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Infertile ovotestis (mixture of ovary and testis) often occurs in intersex individuals under certain pathological and physiological conditions. However, how ovotestis is formed remains largely unknown. Here, we report the first comprehensive single-cell developmental atlas of the model ovotestis. We provide an overview of cell identities and a roadmap of germline, niche, and stem cell development in ovotestis by cell lineage reconstruction and a uniform manifold approximation and projection. We identify common progenitors of germline stem cells with two states, which reveal their bipotential nature to differentiate into both spermatogonial stem cells and female germline stem cells. Moreover, we found that ovotestis infertility was caused by degradation of female germline cells via liquid–liquid phase separation of the proteasomes in the nucleus, and impaired histone-to-protamine replacement in spermatid differentiation. Notably, signaling pathways in gonadal niche cells and their interaction with germlines synergistically determined distinct cell fate of both male and female germlines. Overall, we reveal a cellular fate map of germline and niche cell development that shapes cell differentiation direction of ovotestis, and provide novel insights into ovotestis development.

Gonadal trans-differentiation from ovary to testis occurs in a same individual, suggesting a role of epigenetic regulation. However, histone modifications concerning the sex reversal process remain elusive. We analyzed histone modifications using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology was used to test chromatin immunoprecipitation of gonads. Western blot analysis was performed to analyze protein expression. Immunofluorescence analysis was conducted to localize proteins in gonadal tissues. Here, we report a developmental atlas of histone modifications in the gonadal differentiation, including acetylation, methylation, and ubiquitination. We provided a detail distribution map of these modification sites including novel histone modifications along histones H2a, H2b, H3, and H4, and revealed their relationship with types of gonadal differentiation. We then determined a testis-enriched histone modification site, H2b monoubiquitination at K120, and its association with spermatogenesis. ChIP-seq demonstrated that the modification was highly enriched in the male sex-determining gene dmrt1 (doublesex and mab-3 related transcription factor 1), in particular, in its exon regions, suggesting its role in transcriptional regulation of dmrt1 in testis. Together, these data not only provide a new resource for epigenetic study in gonadal development, but also define an association of histone modifications with gonadal differentiation from ovary to testis.

Selective autophagic degradation of mitochondria (mitophagy) is important in maintaining proper cellular homeostasis. Here, we found that SPATA33 is a novel autophagy mediator for mitophagy in testis. The SPATA33 protein localizes on mitochondria via its binding of the carboxyl terminal with the outer mitochondrial membrane protein VDAC2. Upon starvation induction, SPATA33 is recruited to autophagosome by binding the autophagy machinery ATG16L1 via its N-terminal along with mitochondria. Notably, Spata33 knockout inhibited autophagy and overexpression can promote autophagosome formation for mitochondrial sequestration. Therefore, SPATA33 confers selectivity for mitochondrial degradation and promotes mitophagy in male germline cells.

Spermatogenesis is an essential process for producing sperm cells. Reproductive strategy is successfully evolved for a species to adapt to a certain ecological system. However, roles of newly evolved genes in testis autophagy remain unclear. In this study, we found that a newly evolved gene srag (Sox9-regulated autophagy gene) plays an important role in promoting autophagy in testis in the lineage of the teleost Monopterus albus. The gene integrated into an interaction network through a two-way strategy of evolution, via Sox9-binding in its promoter and interaction with Becn1 in the coding region. Its promoter region evolved a cis element for binding of Sox9, a transcription factor for male sex determination. Both in vitro and in vivo analyses demonstrated that transcription factor Sox9 could bind to and activate the srag promoter. Its coding region acquired ability to interact with key autophagy initiation factor Becn1 via the conserved C-terminal, indicating that srag integrated into preexisting autophagy network. Moreover, we determined that Srag enhanced autophagy by interacting with Becn1. Notably, srag transgenic zebrafish revealed that Srag exerted the same function by enhancing autophagy through the Srag–Becn1 pathway. Thus, the new gene srag regulated autophagy in testis by integrated into preexisting autophagy network.

BackgroundTeleost fishes account for over half of extant vertebrate species. A core question in biology is how genomic changes drive phenotypic diversity that relates to the origin of teleost fishes.ResultsHere, we used comparative genomic analyses with chromosome assemblies of diverse lineages of vertebrates and reconstructed an ancestral vertebrate genome, which revealed phylogenomic trajectories in vertebrates. We found that the whole-genome-wide chromosome fission/fusions took place in theMonopterus albuslineage after the 3-round whole-genome duplication. Four times of genomic fission/fusions events resulted in the whole genome-wide chromosome fusions in the genomic history of the lineage. In addition, abundant recently evolved new genes for reproduction emerged in theMonopterus albusafter separated from medaka. Notably, we described evolutionary trajectories of conserved blocks related to sex determination genes in teleosts.ConclusionsThese data pave the way for a better understanding of genomic evolution in extant teleosts.

Detecting selection signatures in genomes that relates to transcription regulation has been challenges in genetic analysis. Here, we report a set of transcription factors EBF1, E2F1 and EGR2 for transcription activation of RAB37 promoter by a comparative analysis of promoter activities of RAB37 in humans, mice, and pigs. Two of the transcription factors bound to and co-regulated RAB37 promoter in each species. SNPs were further screened in pig RAB37 gene by population genomics in pig populations from both China and Europe. Three SNPs were identified in second CpG island upstream of core promoter of RAB37. These SNP variations led to at least 5 haplotypes, representing 5 multiple alleles of RAB37 in pig population. Distribution of these alleles in different genetic background of breeds showed a role of artificial selection for the variations of these multiple alleles. Of them, RAB37-c acquired the highest ability to activate gene expression in comparison with the other promoters, thus enhanced autophagy efficiently. These findings provide better understanding of transcription activation of RAB37 and artificial selection via RAB37 for autophagy regulation.

Over the past 440 years since the discovery of the medicinal value of swamp eels, much progress has been made in the study of their biology. The fish is emerging as an important model animal in sexual development, in addition to economic and pharmaceutical implications. Tracing genomic history that shapes speciation of the fish has led to discovery of the whole genome-wide chromosome fission/fusion events. Natural intersex differentiation is a compelling feature for sexual development research. Notably, identification of progenitors of germline stem cells that have bipotential to differentiate into either male or female germline stem cells provides new insight into sex reversal. Here, we review these advances that have propelled the field forwards and present unsolved issues that will guide future investigations to finally elucidate vertebrate sexual development using the new model.

Photoreceptor degeneration and damages often lead to blindness, and the underlying molecular mechanisms are largely unknown. There is also a lot of missing information for establishing the role of macroautophagy/autophagy in the retinopathy. We recently generated knockout mouse lines of the essential gene Tubgcp4 (tubulin, gamma complex associated protein 4) and revealed an interplay between essential genes and autophagy regulation. Complete knockout of Tubgcp4 in mice results in early embryonic lethality due to abnormal spindle assembly, whereas heterozygotes are viable through dosage compensation from one wild-type allele, suggesting a dosage effect of the essential gene. However, haploinsufficiency of TUBGCP4 impairs assembly of TUBG/γ-tubulin ring complexes and disturbs autophagy homeostasis of the retina, with pathological phenotypes of photoreceptor degeneration and a decrease of electroretinography responses. TUBGCP4 can inhibit autophagy by competing with ATG3 to interact with ATG7, thus interfering with lipidation of LC3B. Taken together, these findings demonstrate dosage effect of the essential gene Tubgcp4 for viability of mutant mice, and suggest key roles of TUBGCP4 in embryo development and retinal homeostasis by autophagy regulation. Abbreviations: ATG3: autophagy related 3; ATG7: autophagy related 7; CRISPR: clustered regularly interspaced short palindromic repeats; ERG: electroretinography; HCQ: hydroxychloroquine; LC3B: microtubule-associated protein 1 light chain 3 beta; NFE2L2: nuclear factor, erythroid 2 like 2; ONL: outer nuclear layer; PPARGC1A: peroxisome proliferator-activated receptor gamma coactivator-1 alpha; RB1CC1: RB1 inducible coiled-coil 1; SQSTM1: sequestosome 1; TUBGCP: tubulin, gamma complex associated protein; TUBGRC/γ: TuRCs gamma-tubulin ring complexes.

Retinopathy, owing to damage to the retina, often causes vision impairment, and the underlying molecular mechanisms are largely unknown. Using a gene targeting strategy, we generated mice with the essential gene Tubgcp4 knocked out. Homozygous mutation of Tubgcp4 resulted in early embryonic lethality due to abnormal spindle assembly caused by GCP4 (gamma-tubulin complex protein 4, encoded by Tubgcp4) depletion. Heterozygotes were viable through dosage compensation of one wild-type allele. However, haploinsufficiency of GCP4 affected the assembly of γ-TuRCs (γ-tubulin ring complexes) and disrupted autophagy homeostasis in retina, thus leading to photoreceptor degeneration and retinopathy. Notably, GCP4 exerted autophagy inhibition by competing with ATG3 for interaction with ATG7, thus interfering with lipidation of LC3B. Our findings justify dosage effects of essential genes that compensate for null alleles in viability of mutant mice and uncover dosage-dependent roles of GCP4 in embryo development and retinal homeostasis. These data have also clinical implications in genetic counseling on embryonic lethality and in development of potential therapeutic targets associated with retinopathy.

Although depression is associated with anxiety and memory deficit in humans, the molecular mechanisms of the complication remain largely unknown. In this study, we generated P11 knockout mice using CRISPR/Cas9 technology, as well as P11 knockout MEF cell lines, and confirmed depression-like phenotype. We observed that knockout of P11 in MEFs led to a decreased cell proliferation compared with P11 +/+ MEFs. Moreover, P11 knockout resulted in a larger cell size, which resulted probably from accumulated F-actin stress fibers. The number of proliferating cells was decreased in the hippocampus of P11 KO mice. We observed anxiety-like disorder in addition to depression phenotype in the knockout mice. In addition, knockout of P11 led to memory deficit in female mice, but not in males. These data indicated that P11 is involved in regulating cell proliferation and cell size. The molecular associations of depression behavior with anxiety and memory deficit suggested a potential approach to improve therapeutic intervention through P11 in these disorders.

Selective autophagic degradation of mitochondria (mitophagy) is important in maintaining proper cellular homeostasis. Here, we found that SPATA33 is a novel autophagy mediator for mitophagy in testis. The SPATA33 protein localizes on mitochondria via its binding of the carboxyl terminal with the outer mitochondrial membrane protein VDAC2. Upon starvation induction, SPATA33 is recruited to autophagosome by binding the autophagy machinery ATG16L1 via its N-terminal along with mitochondria. Notably, Spata33 knockout inhibited autophagy and overexpression can promote autophagosome formation for mitochondrial sequestration. Therefore, SPATA33 confers selectivity for mitochondrial degradation and promotes mitophagy in male germline cells.

BackgroundZebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors.ResultsHere, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS).ConclusionsOverall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

Nearly all diseases in humans, to a certain extent, exhibit sex differences, including differences in the onset, progression, prevention, therapy, and prognosis of diseases. Accumulating evidence shows that macroautophagy/autophagy, as a mechanism for development, differentiation, survival, and homeostasis, is involved in numerous aspects of sex differences in diseases such as cancer, neurodegeneration, and cardiovascular diseases. Advances in our knowledge regarding sex differences in autophagy-mediated diseases have enabled an understanding of their roles in human diseases, although the underlying molecular mechanisms of sex differences in autophagy remain largely unexplored. In this review, we discuss current advances in our insight into the biology of sex differences in autophagy and disease, information that will facilitate precision medicine.Abbreviations: AD: Azheimer disease; AMBRA1: autophagy and beclin 1 regulator 1; APP: amyloid beta precursor protein; AR: androgen receptor; AMPK: AMP-activated protein kinase; ATG: autophagy related; ATP6AP2: ATPase H+ transporting accessory protein 2; BCL2L1: BCL2 like 1; BECN1: beclin 1; CTSD: cathepsin D; CYP19A1: cytochrome P450 family 19 subfamily A member 1; DSD: disorders of sex development; eALDI: enhancer alternate long-distance initiator; ESR1: estrogen receptor 1; ESR2: estrogen receptor 2; FYCO1: FYVE and coiled-coil domain autophagy adaptor 1; GABARAP: GABA type A receptor-associated protein; GLA: galactosidase alpha; GTEx: genotype-tissue expression; HDAC6: histone deacetylase 6; I-R: ischemia-reperfusion; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; m6A: N6-methyladenosine; MYBL2: MYB proto-oncogene like 2; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PSEN1: presenilin 1; PSEN2: presenilin 2; RAB9A, RAB9A: member RAS oncogene family; RAB9B, RAB9B: member RAS oncogene family; RAB40AL: RAB40A like; SF1: splicing factor 1; SOX9: SRY-box transcription factor 9; SRY: sex determining region Y; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; VDAC2: voltage dependent anion channel 2; WDR45: WD repeat domain 45; XPDS: X-linked parkinsonism and spasticity; YTHDF2: YTH N6-methyladenosine RNA binding protein 2.

Additional file 1. Table S1. Primer sequences and PCR conditions. Table S2. Linearities of m5dC and hm5dC by LC-MS analysis. Table S3. Mass spectrometry parameters for the analysis of nucleosides.

Additional file 1. Additional figures and tables.

Gene expansion and contraction are important evolution events. Some tdrd genes, especially multi-Tudor members, participate in Piwi-interacting RNA pathway and spermatogenesis. However, tdrd evolution and their functions in teleost fish are poorly understood. Here, we identified 14 tdrds in the teleost fish, swamp eel, which were clustered into 12 tdrd branches. Comparative synteny showed biased duplications and loss of members in the tdrd family. Both tdrd6 and tdrd7 were duplicated in the teleost fish, whereas tdrd8 was lost from the original locus. Expression analysis at both RNA and protein levels showed that tdrd6l, a duplicated multi-Tudor member, was gonad enriched. Expression pattern of tdrd6l in follicular epithelium and seminiferous epithelium during sex reversal supports its potential role in genome defense in germline.

We described genome-wide screening and characterization of microsatellites in the swamp eel genome. A total of 99,293 microsatellite loci were identified in the genome with an overall density of 179 microsatellites per megabase of genomic sequences. The dinucleotide microsatellites were the most abundant type representing 71% of the total microsatellite loci and the AC-rich motifs were the most recurrent in all repeat types. Microsatellite frequency decreased as numbers of repeat units increased, which was more obvious in long than short microsatellite motifs. Most of microsatellites were located in non-coding regions, whereas only approximately 1% of the microsatellites were detected in coding regions. Trinucleotide repeats were most abundant microsatellites in the coding regions, which represented amino acid repeats in proteins. There was a chromosome-biased distribution of microsatellites in non-coding regions, with the highest density of 203.95/Mb on chromosome 8 and the least on chromosome 7 (164.06/Mb). The most abundant dinucleotides (AC)n was mainly located on chromosome 8. Notably, genomic mapping showed that there was a chromosome-biased association of genomic distributions between microsatellites and transposon elements. Thus, the novel dataset of microsatellites in swamp eel provides a valuable resource for further studies on QTL-based selection breeding, genetic resource conservation and evolutionary genetics.

Macroautophagy/autophagy is a catabolic process that is essential for cellular homeostasis. How autophagosomal vesicle forms in a spatio-temporally regulated manner remains elusive. Our recent study revealed that small GTPase, RAB37 (RAB37, member RAS oncogene family), functions as a key organizer of autophagosomal membrane biogenesis. RAB37 interacts with ATG5 (autophagy related 5) and promotes autophagosome formation by modulating ATG12–ATG5-ATG16L1 complex assembly. These findings provide new insights into autophagy regulation.

Photoreceptor degeneration and damages often lead to blindness, and the underlying molecular mechanisms are largely unknown. There is also a lot of missing information for establishing the role of macroautophagy/autophagy in the retinopathy. We recently generated knockout mouse lines of the essential gene Tubgcp4 (tubulin, gamma complex associated protein 4) and revealed an interplay between essential genes and autophagy regulation. Complete knockout of Tubgcp4 in mice results in early embryonic lethality due to abnormal spindle assembly, whereas heterozygotes are viable through dosage compensation from one wild-type allele, suggesting a dosage effect of the essential gene. However, haploinsufficiency of TUBGCP4 impairs assembly of TUBG/γ-tubulin ring complexes and disturbs autophagy homeostasis of the retina, with pathological phenotypes of photoreceptor degeneration and a decrease of electroretinography responses. TUBGCP4 can inhibit autophagy by competing with ATG3 to interact with ATG7, thus interfering with lipidation of LC3B. Taken together, these findings demonstrate dosage effect of the essential gene Tubgcp4 for viability of mutant mice, and suggest key roles of TUBGCP4 in embryo development and retinal homeostasis by autophagy regulation. Abbreviations: ATG3: autophagy related 3; ATG7: autophagy related 7; CRISPR: clustered regularly interspaced short palindromic repeats; ERG: electroretinography; HCQ: hydroxychloroquine; LC3B: microtubule-associated protein 1 light chain 3 beta; NFE2L2: nuclear factor, erythroid 2 like 2; ONL: outer nuclear layer; PPARGC1A: peroxisome proliferator-activated receptor gamma coactivator-1 alpha; RB1CC1: RB1 inducible coiled-coil 1; SQSTM1: sequestosome 1; TUBGCP: tubulin, gamma complex associated protein; TUBGRC/γ: TuRCs gamma-tubulin ring complexes

Summary A cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents., Graphical Abstract, Highlights • Zebrafish embryos may start to exit from pluripotency shortly after the oblong stage • Beating cell clusters are efficiently generated from zebrafish blastomeres • Beating cell clusters contain specific cardiomyocyte subtypes • Induced cardiomyocytes possess normal electrophysiological features, In this article, Zhou, Cheng, and colleagues describe a method of cardiomyocyte differentiation from primary embryonic cells in zebrafish after determining a pluripotency window of embryos. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes, which exhibited typical contractile kinetics and electrophysiological features. The technology provides a new platform for the study of heart development and regeneration.

Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint.

Insertion and deletion (indel) mutations, which can trigger single nucleotide substitutions on the flanking regions of genes, may generate abundant materials for disease defense, reproduction, species survival and evolution. However, genetic and evolutionary mechanisms of indels remain elusive. We establish a comparative genome-transcriptome-alignment approach for a large-scale identification of indels in Monopterus population. Over 2000 indels in 1738 indel genes, including 1-21 bp deletions and 1-15 bp insertions, were detected. Each indel gene had ∼1.1 deletions/insertions, and 2-4 alleles in population. Frequencies of deletions were prominently higher than those of insertions on both genome and population levels. Most of the indels led to in frame mutations with multiples of three and majorly occurred in non-domain regions, indicating functional constraint or tolerance of the indels. All indel genes showed higher expression levels than non-indel genes during sex reversal. Slide window analysis of global expression levels in gonads showed a significant positive correlation with indel density in the genome. Moreover, indel genes were evolutionarily conserved and evolved slowly compared to non-indel genes. Notably, population genetic structure of indels revealed divergent evolution of Monopterus population, as bottleneck effect of biogeographic isolation by Taiwan Strait, China.

Intracellular membrane trafficking is essential for eukaryotic cell existence. Here, we show that RAB37 activation through GTP binding recruits ATG5-12 to isolation membrane and promotes autophagosome formation through the ATG5-ATG12-ATG16L1 complex. RAB37 is localized on the isolation membrane. It can bind directly with ATG5 and promotes formation of the ATG5-12-16 complex. Mutation analysis reveals that GTP-bound RAB37 exhibits an enhanced interaction with ATG5-12 and GDP-stabilised mutation impairs the interaction. RAB37 promotes ATG5-12 interaction with ATG16L1, thus facilitates lipidation of LC3B in a GTP-dependent manner to enhance autophagy. Notably, ablation of RAB37 expression affects the complex formation and decreases autophagy, whereas forced RAB37 expression promotes autophagy and also suppresses cell proliferation. Our results demonstrate a role of RAB37 in autophagosome formation through a molecular connection of RAB37, ATG5-12, ATG16L1 up to LC3B, suggesting an organiser role of RAB37 during autophagosomal membrane biogenesis. These findings have broad implications for understanding the role of RAB vesicle transport in autophagy and cancer.

Germline stem cells are essential in the generation of both male and female gametes. In mammals, the male testis produces sperm throughout the entire lifetime, facilitated by testicular germline stem cells. Oocyte renewal ceases in postnatal or adult life in mammalian females, suggesting that germline stem cells are absent from the mammalian ovary. However, studies in mice, rats, and humans have recently provided evidence for ovarian female germline stem cells (FGSCs). A better understanding of the role of FGSCs in ovaries could help improve fertility treatments. Here, we developed a rapid and efficient method for isolating FGSCs from ovaries of neonatal mice. Notably, our FGSC isolation method could efficiently isolate on average 15 cell "strings" per ovary from mice at 1-3 days postpartum. FGSCs isolated from neonatal mice displayed the string-forming cell configuration at mitosis (i.e. a "stringing" FGSC (sFGSC) phenotype) and a disperse phenotype in postnatal mice. We also found that sFGSCs undergo vigorous mitosis especially at 1-3 days postpartum. After cell division, the sFGSC membranes tended to be connected to form sFGSCs. Moreover, F-actin filaments exhibited a cell-cortex distribution in sFGSCs, and E-cadherin converged in cell-cell connection regions, resulting in the string-forming morphology. Our new method provides a platform for isolating FGSCs from the neonatal ovary, and our findings indicate that FGCSs exhibit string-forming features in neonatal mice. The sFGSCs represent a valuable resource for analysis of ovary function and an in vitro model for future clinical use to address ovarian dysfunction.

Gene expansion and contraction are important evolution events. Some tdrd genes, especially multi-Tudor members, participate in Piwi-interacting RNA pathway and spermatogenesis. However, tdrd evolution and their functions in teleost fish are poorly understood. Here, we identified 14 tdrds in the teleost fish, swamp eel, which were clustered into 12 tdrd branches. Comparative synteny showed biased duplications and loss of members in the tdrd family. Both tdrd6 and tdrd7 were duplicated in the teleost fish, whereas tdrd8 was lost from the original locus. Expression analysis at both RNA and protein levels showed that tdrd6l, a duplicated multi-Tudor member, was gonad enriched. Expression pattern of tdrd6l in follicular epithelium and seminiferous epithelium during sex reversal supports its potential role in genome defense in germline.

The swamp eel is a teleost fish with a characteristic of natural sex reversal and an ideal model for vertebrate sexual development. However, underlying molecular mechanisms are poorly understood. We report the identification of five DM (doublesex and mab-3) domain genes in the swamp eel that include Dmrt2, Dmrt2b, Dmrt3, Dmrt4 and Dmrt5, which encode putative proteins of 527, 373, 471, 420 and 448 amino acids, respectively. Phylogenetic tree showed that these genes are clustered into corresponding branches of the DM genes in vertebrates. Southern blot analysis indicated that the Dmrt1-Dmrt3-Dmrt2 genes are tightly linked in a conserved gene cluster. Notably, these Dmrt genes are up-regulated during gonad transformation. Furthermore, mRNA in situ hybridisation showed that Dmrt2, Dmrt3, Dmrt4 and Dmrt5 are expressed in developing germ cells. These results are evidence that the DM genes are involved in sexual differentiation in the swamp eel.

Loss and/or inactivation of the VHL (von Hippel–Lindau) tumour suppressor causes various tumours. Using a yeast two-hybrid system, we have identified the AR (androgen receptor) co-activator UXT (ubiquitously expressed transcript), as a VHL-interacting protein. GST pull-down and co-immunoprecipitation assays show that UXT interacts with VHL. In addition, UXT recruits VHL to the nucleus. VHL associates with the DBD (DNA-binding domain) and hinge domains of the AR and induces AR ubiquitination. Moreover, VHL interaction with the AR activates AR transactivation upon DHT (dihydrotestosterone) treatment. VHL knockdown inhibits AR ubiquitination and decreases transcriptional activation of the AR. Our data suggest that the VHL–UXT interaction and VHL-induced ubiquitination of AR regulate transcriptional activation of the AR.

Background information. The translationally controlled tumour protein (TCTP) plays an important role in maintaining cell proliferation and its high expression is associated with many tumours. The tumour suppressor von Hippel– Lindau protein (VHL) has been shown to function as an E3 ubiquitin ligase. Although great progress has been made, biological roles of these factors and relevant molecular mechanisms remain largely unknown. Results. In this study, we have shown that TCTP specifically binds to VHL through its β domain and competes with hypoxia-inducible factor-1α (HIF1α). TCTP over-expression decreased the protein level of VHL and the inhibition of TCTP expression by miRNA resulted in an increase of the VHL protein level. Moreover, TCTP over-expression promoted the K48-linked ubiquitination of VHL, thus degradation through the ubiquitin–proteasome pathway. In addition, we showed that TCTP increased the protein level of HIF1α, which promoted both vascular endothelial growth factor–hypoxic response element-promoter-driven luciferase reporter and endogenous VEGF expression. Conclusions. These data have demonstrated that TCTP binds to the β domain of VHL through competition with HIF1α, which promotes VHL degradation by the ubiquitin–proteasome system and HIF1α stability. Additional supporting information may be found in the online version of this article at the publisher’s web-site.

Macroautophagy/autophagy is a catabolic process that is essential for cellular homeostasis. Studies on autophagic degradation of cytoplasmic components have generated interest in nuclear autophagy. Although its mechanisms and roles have remained elusive, tremendous progress has been made toward understanding nuclear autophagy. Nuclear autophagy is evolutionarily conserved in eukaryotes that may target various nuclear components through a series of processes, including nuclear sensing, nuclear export, autophagic substrate encapsulation and autophagic degradation in the cytoplasm. However, the molecular processes and regulatory mechanisms involved in nuclear autophagy remain largely unknown. Numerous studies have highlighted the importance of nuclear autophagy in physiological and pathological processes such as cancer. This review focuses on current advances in nuclear autophagy and provides a summary of its research history and landmark discoveries to offer new perspectives.

Spermatogenesis is a highly regulated process during which haploid sperm cells are generated. Although autophagy is involved in the spermatogenesis process, the molecular pathways and regulations of autophagy in germ cell development remain elusive. Here, we showed that Ppp1r36, a regulatory subunit of protein phosphatase 1, is expressed during gonadal development, mainly in testes during spermatogenesis. Autophagy protein LC3 (microtubule associated protein 1 light chain 3), especially its active form LC3-II, had a similar expression pattern to Ppp1r36. Moreover, LC3-II level and puncta analysis showed that autophagy is up-regulated around 21 dpp (day postpartum) in postnatal testis, indicating a potential role of autophagy during the first wave of spermatogenesis. We demonstrated that Ppp1r36 promotes autophagosome formation upon starvation induction. Further autophagy flux analysis using a tandem fluorescent indicator, mCherry-GFP-LC3, confirmed that Ppp1r36 participated in autophagy. We further determined that Ppp1r36 is associated with Atg16L1 (autophagy related 16-like 1) in autophagy of starvation induction. Thus, our results uncover a potential role of the regulatory subunit Ppp1r36 of protein phosphatase 1 in enhancing autophagy during spermatogenesis.