acids. For BMPR1B, the ORF was 1599 bp encoding 532 amino acids, and an ORF of 906 bp encoding 301 amino acids was identified in FOXL2. The effects of maternal age and size on the expression of ESR1, BMPR1B, and FOXL2 in the ovary, brain, and uterus showed that ESR1 expression in large females was significantly lower than that in small females in the brain, but body size did not affect ESR1 expression in the ovary. The expression of ESR1 was significantly different in the different age groups and size groups, and there was interaction detected between maternal age and body size. However, BMPR1B expression in the ovary, brain, and uterus was independent of maternal age and size. In addition, we found different FOXL2 expression patterns between the brain and uterus, while detected interaction of female age and size in the brain and ovary. Our results imply the complexity and diversity of maternal age and size in regulating the expression of genes related to reproduction. These results provide more information for the maternal effects on the reproduction-related gene expression. Graphical abstract Unlabelled Image Highlights • The cDNA full-length of ESR1 , BMPR1B and FOXL2 were cloned and characterized. • Maternal size had a negative effect on the expression of ESR1. • Female age and size had no effect on expression level of BMPR1B. • Expression level of FOXL2 was related to maternal age and size. [ABSTRACT FROM AUTHOR]

In order to understand the endocrine mechanism associated with fecundity of seasonally breeding animals, we investigated the plasma reproductive hormones levels and detected the differences in steroidogenic enzymes and sex hormone receptor mRNA levels in female Mauremys mutica. These turtles were divided into higher fecundity (HF) group than those in lower fecundity (LF) group based on paternity identification in our previous research. The plasma estrogen (E2), testosterone (T) and progesterone (P4) levels were significantly higher in pre-breeding season (PBS) than those in non-breeding season (NBS) and were markedly higher in the HF group than those in LF group. In the hypothalamus, there was significantly higher mRNA abundance of P450-cholesterol side-chain cleavage enzyme (P450Scc) encoded by Cyp11α1, aromatase (Cyp19α1) and 5-reductase (5α-R), but significantly lower mRNA levels of follicular stimulating hormone receptor (FSHR) and progesterone receptor (PR) detected in PBS than those in NBS. The pituitary steroidogenic acute regulatory protein (StAR), cytochrome P450-17alpha-hydroxylase (Cyp17α1), 3-hydroxy-steroid dehydrogenase (3βHSD), 17-hydroxy-steroid dehydrogenase 3 (17βHSD3), Cyp19α1, 5α-R, FSHR, estrogen receptor 1 (ESR1), androgen receptor (AR) and PR transcriptional levels in HF group were up-regulated significantly compared with the LF group. In the ovary, Cyp17α1 and 17βHSD3 transcriptional levels were markedly higher in PBS than those in NBS. We detected significantly increased expression levels of all steroidogenic enzymes, but notably lower mRNA levels of FSHR and PR in uterus during the PBS, and the HF group has significantly higher expression levels of StAR, Cyp17α1, 5α-R and AR than LF group. Our work reveals seasonal variations in hormone regulation as well as gene regulation in turtles, providing reliable information to understand the mechanisms underlying the different reproductive capacity of reptiles. [Display omitted] • Plasma T level can be an indicator for screening high-fecundity females. • Steroidogenic enzymes in uterus were significantly higher in PBS than those in NBS. • Pituitary sex hormone receptors expressed significantly more in HF than that in LF. • Uterus plays a critical role in regulating turtle's reproduction. [ABSTRACT FROM AUTHOR]

• We identified 8,237 DE mRNAs, 9,573 DE lncRNAs, 84 DE circRNAs and 665 DE miRNAs. • We identified DE mRNAs majorly involved in reproduction, reproductive process. • dmrt3a etc were closely associated with the levels of their corresponding ncRNA. Noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) have been extensively studied in biological processes such as disease development, cell proliferation but remained unclear in sex differentiation in organisms. In this study, the transcriptome profiles were comparatively analyzed between male and female gonads in Mauremys mutica. A total of 8237 differentially expressed genes (DEGs), 9573 DE lncRNAs, 84 DE circRNAs and 665 DE miRNAs were identified between male and female gonads. Through gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) of the DE RNAs, it was revealed that the DE mRNAs were majorly involved in GO terms, such as 'reproduction', 'reproductive process' and the pathways of 'focal adhesion' and 'oocyte meiosis'. In addition, a co-expression network showed that the expression of gametogenesis and sex differentiation related genes, including dmrt3a , tdrd7 , sox14 , etc were closely associated with the levels of their corresponding ncRNAs. Intriguingly, the dmrt1 circRNA and its target mRNA were detected upregulated both in adult testis and male producing temperature (MPT) embryos. Our findings demonstrated the sexually dimorphic expression profiles of mRNAs and ncRNAs in turtle gonads, which will provide the index to find out the molecular mechanisms behind the sex differentiation in turtles, even in other environmental sex determination (ESD) species. [ABSTRACT FROM AUTHOR]