Srag Regulates Autophagy via Integrating into a Preexisting Autophagy Pathway in Testis.

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.

Biased Duplications and Loss of Members in Tdrd Family in Teleost Fish.

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.

RNF20 is required for male fertility through regulation of H2B ubiquitination in the Sertoli cells.

BACKGROUND: Spermatogenesis depends on the supporting of the Sertoli cells and their communications with germ cells. However, the regulation of crosstalk between the Sertoli cells and germ cells remains unclear. RESULTS: In this report, we used conditional knockout technology to generate the Sertoli cells-specific knockout of Rnf20 in mice. The Amh-Rnf20-/- male mice were infertile owing to spermatogenic failure that mimic the Sertoli cell-only syndrome (SCOS) in humans. Knockout of Rnf20 resulted in the H2BK120ub loss in the Sertoli cells and impaired the transcription elongation of the Cldn11, a gene encoding a component of tight junction. Notably, RNF20 deficiency disrupted the cell adhesion, caused disorganization of the seminiferous tubules, and led to the apoptotic cell death of both spermatogonia and spermatocytes in the seminiferous tubules. CONCLUSIONS: This study describes a Rnf20 knockout mouse model that recapitulates the Sertoli cell-only syndrome in humans and demonstrates that RNF20 is required for male fertility through regulation of H2B ubiquitination in the Sertoli cells.

Cellular fate of intersex differentiation.

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.

Identification of Histone Modifications Reveals a Role of H2b Monoubiquitination in Transcriptional Regulation of dmrt1 in Monopterus albus.

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.

DNA methylation modification is associated with gonadal differentiation in Monopterus albus.

BACKGROUND: Both testis and ovary can be produced sequentially in an individual with the same genome when sex reversal occurs in the teleost Monopterus albus, and epigenetic modification is supposed to be involved in gonadal differentiation. However, DNA methylation regulation mechanism underlying the gonadal differentiation remains unclear. RESULTS: Here, we used liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to simultaneously determine endogenous levels of both 5-methyl-2'-deoxycytidine (m5dC) and 5-hydroxymethyl-2'-deoxycytidine (hm5dC) during gonadal differentiation. Overall DNA methylation level was upregulated from ovary to testis via ovotestis. As a de novo methylase, dnmt3aa expression was also upregulated in the process. Notably, we determined transcription factor Foxa1 for dnmt3aa gene expression. Site-specific mutations and chromatin immunoprecipitation showed that Foxa1 can bind to and activate the dnmt3aa promoter. Furthermore, DNA methylation levels of key genes foxl2 (forkhead box L2) and cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) in regulation of female hormone synthesis were consistently upregulated during gonadal differentiation. CONCLUSIONS: These data suggested that dynamic change of DNA methylation modification is associated with gonadal differentiation.

Whole genome-wide chromosome fusion and new gene birth in the Monopterus albus genome.

BACKGROUND: Teleost 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. RESULTS: Here, 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 the Monopterus albus lineage 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 the Monopterus albus after separated from medaka. Notably, we described evolutionary trajectories of conserved blocks related to sex determination genes in teleosts. CONCLUSIONS: These data pave the way for a better understanding of genomic evolution in extant teleosts.

Potential of the glasses-free three-dimensional display system in shortening the learning curve of video-assisted endoscopic surgery: a self-controlled ex-vivo study.

BACKGROUND: One of the largest challenges in endoscopic surgical training is adapting to a two-dimensional (2D) view. The glasses-free three-dimensional (GF-3D) display system was designed to integrate the merits of both 2D and conventional 3D (C-3D) displays, allowing surgeons to perform video-assisted endoscopic surgery under a stereoscopic view without heavy and cumbersome 3D glasses. METHODS: In this study, 15 junior thoracic surgeons were divided to test one routine and one complex task three times each via traditional high-definition 2D (HD-2D) and GF-3D to determine whether there was any advantage when using the GF-3D system to acquire endoscopic skills. The duration, numbers of stitches, and distance between every two stitches were recorded for every procedure. RESULTS: Seven participants were enrolled in the HD-2D group and eight participants were enrolled in the GF-3D group. All 15 participants successfully completed porcine skin continuous suture and tracheal continuous anastomosis procedures three times each. For skin continuous suture, there was no significant difference between the two groups in terms of the learning curve for speed (P=0.683) and accuracy (P=0.556). For tracheal continuous anastomosis, there was a significant difference between the two groups in terms of the learning curve for speed (P=0.001), but no significant difference was observed between the two groups in terms of the learning curve for accuracy (P=0.211). CONCLUSIONS: In summary, both HD-2D and GF-3D display systems are efficient for routine and complex endoscopic surgery. With the help of GF-3D, surgeons can acquire new complex endoscopic skills faster than HD-2D and be free from burdensome polarized glasses. More comparative studies in a clinical setting are needed to further explore the feasibility, necessity, and economic aspects of the GF-3D display system.

Chromosome-scale assembly of the Monopterus genome.

Background: The teleost fish Monopterus albus is emerging as a new model for biological studies due to its natural sex transition and small genome, in addition to its enormous economic and potential medical value. However, no genomic information for the Monopterus is currently available. Findings: Here, we sequenced and de novo assembled the genome of M. albus and report the de novochromosome assembly by FISH walking assisted by conserved synteny (Cafs). Using Cafs, 328 scaffolds were assembled into 12 chromosomes, which covered genomic sequences of 555 Mb, accounting for 81.3% of the sequences assembled in scaffolds (∼689 Mb). A total of 18 ,660 genes were mapped on the chromosomes and showed a nonrandom distribution along chromosomes. Conclusions: We report the first reference genome of the Monopterus and provide an efficient Cafs strategy for a de novo chromosome-level assembly of the Monopterus genome, which provides a valuable resource, not only for further studies in genetics, evolution, and development, particularly sex determination, but also for breed improvement of the species.