Xiao-Juan Zhang

Evolutionary history of two divergent Dmrt1 genes reveals two rounds of polyploidy origins in gibel carp

Polyploidy lineages, despite very rare in vertebrates, have been proposed to play significant role in speciation and evolutionary success, but the occurrence history and consequences are still largely unknown. In this study, we used the conserved Dmrt1 to analyze polyploidy occurrence and evolutionary process in polyploid gibel carp. We identified two divergent Dmrt1 genes and respectively localized the two genes on three homologous chromosomes. Subsequently, the corresponding full-length cDNAs and genomic sequences of Dmrt1 genes were also characterized from the closely related species including Carassius auratus auratus and Cyprinus carpio, and their two Dmrt1 genes were respectively localized on two homologous chromosomes. Significantly, the evolutionary relationship analyses among cDNA and genomic DNA sequences of these Dmrt1 genes revealed two rounds of polyploidy origins in the gibel carp: an early polyploidy might result in an common tetraploid ancestor of Carassius auratus gibelio, Carassius auratus auratus and Cyprinus carpio before 18.49 million years ago (Mya), and an late polyploidy might occur from evolutionary branch of Carassius auratus at around 0.51 Mya, which lead to the occurrence of the hexaploid gibel carp. Therefore, this study provides clear genetic evidence for understanding occurrence time and historical process of polyploidy in polyploid vertebrates.

Expression characterization of testicular DMRT1 in both Sertoli cells and spermatogenic cells of polyploid gibel carp

Dmrt1 has been suggested to play significant roles in sex determination and differentiation, but various expression patterns and cell types have been observed in the testis of vertebrates. Polyploid gibel carp, because of the multiple modes of unisexual gynogenesis and sexual reproduction, has become a unique case to explore the evolution of sex determination and differentiation. However, the sex-determination related genes in gibel carp have remained unknown. In this study, we identified and characterized 4 cDNAs of Dmrt1 genes. Subsequently, a polyclonal antibody specific to CagDMRT1 was prepared to examine its expression and distribution patterns at protein level. Significantly, both relative real-time PCR and Western blot detection confirmed predominant expression of CagDmrt1 in the adult testis of gibel carp. Moreover, the intensive expression of CagDMRT1 around spermatogenic cysts was revealed during spermatogenesis. And, following immunofluorescence co-localization of CagDMRT1 and CagVASA, a prominent CagDMRT1 expression in Sertoli cells and a mild CagDMRT1 expression in spermatogenic cells including spermatogonia and primary spermatocytes were clearly characterized. The CagDMRT1 signal in Sertoli cells is extensively distributed in both nuclei and cytoplasm, while the CagDMRT1 in spermatogonia and primary spermatocytes is mainly expressed in nuclei, and there is only the remained CagDMRT1 signal in the cytoplasm of secondary spermatocytes. These findings suggest that DMRT1 should be related to testis differentiation and spermatogenesis in gibel carp.

Extra Microchromosomes Play Male Determination Role in Polyploid Gibel Carp

Sex is generally determined by sex chromosomes in vertebrates, and sex chromosomes exhibit the most rapidly-evolving traits. Sex chromosome evolution has been revealed previously in numerous cases, but the association between sex chromosome origin and the reproduction mode transition from unisexual to sexual reproduction remains unclear. Here, we have isolated a male-specific sequence via analysis of amplified fragment length polymorphism from polyploid gibel carp (Carassius gibelio), a species that not only has the ability to reproduce unisexually but also contains males in wild populations. Subsequently, we have found through FISH analysis that males have several extra microchromosomes with repetitive sequences and transposable elements when compared to females. Moreover, we produced sex-reversed physiological females with a male-specific marker by using estradiol hormone treatment, and two gynogenetic families were established from them. In addition, the male incidence rates of two gynogenetic families were revealed to be closely associated with the extra microchromosome number of the sex-reversed physiological females. These results suggest that the extra microchromosomes in males might resemble a common feature of sex chromosomes and might play a significant role in male determination during the evolutionary trajectory of the reproduction mode transition from unisexual to sexual reproduction in the polyploid fish.

Complete depletion of primordial germ cells in an All-female fish leads to Sex-biased gene expression alteration and sterile All-male occurrence

BackgroundGynogenesis is one of unisexual reproduction modes in vertebrates, and produces all-female individuals with identical genetic background. In sexual reproduction vertebrates, the roles of primordial germ cells on sexual dimorphism and gonadal differentiation have been largely studied, and two distinct functional models have been proposed. However, the role of primordial germ cells remains unknown in unisexual animals, and it is also unclear whether the functional models in sexual reproduction animals are common in unisexual animals.ResultsTo solve these puzzles, we attempt to utilize the gynogenetic superiority of polyploid Carassius gibelio to create a complete germ cell-depleted gonad model by a similar morpholino-mediated knockdown approach used in other examined sexual reproduction fishes. Through the germ cell-depleted gonad model, we have performed comprehensive and comparative transcriptome analysis, and revealed a complete alteration of sex-biased gene expression. Moreover, the expression alteration leads to up-regulation of testis-biased genes and down-regulation of ovary-biased genes, and results in the occurrence of sterile all-males with testis-like gonads and secondary sex characteristics in the germ cell-depleted gynogenetic Carassius gibelio.ConclusionsOur current results have demonstrated that unisexual gynogenetic embryos remain keeping male sex determination information in the genome, and the complete depletion of primordial germ cells in the all-female fish leads to sex-biased gene expression alteration and sterile all-male occurrence.

Type-IV Antifreeze Proteins are Essential for Epiboly and Convergence in Gastrulation of Zebrafish Embryos

Many organisms in extremely cold environments such as the Antarctic Pole have evolved antifreeze molecules to prevent ice formation. There are four types of antifreeze proteins (AFPs). Type-IV antifreeze proteins (AFP4s) are present also in certain temperate and even tropical fish, which has raised a question as to whether these AFP4s have important functions in addition to antifreeze activity. Here we report the identification and functional analyses of AFP4s in cyprinid fish. Two genes, namely afp4a and afp4b coding for AFP4s, were identified in gibel carp (Carassius auratus gibelio) and zebrafish (Danio rerio). In both species, afp4a and afp4b display a head-to-tail tandem arrangement and share a common 4-exonic gene structure. In zebrafish, both afp4a and afp4b were found to express specifically in the yolk syncytial layer (YSL). Interestingly, afp4a expression continues in YSL and digestive system from early embryos to adults, whereas afp4b expression is restricted to embryogenesis. Importantly, we have shown by using afp4a-specific and afp4b-specifc morpholino knockdown and cell lineage tracing approaches that AFP4a participates in epiboly progression by stabilizing yolk cytoplasmic layer microtubules, and AFP4b is primarily related to convergence movement. Therefore, both AFP4 proteins are essential for gastrulation of zebrafish embryos. Our current results provide first evidence that AFP such as AFP4 has important roles in regulating developmental processes besides its well-known function as antifreeze factors.

Wider geographic distribution and higher diversity of hexaploids than tetraploids in Carassius species complex reveal recurrent polyploidy effects on adaptive evolution

Polyploidy roles on adaptive evolution and ecological novelty have been extensively studied in plants but remained unclear in vertebrates owing to the rare polyploidy incidences. Here, a huge number of 3105 specimens in Carassius species complex including 2211 hexaploids and 894 tetraploids were sampled from 34 locations through mainland China. And hexaploids had wider geographic distribution than tetraploids especially in the areas with high altitude, high latitude and low annual precipitation. Then, an approximate 1050 bp transferrin (tf) fragments were amplified from all the samples, and 526 tf alleles were identified from a total of 37260 sequences at last. Intriguingly, higher nucleotide diversity of tf alleles in hexaploids than in tetraploids was revealed. Moreover, via phylogenetic analysis of tf alleles, potential origin center of Carassius species complex was deduced to be Yangtze River basin and hexaploids should undergo multiple independent polyploidy origins from sympatric tetraploids. These findings indicate that the hexaploids might possess stronger environmental adaptation and ecological novelty than the tetraploids, which provide an association paradigm of recurrent polyploidy and ecological context in polyploid vertebrates.

Numerous mitochondrial DNA haplotypes reveal multiple independent polyploidy origins of hexaploids in Carassius species complex

Abstract Evolutionary trajectory and occurrence history of polyploidy have been extensively studied in plants, but they remain quite elusive in vertebrates. Here, we sampled and gathered 4,159 specimens of polyploid Carassius species complex including 1,336 tetraploids and 2,823 hexaploids from a large geographic scale (49 localities) across East Asia, and identified a huge number of 427 diverse haplotypes of mitochondrial control region, in which 74 haplotypes with total occurrence frequency up to 75.498% were shared by hexaploids and tetraploids. Significantly, these diverse haplotypes were clustered into four major lineages, and many haplotypes of hexaploids and tetraploids were intermixed in every lineage. Moreover, the evolutionary trajectory and occurrence history of four different lineages were revealed by a simplified time‐calibrated phylogenetic tree, and their geographic distribution frequencies and haplotype diversity were also analyzed. Furthermore, lineage C and D were revealed to undergo population expansion throughout mainland China. Therefore, our current data indicate that hexaploids should undergo multiple independent polyploidy origins from sympatric tetraploids in the polyploid Carassius species complex across East Asia.

Origin and transition of sex determination mechanisms in a gynogenetic hexaploid fish

Most vertebrates reproduce sexually, and plastic sex determination mechanisms including genotypic sex determination (GSD) and environmental sex determination (ESD) have been extensively revealed. However, why sex determination mechanisms evolve diversely and how they correlate with diverse reproduction strategies remain largely unclear. Here, we utilize the superiority of a hexaploid gibel carp (Carassius gibelio) that is able to reproduce by unisexual gynogenesis and contains a rare but diverse proportion of males to investigate these puzzles. A total of 2248 hexaploid specimens were collected from 34 geographic wild populations throughout mainland China, in which 24 populations were revealed to contain 186 males with various incidences ranging from 1.2 to 26.5%. Subsequently, the proportion of temperature-dependent sex determination (TSD) was revealed to be positively correlated to average annual temperature in wild populations, and male incidence in lab gynogenetic progenies was demonstrated to increase with the increasing of larval rearing temperature. Meanwhile, extra microchromosomes were confirmed to play genotypic male determination role as previously reported. Thereby, GSD and TSD were found to coexist in gibel carp, and the proportions of GSD were observed to be much higher than that of TSD in sympatric wild populations. Our findings uncover a potential new mechanism in the evolution of sex determination system in polyploid vertebrates with unisexual gynogenesis ability, and also reveal a possible association of sex determination mechanism transition between TSD and GSD and reproduction mode transition between unisexual gynogenesis and bisexual reproduction.

Molecular characterization and expression of an oocyte-specific histone stem-loop binding protein in Carassius gibelio.

Stem-loop-binding proteins (SLBPs) have been revealed to interact with stem-loop of histones, and oocyte-specific and oocyte-preferential SLBP2 have been identified in vertebrates including Xenopus (S. tropicalis) and B. taurus to play a key role in histone translation regulation, but no oocyte-specific SLBPs have been characterized in fish. Here, we have identified and characterized the first fish oocyte-specific SLBP2 in Carassius gibelio. Its full-length cDNA contains 975 bp ORF encoding 324 amino acids. Firstly, the polyclonal antibody specific to C. gibelio SLBP2 was prepared. Then, RT-PCR analysis and Western blot detection revealed its oocyte-specific and dynamic expression pattern during oogenesis and embryogenesis of C. gibelio. Moreover, in situ hybridization and immunofluorescence localization observed its abundant expression in cortical alveolar stage oocytes and dynamic distribution in different stage oocytes. Altogether, our current data suggest that C. gibelio SLBP2 might play significant role in the early oogenesis and oocyte growth.

Distinct sperm nucleus behaviors between genotypic and temperature-dependent sex determination males are associated with replication and expression-related pathways in a gynogenetic fish

BackgroundCoexistence and transition of diverse sex determination strategies have been revealed in some ectothermic species, but the variation between males caused by different sex determination strategies and the underlying mechanism remain unclear. Here, we used the gynogenetic gibel carp (Carassius gibelio) with both genotypic sex determination (GSD) and temperature-dependent sex determination (TSD) strategies to illustrate this issue.ResultsWe found out that males of GSD and TSD in gibel carp had similar morphology, testicular histology, sperm structure and sperm vitality. However, when maternal individuals were mated with males of GSD, sperm nucleus swelling and fusing with the female pronucleus were observed in the fertilized eggs. On the contrary, when maternal individuals were mated with males of TSD, sperm nucleus remained in the condensed status throughout the whole process. Subsequently, semen proteomics analysis unveiled that DNA replication and gene expression-related pathways were inhibited in the sperm from males of TSD compared to males of GSD, and most differentially expressed proteins associated with DNA replication, transcription and translation were down-regulated. Moreover, via BrdU incorporation and immunofluorescence detection, male nucleus replication was revealed to be present in the fertilized eggs by the sperm from males of GSD, but absent in the fertilized eggs by the sperm from males of TSD.ConclusionsThese findings indicate that DNA replication and gene expression-related pathways are associated with the distinct sperm nucleus development behaviors in fertilized eggs in response to the sperm from males of GSD and TSD. And this study is the first attempt to screen the differences between males determined via GSD and TSD in gynogenetic species, which might give a hint for understanding evolutionary adaption of diverse sex determination mechanisms in unisexual vertebrates.