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.

An optimized base editor with efficient C-to-T base editing in zebrafish.

BACKGROUND: Zebrafish 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. RESULTS: Here, 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). CONCLUSIONS: Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

Teaching practice in human genetics integrated into general education.

General education is an important part in higher education, which emphasizes the educational idea of integration of generality with specialty, and practices people-oriented education concept. However, there are some difficulties and puzzles in general education. Now the general education system with Chinese characteristics is needed to be established through practice and development. In this paper, we enumerate how to integrate knowledge of human genetics in practice of general education, teaching cases, and relevant analysis with concepts of general education. Using questions as "what are human beings?" as a leverage, we introduce teaching contents closely related to daily life. For example, we explain the past, present and future of human beings through contemporary evolutionary genomics teaching. In addition, we introduce problem-based deep thinking for students, thus integrating classical attributes of human beings into general education.

Isolation and characterization of string-forming female germline stem cells from ovaries of neonatal mice.

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.

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.

Long-term artificial selection reveals a role of TCTP in autophagy in mammalian cells.

Understanding genomic variation and detecting selection signatures in a genome under selection have been great challenges for a century. Activation, development/exhaustion of primordial follicles in mammalian ovary determines reproductive success, menopause/end of female reproductive life. However, molecular mechanisms underlying oogenesis, particularly under artificial selection, are largely unknown. We report that a proteome-wide scan for selection signatures in the genome over 9,000 years of artificial pressure on the ovary revealed a general picture of selection signatures in the genome, especially genomic variations through artificial selection were detected in promoter and intron regions. Crossbreeding between domestic and wild species results in more than half of the protein spots exhibiting heterosis. Translationally controlled tumor protein (TCTP) is upregulated by artificial selection and positively regulates autophagy through the AMP-activated protein kinase pathway. Notably, TCTP interacts with ATG16 complex. In addition to cytoplasmic autophagy, nucleophagy occurs in the nuclei of granulosa and cumulus cells in ovaries, indicating an importance of the nuclear material for degradation by nucleophagy. Our findings provide insight into cellular and molecular mechanisms relevant for improvement of ovary functions, and identify selection signatures in the genome for ovary function over long-term artificial selection pressure.

Loss-of-function mutation in the X-linked TBX22 promoter disrupts an ETS-1 binding site and leads to cleft palate.

The cleft palate only (CPO) is a common congenital defect with complex etiology in humans. The molecular etiology of the CPO remains unknown. Here, we report a loss-of-function mutation in X-linked TBX22 gene (T-box 22) in a six-generation family of the CPO with obvious phenotypes of both cleft palate and hyper-nasal speech. We identify a functional -73G>A mutation in the promoter of TBX22, which is located at the core-binding site of transcription factor ETS-1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1). Phylogenetic analysis showed that the sequence around the -73G>A mutation site is specific in primates. The mutation was detected in all five affected male members cosegregating with the affected phenotype and heterozygote occurred only in some unaffected females of the family, suggesting an X-linked transmission of the mutation in the family. The -73G>A variant is a novel single nucleotide mutation. Cell co-transfections indicated that ETS-1 could activate the TBX22 promoter. Moreover, EMSA and ChIP assays demonstrated that the allele A disrupts the binding site of ETS-1, thus markedly decreases the activity of the TBX22 promoter, which is likely to lead to the birth defect of the CPO without ankyloglossia. These results suggest that a loss-of-function mutation in the X-linked TBX22 promoter may cause the cleft palate through disruption of TBX22-ETS-1 pathway.

DNA demethylation and USF regulate the meiosis-specific expression of the mouse Miwi.

Miwi, a member of the Argonaute family, is required for initiating spermiogenesis; however, the mechanisms that regulate the expression of the Miwi gene remain unknown. By mutation analysis and transgenic models, we identified a 303 bp proximal promoter region of the mouse Miwi gene, which controls specific expression from midpachytene spermatocytes to round spermatids during meiosis. We characterized the binding sites of transcription factors NF-Y (Nuclear Factor Y) and USF (Upstream Stimulatory Factor) within the core promoter and found that both factors specifically bind to and activate the Miwi promoter. Methylation profiling of three CpG islands within the proximal promoter reveals a markedly inverse correlation between the methylation status of the CpG islands and germ cell type-specific expression of Miwi. CpG methylation at the USF-binding site within the E2 box in the promoter inhibits the binding of USF. Transgenic Miwi-EGFP and endogenous Miwi reveal a subcellular co-localization pattern in the germ cells of the Miwi-EGFP transgenic mouse. Furthermore, the DNA methylation profile of the Miwi promoter-driven transgene is consistent with that of the endogenous Miwi promoter, indicating that Miwi transgene is epigenetically modified through methylation in vivo to ensure its spatio-temporal expression. Our findings suggest that USF controls Miwi expression from midpachytene spermatocytes to round spermatids through methylation-mediated regulation. This work identifies an epigenetic regulation mechanism for the spatio-temporal expression of mouse Miwi during spermatogenesis.

Genome-wide analysis reveals origin of transfer RNA genes from tRNA halves.

Transfer RNAs (tRNAs) play an important role linking mitochondrial RNA and amino acids during protein biogenesis. Four types of tRNA genes have been identified in living organisms. However, the evolutionary origin of tRNAs remains largely unknown. In this article, we conduct a deep sequence analysis of diverse genomes that cover all three domains of life to unveil the evolutionary history of tRNA genes from tRNA halves. tRNA half homologs were detected in diverse organisms, and some of them were expressed in mouse tissues. Continuous tRNA genes have a conserved pattern similar to indels, which is, more closely flanking regions have higher single nucleotide substitution rates, whereas tRNA half homologs do not have this pattern. In addition, tRNAs tend to break into tRNA halves when tissues are incubated in vitro, the tendency of tRNA to break into tRNA halves may be a "side-effect" of tRNA genes evolving from tRNA halves. These results suggest that modern tRNAs originated from tRNA halves through a repeat element-mediated mechanism. These findings provide insight into the evolutionary origin of tRNA genes.

Identification of Dmrt genes and their up-regulation during gonad transformation in the swamp eel (Monopterus albus).

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.

TCTP increases stability of hypoxia-inducible factor 1α by interaction with and degradation of the tumour suppressor VHL.

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 ß 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 ß domain of VHL through competition with HIF1α, which promotes VHL degradation by the ubiquitin-proteasome system and HIF1α stability.

Regulation of the transcriptional activation of the androgen receptor by the UXT-binding protein VHL.

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.

RAB37-mediated autophagy guards ovarian homeostasis and function.

Loss of ovarian homeostasis is associated with ovary dysfunction and female diseases; however, the underlying mechanisms responsible for the establishment of homeostasis and its function in the ovary have not been fully elucidated. Here, we showed that conditional knockout of Rab37 in oocytes impaired macroautophagy/autophagy proficiency in the ovary and interfered with follicular homeostasis and ovary development in mice. Flunarizine treatment upregulated autophagy, thus rescuing the impairment of follicular homeostasis and ovarian dysfunction in rab37 knockout mice by reprogramming of homeostasis. Notably, both the E2F1 and EGR2 transcription factors synergistically activated Rab37 transcription and promoted autophagy. Thus, RAB37-mediated autophagy ensures ovary function by maintaining ovarian homeostasis.Abbreviations: AMH: anti-Mullerian hormone; ATG: autophagy related; BECN1: beclin 1; cKO: conditional knockout; Cre: cyclization recombination enzyme; dpp: days postpartum; E2: estradiol; E2F1: E2F transcription factor 1; EBF1: EBF transcription factor 1; EGR2: early growth response 2; FSH: follicle stimulating hormone; LH: luteinizing hormone; mpp: months postpartum; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; RAB37: RAB37, member RAS oncogene family; SQSTM1: sequestosome 1; TFEB: transcription factor EB; Zp3: zona pellucida glycoprotein 3.

RPGR is a guanine nucleotide exchange factor for the small GTPase RAB37 required for retinal function via autophagy regulation.

Although the small GTPase RAB37 acts as an organizer of autophagosome biogenesis, the upstream regulatory mechanism of autophagy via guanosine diphosphate (GDP)-guanosine triphosphate (GTP) exchange in maintaining retinal function has not been determined. We found that retinitis pigmentosa GTPase regulator (RPGR) is a guanine nucleotide exchange factor that activates RAB37 by accelerating GDP-to-GTP exchange. RPGR directly interacts with RAB37 via the RPGR-RCC1-like domain to promote autophagy through stimulating exchange. Rpgr knockout (KO) in mice leads to photoreceptor degeneration owing to autophagy impairment in the retina. Notably, the retinopathy phenotypes of Rpgr KO retinas are rescued by the adeno-associated virus-mediated transfer of pre-trans-splicing molecules, which produce normal Rpgr mRNAs via trans-splicing in the Rpgr KO retinas. This rescue upregulates autophagy through the re-expression of RPGR in KO retinas to accelerate GDP-to-GTP exchange; thus, retinal homeostasis reverts to normal. Taken together, these findings provide an important missing link for coordinating RAB37 GDP-GTP exchange via the RPGR and retinal homeostasis by autophagy regulation.

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.

EST dataset of pituitary and identification of somatolactin and novel genes in Chinese sturgeon, Acipenser sinensis.

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry, however, a few genes have been identified in this species. We report here construction of a pituitary cDNA library from a 24 years old female Chinese sturgeon just after its spawning, and obtained 2,025 ESTs from the library. 885 unique sequences were identified, which were categorized into 12 functional groups. More than half of the unique sequences (57%) do not match with annotated sequences in the public databases. Three of these novel genes were further identified. Notably, a full-length of cDNA (1,143 bp) encoding somatolactin of 232 amino acids was identified. Phylogenetic analysis showed 97% amino acid identity with White sturgeon somatolactin. RT-PCR analysis indicated that the somatolactin mRNA was only detected in pituitary. Pituitary-specific expression of the somatolactin suggested that the protein may play important physiological functions in pituitary-endocrine system of the Chinese sturgeon.

[Molecular mechanisms and functions of autophagy and the ubiquitin-proteasome pathway].

All proteins in eukaryotic cells are continually being degraded and replaced. Autophagy and the ubiquitin-proteasome system are two mechanisms for intracellular protein degradation. Autophagy is mediated by lysosome, and is further divided into chaperone-mediated autophagy, microautophagy and macroautophagy. The ubiquitin-proteasome system is highly complex and mediated by ubiquitin, which participates in intracellular protein degradation in a specific manner. It is now known that degradation of intracellular proteins is involved in regulation of a series of cellular processes, including cell-cycle division, DNA repair, cell growth and differentiation, quality control, pathogen infection, and apoptosis. The aberrations in the protein degradation systems are involved in many serious human diseases. The present review summarizes the mechanisms of protein degradation and related human diseases.

Decoding genome recombination and sex reversal.

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.

Germline stem cells in human.

The germline cells are essential for the propagation of human beings, thus essential for the survival of mankind. The germline stem cells, as a unique cell type, generate various states of germ stem cells and then differentiate into specialized cells, spermatozoa and ova, for producing offspring, while self-renew to generate more stem cells. Abnormal development of germline stem cells often causes severe diseases in humans, including infertility and cancer. Primordial germ cells (PGCs) first emerge during early embryonic development, migrate into the gentile ridge, and then join in the formation of gonads. In males, they differentiate into spermatogonial stem cells, which give rise to spermatozoa via meiosis from the onset of puberty, while in females, the female germline stem cells (FGSCs) retain stemness in the ovary and initiate meiosis to generate oocytes. Primordial germ cell-like cells (PGCLCs) can be induced in vitro from embryonic stem cells or induced pluripotent stem cells. In this review, we focus on current advances in these embryonic and adult germline stem cells, and the induced PGCLCs in humans, provide an overview of molecular mechanisms underlying the development and differentiation of the germline stem cells and outline their physiological functions, pathological implications, and clinical applications.