DDX20 is required for cell-cycle reentry of prospermatogonia and establishment of spermatogonial stem cell pool during testicular development in mice.

RNA-binding proteins (RBPs), as key regulators of mRNA fate, are abundantly expressed in the testis. However, RBPs associated with human male infertility remain largely unknown. Through bioinformatic analyses, we identified 62 such RBPs, including an evolutionarily conserved RBP, DEAD-box helicase 20 (DDX20). Male germ-cell-specific inactivation of Ddx20 at E15.5 caused T1-propsermatogonia (T1-ProSG) to fail to reenter cell cycle during the first week of testicular development in mice. Consequently, neither the foundational spermatogonial stem cell (SSC) pool nor progenitor spermatogonia were ever formed in the knockout testes. Mechanistically, DDX20 functions to control the translation of its target mRNAs, many of which encode cell-cycle-related regulators, by interacting with key components of the translational machinery in prospermatogonia. Our data demonstrate a previously unreported function of DDX20 as a translational regulator of critical cell-cycle-related genes, which is essential for cell-cycle reentry of T1-ProSG and formation of the SSC pool.

Decoding the spermatogonial stem cell niche under physiological and recovery conditions in adult mice and humans.

The intricate interaction between spermatogonial stem cell (SSC) and testicular niche is essential for maintaining SSC homeostasis; however, this interaction remains largely uncharacterized. In this study, to characterize the underlying signaling pathways and related paracrine factors, we delineated the intercellular interactions between SSC and niche cell in both adult mice and humans under physiological conditions and dissected the niche-derived regulation of SSC maintenance under recovery conditions, thus uncovering the essential role of C-C motif chemokine ligand 24 and insulin-like growth factor binding protein 7 in SSC maintenance. We also established the clinical relevance of specific paracrine factors in human fertility. Collectively, our work on decoding the adult SSC niche serves as a valuable reference for future studies on the aetiology, diagnosis, and treatment of male infertility.

Identification of quiescent FOXC2+ spermatogonial stem cells in adult mammals.

In adult mammals, spermatogenesis embodies the complex developmental process from spermatogonial stem cells (SSCs) to spermatozoa. At the top of this developmental hierarchy lie a series of SSC subpopulations. Their individual identities as well as the relationships with each other, however, remain largely elusive. Using single-cell analysis and lineage tracing, we discovered both in mice and humans the quiescent adult SSC subpopulation marked specifically by forkhead box protein C2 (FOXC2). All spermatogenic progenies can be derived from FOXC2+ SSCs and the ablation of FOXC2+ SSCs led to the depletion of the undifferentiated spermatogonia pool. During germline regeneration, FOXC2+ SSCs were activated and able to completely restore the process. Germ cell-specific Foxc2 knockout resulted in an accelerated exhaustion of SSCs and eventually led to male infertility. Furthermore, FOXC2 prompts the expressions of negative regulators of cell cycle thereby ensures the SSCs reside in quiescence. Thus, this work proposes that the quiescent FOXC2+ SSCs are essential for maintaining the homeostasis and regeneration of spermatogenesis in adult mammals.

CHD4 acts as a critical regulator in the survival of spermatogonial stem cells in mice†.

Spermatogenesis is sustained by homeostatic balance between the self-renewal and differentiation of spermatogonial stem cells, which is dependent on the strict regulation of transcription factor and chromatin modulator gene expression. Chromodomain helicase DNA-binding protein 4 is highly expressed in spermatogonial stem cells but roles in mouse spermatogenesis are not fully understood. Here, we report that the germ-cell-specific deletion of chromodomain helicase DNA-binding protein 4 resulted in complete infertility in male mice, with rapid loss of spermatogonial stem cells and excessive germ cell apoptosis. Chromodomain helicase DNA-binding protein 4-knockdown in cultured spermatogonial stem cells also promoted the expression of apoptosis-related genes and thereby activated the tumor necrosis factor signaling pathway. Mechanistically, chromodomain helicase DNA-binding protein 4 occupies the genomic regulatory region of key apoptosis-related genes, including Jun and Nfkb1. Together, our findings reveal the determinant role of chromodomain helicase DNA-binding protein 4 in spermatogonial stem cells survival in vivo, which will offer insight into the pathogenesis of male sterility and potential novel therapeutic targets.

A Novel HCC Prognosis Predictor EEF1E1 Is Related to Immune Infiltration and May Be Involved in EEF1E1/ATM/p53 Signaling.

BACKGROUND: EEF1E1 has been reported to play a role in ovarian cancer, breast cancer, non-small cell lung cancer and other cancers, but its role and mechanism in hepatocellular carcinoma (HCC) are still unknown. METHODS: EEF1E1 expression in human HCC was analyzed via the GTEx and TCGA database. Logistic regression analysis was used to analyze the clinicopathological correlation of EEF1E1 expression. The correlation between EEF1E1 expression and patients' prognosis was analyzed in HCC, shown by forest plots, nomogram and Kaplan-Meier curves. Hazard ratio (HR) with 95% confidence intervals and log-rank p-value were calculated via multivariate/univariate survival analyses. Moreover, the correlation between EEF1E1 and tumor immune infiltration was analyzed using the gsva package with the ssgsea algorithm. Pearson correlation was used to investigate the correlation between EEF1E1 expression and p53 pathway genes expression. Two third-party databases were used to validate the content of EEF1E1 protein and mRNA expression patterns and prognosis analysis. The immunohistochemistry and multiplex immunohistochemistry was used to verify the bio-informatics results. RESULTS: EEF1E1 mRNA and protein expression in tumor was statistically higher than normal (EEF1E1 mRNA: p < 0.001; EEF1E1 protein: p < 0.01). Results from paired t-test (cancer and adjacent tissues) exhibited consistent trend (t = 7.572, p < 0.001). Immunohistochemistry showed that EEF1E1 is highly expressed in cancer. The expression of EEF1E1 was positively correlated with body weight, BMI, tumor status, vascular invasion, AFP, logistic grade, T stage and pathological stage. The univariate Cox model revealed that high EEF1E1 expression was strongly associated with worse OS (HR=2.581; 95% CI: 1.782-3.739; p < 0.001), as was T stage, pathologic stage, Histologic grade. High EEF1E1 expression was the only independent prognostic factor associated with OS (HR=2.57; 95% CI: 1.715-3.851; p < 0.001) in the multivariate analysis. EEF1E1 was significantly correlated with various immune cells, including cytotoxic cells, DC, macrophages, neutrophils, NK cd56bright, TFH, Tgd, Th17, Th2, Treg. Multiplex immunohistochemistry showed that the EEF1E1 protein level is positively correlated to the CD3, CD4, PD1 and is negatively correlated to the CD8. The expression level of EEF1E1 in HCC was significantly correlated with the key genes involved in the p53 pathway. The expression of EEF1E1, ATM, p53 and CASPASE3 in HCC tissues was significantly higher than that in adjacent tissues. CONCLUSION: EEF1E1 is highly expressed in cancer tissues in HCC. EEF1E1's high expression is significantly correlated with worse prognosis and immune cell infiltration of HCC. EEF1E1 may be participating in EEF1E1/ATM/p53 signaling pathway in HCC.

Panoramic transcriptome analysis and functional screening of long noncoding RNAs in mouse spermatogenesis.

Long noncoding RNAs (lncRNAs) have emerged as diverse functional regulators involved in mammalian development; however, large-scale functional investigation of lncRNAs in mammalian spermatogenesis in vivo is lacking. Here, we delineated the global lncRNA expression landscape in mouse spermatogenesis and identified 968 germ cell signature lncRNAs. By combining bioinformatics and functional screening, we identified three functional lncRNAs (Gm4665, 1700027A15Rik, and 1700052I22Rik) that directly influence spermatogenesis in vivo. Knocking down Gm4665 hampered the development of round spermatids into elongating spermatids and disrupted key spermatogenic gene expression. Mechanistically, lncRNA Gm4665 localized in the nucleus of round spermatids and occupied the genomic regulatory region of important spermatogenic genes including Ip6k1 and Akap3 These findings provide a valuable resource and framework for future functional analysis of lncRNAs in spermatogenesis and their potential roles in other biological processes.

A long noncoding RNA binding to QKI-5 regulates germ cell apoptosis via p38 MAPK signaling pathway.

Spermatogenesis is the complex process of male germline development and requires coordinated interactions by multiple gene products that undergo strict developmental regulations. Increasing evidence has suggested that a number of long noncoding RNAs (lncRNAs) may function as important regulatory molecules in various physiological and pathological processes by binding to specific proteins. Here, we identified a subset of QKI-5-binding lncRNAs in the mouse testis through the integrated analyses of RNA immunoprecipitation (RIP)-microarray and biological verification. Among the lncRNAs, we revealed that NONMMUT074098.2 (Lnc10), which was highly expressed in the spermatogonia and spermatocytes of the testis, interacted with QKI-5. Furthermore, Lnc10 depletion promoted germ cell apoptosis via the activation of p38 MAPK, whereas the simultaneous knockdown of QKI-5 could rescue the apoptotic phenotype and the activation of p38 MAPK, which were induced by the loss of Lnc10. These data indicated that the Lnc10-QKI-5 interaction was associated with the regulatory roles of QKI-5 and that the Lnc10-QKI-5 interaction inhibited the regulation of QKI-5 on the downstream p38 MAPK signaling pathway. Additionally, we functionally characterized the biological roles of Lnc10 and found that the knockdown of Lnc10 promoted the apoptosis of spermatogenic cells in vivo; this suggested that Lnc10 had an important biological role in mouse spermatogenesis. Thus, our study provides a potential strategy to investigate the biological significance of lncRNA-RBP interactions during male germline development.