Histone methyltransferase DOT1L is essential for self-renewal of germline stem cells.

Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at HoxC9 and HoxC10 genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.

The DOT1L-MLLT10 complex regulates male fertility and promotes histone removal during spermiogenesis.

Histone modifications regulate chromatin remodeling and gene expression in development and diseases. DOT1L, the sole histone H3K79 methyltransferase, is essential for embryonic development. Here, we report that DOT1L regulates male fertility in mouse. DOT1L associates with MLLT10 in testis. DOT1L and MLLT10 localize to the sex chromatin in meiotic and post-meiotic germ cells in an inter-dependent manner. Loss of either DOT1L or MLLT10 leads to reduced testis weight, decreased sperm count and male subfertility. H3K79me2 is abundant in elongating spermatids, which undergo the dramatic histone-to-protamine transition. Both DOT1L and MLLT10 are essential for H3K79me2 modification in germ cells. Strikingly, histones are substantially retained in epididymal sperm from either DOT1L- or MLLT10-deficient mice. These results demonstrate that H3K79 methylation promotes histone replacement during spermiogenesis.

Paternal USP26 mutations raise Klinefelter syndrome risk in the offspring of mice and humans.

Current understanding holds that Klinefelter syndrome (KS) is not inherited, but arises randomly during meiosis. Whether there is any genetic basis for the origin of KS is unknown. Here, guided by our identification of some USP26 variations apparently associated with KS, we found that knockout of Usp26 in male mice resulted in the production of 41, XXY offspring. USP26 protein is localized at the XY body, and the disruption of Usp26 causes incomplete sex chromosome pairing by destabilizing TEX11. The unpaired sex chromosomes then result in XY aneuploid spermatozoa. Consistent with our mouse results, a clinical study shows that some USP26 variations increase the proportion of XY aneuploid spermatozoa in fertile men, and we identified two families with KS offspring wherein the father of the KS patient harbored a USP26-mutated haplotype, further supporting that paternal USP26 mutation can cause KS offspring production. Thus, some KS should originate from XY spermatozoa, and paternal USP26 mutations increase the risk of producing KS offspring.

RNase H1 facilitates recombinase recruitment by degrading DNA-RNA hybrids during meiosis.

DNA-RNA hybrids play various roles in many physiological progresses, but how this chromatin structure is dynamically regulated during spermatogenesis remains largely unknown. Here, we show that germ cell-specific knockout of Rnaseh1, a specialized enzyme that degrades the RNA within DNA-RNA hybrids, impairs spermatogenesis and causes male infertility. Notably, Rnaseh1 knockout results in incomplete DNA repair and meiotic prophase I arrest. These defects arise from the altered RAD51 and DMC1 recruitment in zygotene spermatocytes. Furthermore, single-molecule experiments show that RNase H1 promotes recombinase recruitment to DNA by degrading RNA within DNA-RNA hybrids and allows nucleoprotein filaments formation. Overall, we uncover a function of RNase H1 in meiotic recombination, during which it processes DNA-RNA hybrids and facilitates recombinase recruitment.

MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage.

MicroRNAs (miRNAs) are believed to play important roles in mammalian spermatogenesis but the in vivo functions of single miRNAs in this highly complex developmental process remain unclear. Here, we report that miR-202, a member of the let-7 family, plays an important role in spermatogenesis by phenotypic evaluation of miR-202 knockout (KO) mice. Loss of miR-202 results in spermatocyte apoptosis and perturbation of the zygonema-to-pachynema transition. Multiple processes during meiosis prophase I including synapsis and crossover formation are disrupted, and inter-sister chromatid synapses are detected. Moreover, we demonstrate that Separase mRNA is a miR-202 direct target and provides evidence that miR-202 upregulates REC8 by repressing Separase expression. Therefore, we have identified miR-202 as a new regulating noncoding gene that acts on the established SEPARASE-REC8 axis in meiosis.

SCF ubiquitin E3 ligase regulates DNA double-strand breaks in early meiotic recombination.

Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.

Berberine alters the gut microbiota metabolism and impairs spermatogenesis.

Berberine (BBR) is used to treat diarrhea clinically. However, its reproductive toxicity is unclear. This study aims to investigate the impact of BBR on the male reproductive system. Intragastric BBR administration for 14 consecutive days results in a significant decrease in the serum testosterone concentration, epididymal sperm concentration, mating rate and fecundity of male mice. Testicular treatment with testosterone propionate (TP) partially reverses the damage caused by BBR to the male reproductive system. Mechanistically, the decrease in Muribaculaceae abundance in the gut microbiota of mice is the principal cause of the BBR-induced decrease in the sperm concentration. Both fecal microbiota transplantation (FMT) and polyethylene glycol (PEG) treatment demonstrate that Muribaculaceae is necessary for spermatogenesis. The intragastric administration of Muribaculaceae intestinale to BBR-treated mice restores the sperm concentration and testosterone levels. Metabolomic analysis reveals that BBR affects arginine and proline metabolism, of which ornithine level is downregulated. Combined analysis via 16S rRNA metagenomics sequencing and metabolomics shows that Muribaculaceae regulates ornithine level. The transcriptomic results of the testes indicate that the expressions of genes related to the low-density lipoprotein receptor (LDLR)-mediated testosterone synthesis pathway decrease after BBR administration. The transcriptional activity of the Ldlr gene in TM3 cells is increased with increased ornithine supplementation in the culture media, leading to increased testosterone synthesis. Overall, this study reveals an association between a BBR-induced decrease in Muribaculaceae abundance and defective spermatogenesis, providing a prospective therapeutic approach for addressing infertility-related decreases in serum testosterone triggered by changes in the gut microbiota composition.

mRBPome capture identifies the RNA-binding protein TRIM71, an essential regulator of spermatogonial differentiation.

Continual spermatogenesis relies on the actions of an undifferentiated spermatogonial population that is composed of stem cells and progenitors. Here, using mouse models, we explored the role of RNA-binding proteins (RBPs) in regulation of the biological activities of this population. Proteins bound to polyadenylated RNAs in primary cultures of undifferentiated spermatogonia were captured with oligo (dT)-conjugated beads after UV-crosslinking and profiled by proteomics (termed mRBPome capture), yielding a putative repertoire of 473 RBPs. From this database, the RBP TRIM71 was identified and found to be expressed by stem and progenitor spermatogonia in prepubertal and adult mouse testes. Tissue-specific deletion of TRIM71 in the male germline led to reduction of the undifferentiated spermatogonial population and a block in transition to the differentiating state. Collectively, these findings demonstrate a key role of the RBP system in regulation of the spermatogenic lineage and may provide clues about the influence of RBPs on the biology of progenitor cell populations in other lineages.

Bi-allelic variants in SHOC1 cause non-obstructive azoospermia with meiosis arrest in humans and mice.

Meiosis is pivotal to gametogenesis and fertility. Meiotic recombination is a mandatory process that ensures faithful chromosome segregation and generates genetic diversity in gametes. Non-obstructive azoospermia (NOA) caused by meiotic arrest is a common cause of male infertility and has many genetic origins, including chromosome abnormalities, Y chromosome microdeletion and monogenic mutations. However, the genetic causes of the majority of NOA cases remain to be elucidated. Here, we report our findings of three Shortage in chiasmata 1 (SHOC1) bi-allelic variants in three NOA patients, of which two are homozygous for the same loss-of-function variant (c.231_232del: p.L78Sfs*9), and one is heterozygous for two different missense variants (c.1978G>A: p.A660T; c.4274G>A: p.R1425H). Testicular biopsy of one patient revealed impairment of spermatocyte maturation. Both germ-cell-specific and general Shoc1-knockout mice exhibited similar male infertility phenotypes. Subsequent analysis revealed comprehensive defects in homologous pairing and synapsis along with abnormal expression of DMC1, RAD51 and RPA2 in Shoc1-defective spermatocyte spreads. These findings imply that SHOC1 may have a presynaptic function during meiotic recombination apart from its previously identified role in crossover formation. Overall, our results provide strong evidence for the clinical relevance of SHOC1 mutations in patients with NOA and contribute to a deeper mechanistic understanding of the role of SHOC1 during meiotic recombination.

Fbxw17 is dispensable for viability and fertility in mice.

BACKGROUND: Skp1-Cullin-F-box (SCF) E3 ligase complex plays an important role in regulating spermatogenesis and fertility in mice. As a member of F-box proteins, the function of F-box and WD-40 domain protein 17 (Fbxw17) during spermatogenesis and fertility is unclear. In this study, we illustrate its function for spermatogenesis and fertility. METHODS AND RESULTS: Here, we generated the Fbxw17 knockout (KO) mouse model by using the CRISPR/Cas9 system and analyzed the meiotic process and the fertility. Then, our results demonstrated that testis and sperm in the Fbxw17 KO mice had normal morphology. The testis weight, sperm count and fertility of Fbxw17 KO mice showed no significant difference compared with the wild-type mice. Subsequently, histological analysis of Fbxw17 KO mice revealed apparently normal germ cells of all stages and mature spermatozoa. Meanwhile, nuclear spread analysis showed that the synaptonemal complex formation and DSB repair proceeded normally in Fbxw17-deficient spermatocytes. Furthermore, we didn't find defects in the meiotic prophase I spermatocytes and germ cells showed no apparent apoptosis in Fbxw17 KO mice. CONCLUSIONS: Our results show that Fbxw17 is dispensable for fertility in mice.

Nuclear localization of human MEIOB requires its NLS in the OB domain and interaction with SPATA22.

MEIOB is a vital protein in meiotic homologous recombination and plays an indispensable role in human gametogenesis. In mammals, MEIOB and its partner SPATA22 form a heterodimer, ensuring their effective localization on single-strand DNA (ssDNA) and proper synapsis processes. Mutations in human MEIOB (hMEIOB) cause human infertility attributed to the failure of its interaction with human SPATA22 (hSPATA22) and ssDNA binding. However, the detailed mechanism is still unclear. In our study, truncated or full-length hMEIOB and hSPATA22 are traced by fused expression with fluorescent proteins (i.e., copGFP or mCherry), and the live cell imaging system is used to observe the expression and localization of the proteins. When transfected alone, hMEIOB accumulates in the cytoplasm. Interestingly, a covered NLS in the OB domain of hMEIOB is identified, which can be exposed by hSPATA22 and is necessary for the nuclear localization of hMEIOB. When hSPATA22 loses its hMEIOB interacting domain or NLS, the nuclear localization of hMEIOB is aborted. Collectively, our results prove that the NLS in the OB domain of hMEIOB and interaction with hSPATA22 are required for hMEIOB nuclear localization.

Dual functions for the ssDNA-binding protein RPA in meiotic recombination.

Meiotic recombination permits exchange of genetic material between homologous chromosomes. The replication protein A (RPA) complex, the predominant ssDNA-binding complex, is required for nearly all aspects of DNA metabolism, but its role in mammalian meiotic recombination remains unknown due to the embryonic lethality of RPA mutant mice. RPA is a heterotrimer of RPA1, RPA2, and RPA3. We find that loss of RPA1, the largest subunit, leads to disappearance of RPA2 and RPA3, resulting in the absence of the RPA complex. Using an inducible germline-specific inactivation strategy, we find that loss of RPA completely abrogates loading of RAD51/DMC1 recombinases to programmed meiotic DNA double strand breaks, thus blocking strand invasion required for chromosome pairing and synapsis. Surprisingly, loading of MEIOB, SPATA22, and ATR to DNA double strand breaks is RPA-independent and does not promote RAD51/DMC1 recruitment in the absence of RPA. Finally, inactivation of RPA reduces crossover formation. Our results demonstrate that RPA plays two distinct roles in meiotic recombination: an essential role in recombinase recruitment at early stages and an important role in promoting crossover formation at later stages.

SCRE serves as a unique synaptonemal complex fastener and is essential for progression of meiosis prophase I in mice.

Meiosis is a specialized cell division for producing haploid gametes from diploid germ cells. During meiosis, synaptonemal complex (SC) mediates the alignment of homologs and plays essential roles in homologous recombination and therefore in promoting accurate chromosome segregation. In this study, we have identified a novel protein SCRE (synaptonemal complex reinforcing element) as a key molecule in maintaining the integrity of SC during meiosis prophase I in mice. Deletion of Scre (synaptonemal complex reinforcing element) caused germ cell death in both male and female mice, resulting in infertility. Our mechanistic studies showed that the synapses and SCs in Scre knockout mice were unstable due to the lack of the SC reinforcing function of SCRE, which is sparsely localized as discrete foci along the central elements in normal synaptic homologous chromosomes. The lack of Scre leads to meiosis collapse at the late zygotene stage. We further showed that SCRE interacts with synaptonemal complex protein 1 (SYCP1) and synaptonemal complex central element 3 (SYCE3). We conclude that the function of SCRE is to reinforce the integrity of the central elements, thereby stabilizing the SC and ensuring meiotic cell cycle progression. Our study identified SCRE as a novel SC fastener protein that is distinct from other known SC proteins.

PSPC1 regulates CHK1 phosphorylation through phase separation and participates in mouse oocyte maturation.

Liquid-liquid phase separation (LLPS) underlies the formation of membraneless compartments in mammal cells. However, there are few reports that focus on the correlation of mouse oocyte maturation with LLPS. Previous studies have reported that paraspeckle component 1 (PSPC1) is related to the occurrence and development of tumors, but whether PSPC1 functions in mouse oocyte maturation is still unclear. Sequence analysis of PSPC1 protein showed that it contains a prion-like domain (PrLD) that is required for phase separation of proteins. In this study, we found that PSPC1 could undergo phase separation. Moreover, the loss of PrLD domain of PSPC1 could greatly weaken its phase separation ability. The immunofluorescence assays showed that PSPC1 is present in mouse oocytes in the germinal vesicle (GV) stage. Knockdown of PSPC1 significantly impeded the maturation of mouse oocytes in vitro. CHK1 has been reported to play important roles in the GV stage of mouse oocytes. Co-IP experiment revealed that PSPC1 could interact with phosphatase serine/threonine-protein phosphatase 5 (PPP5C), which regulates CHK1 phosphorylation. Western blot analysis revealed that PSPC1 could regulate the phosphorylation of CHK1 through PPP5C; however, PSPC1 without PrLD domain was inactive, suggesting that the lack of phase separation ability led to the abnormal function of PSPC1 in regulating CHK1 phosphorylation. Thus, we conclude that PSPC1 may undergo phase separation to regulate the phosphorylation level of CHK1 via PPP5C and participate in mouse oocyte maturation. Our study provides new insights into the mechanism of mouse oocyte maturation.

Biological and RNA regulatory function of MOV10 in mammalian germ cells.

BACKGROUND: RNA regulation by RNA-binding proteins (RBPs) involve extremely complicated mechanisms. MOV10 and MOV10L1 are two homologous RNA helicases implicated in distinct intracellular pathways. MOV10L1 participates specifically in Piwi-interacting RNA (piRNA) biogenesis and protects mouse male fertility. In contrast, the functional complexity of MOV10 remains incompletely understood, and its role in the mammalian germline is unknown. Here, we report a study of the biological and molecular functions of the RNA helicase MOV10 in mammalian male germ cells. RESULTS: MOV10 is a nucleocytoplasmic protein mainly expressed in spermatogonia. Knockdown and transplantation experiments show that MOV10 deficiency has a negative effect on spermatogonial progenitor cells (SPCs), limiting proliferation and in vivo repopulation capacity. This effect is concurrent with a global disturbance of RNA homeostasis and downregulation of factors critical for SPC proliferation and/or self-renewal. Unexpectedly, microRNA (miRNA) biogenesis is impaired due partially to decrease of miRNA primary transcript levels and/or retention of miRNA via splicing control. Genome-wide analysis of RNA targetome reveals that MOV10 binds preferentially to mRNAs with long 3'-UTR and also interacts with various non-coding RNA species including those in the nucleus. Intriguingly, nuclear MOV10 associates with an array of splicing factors, particularly with SRSF1, and its intronic binding sites tend to reside in proximity to splice sites. CONCLUSIONS: These data expand the landscape of MOV10 function and highlight a previously unidentified role initiated from the nucleus, suggesting that MOV10 is a versatile RBP involved in a broader RNA regulatory network.

A convenient one-pot nanosynthesis of a C(sp2)-C(sp3)-linked 3D grid via an 'A2 + B3' approach.

Fluorene-based 3D-grid-FTPA was synthesised with a total yield of 55% via the one-pot formation of six C(sp2)-C(sp3) bonds through a BF3·Et2O-mediated Friedel-Crafts reaction of A2-type bifluorene tertiary alcohol (BIOH) and two B3-type triphenylamines. At the same time, Un-grid-FTPA (2.7%) and 2D-grid-FTPA (5.6%) were obtained as by-products from this synthesis method. In addition, the effect of stereoisomers of BIOH was evaluated to demonstrate that Rac-BIOH is a better A2-type building block to prepare 3D-grid-FTPA in a relatively high yield. Furthermore, 3D-grid-FTPA showed excellent chemical, thermal, and photo-stabilities.

Polycomb protein SCML2 associates with USP7 and counteracts histone H2A ubiquitination in the XY chromatin during male meiosis.

Polycomb group proteins mediate transcriptional silencing in diverse developmental processes. Sex chromosomes undergo chromosome-wide transcription silencing during male meiosis. Here we report that mouse SCML2 (Sex comb on midleg-like 2), an X chromosome-encoded polycomb protein, is specifically expressed in germ cells, including spermatogonia, spermatocytes, and round spermatids. SCML2 associates with phosphorylated H2AX and localizes to the XY body in spermatocytes. Loss of SCML2 in mice causes defective spermatogenesis, resulting in sharply reduced sperm production. SCML2 interacts with and recruits a deubiquitinase, USP7, to the XY body in spermatocytes. In the absence of SCML2, USP7 fails to accumulate on the XY body, whereas H2A monoubiquitination is dramatically augmented in the XY chromatin. Our results demonstrate that the SCML2/USP7 complex constitutes a novel molecular pathway in modulating the epigenetic state of sex chromosomes during male meiosis.

Type I interferon controls propagation of long interspersed element-1.

Type I interferons (IFN) including IFNα and IFNß are critical for the cellular defense against viruses. Here we report that increased levels of IFNß were found in testes from mice deficient in MOV10L1, a germ cell-specific RNA helicase that plays a key role in limiting the propagation of retrotransposons including Long Interspersed Element-1 (LINE-1). Additional experiments revealed that activation of LINE-1 retrotransposons increases the expression of IFNß and of IFN-stimulated genes. Conversely, pretreatment of cells with IFN suppressed the replication of LINE-1. Furthermore, the efficacy of LINE-1 replication was increased in isogenic cell lines harboring inactivating mutations in diverse elements of the IFN signaling pathway. Knockdown of the IFN receptor chain IFNAR1 also stimulated LINE-1 propagation in vitro. Finally, a greater accumulation of LINE-1 was found in mice that lack IFNAR1 compared with wild type mice. We propose that LINE-1-induced IFN plays an important role in restricting LINE-1 propagation and discuss the putative role of IFN in preserving the genome stability.

Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing.

The increasing emergence of Cas9 variants has attracted broad interest, as these variants were designed to expand CRISPR applications. New Cas9 variants typically feature higher editing efficiency, improved editing specificity, or alternative PAM sequences. To select Cas9 variants and gRNAs for high-fidelity and efficient genome editing, it is crucial to systematically quantify the editing performances of gRNAs and develop prediction models based on high-quality datasets. Using synthetic gRNA-target paired libraries and next-generation sequencing, we compared the activity and specificity of gRNAs of four SpCas9 variants. The nucleotide composition in the PAM-distal region had more influence on the editing efficiency of HiFi Cas9 and LZ3 Cas9. We further developed machine learning models to predict the gRNA efficiency and specificity for the four Cas9 variants. To aid users from broad research areas, the machine learning models for the predictions of gRNA editing efficiency within human genome sites are available on our website.

PWWP3A deficiency accelerates testicular senescence in aged mice.

BACKGROUND: The PWWP domain-containing proteins are involved in chromatin-associated biological processes, including transcriptional regulation and DNA repair, and most of them are significant for gametogenesis and early embryonic development in mammals. PWWP3A, one of the PWWP domain proteins, is a reader of H3K36me2/H3K36me3 and a response factor to DNA damage. However, the physiological role of PWWP3A in spermatogenesis and fertility remains unclear. OBJECTIVE: The goal of this study was to explore the function and mechanism of PWWP3A in the process of spermatogenesis. MATERIALS AND METHODS: We generated V5-Pwwp3a KI mice and PWWP3A polyclonal antibody to observe the localization of PWWP3A in vivo. Meanwhile, Pwwp3a KO mice was used to explore the function in spermatogenesis. RESULTS: We reported that PWWP3A is a predominant expression in the testis of mice. During spermatogenesis, PWWP3A exhibits the temporal expression from early-pachytene to the round spermatids. The results of spermatocyte spreading and immunostaining showed that PWWP3A aggregated on the XY body, which then diffused as the XY chromosome separated at late-diplotene. Although the depletion of PWWP3A had no obvious reproductive defects in young male mice, there were observed morphological abnormalities in sperm heads. Immunoprecipitation demonstrated the interaction of PWWP3A with DNA repair proteins SMC5/6; however, PWWP3A deficiency did not result in any meiotic defects. Notably, the testes of aged male Pwwp3a KO mice displayed pronounced degeneration, and were characterized by the presence of vacuolated seminiferous tubules. Furthermore, RNA-seq analysis revealed an upregulation in the expression of genes which may be involving in immunoregulatory and inflammatory response pathways in aged Pwwp3a KO mice with testicular degeneration. CONCLUSIONS: Our study showed that PWWP3A was highly enriched in the mouse testis, and the Pwwp3a KO mice were fertile. However, the aged Pwwp3a KO male mice displayed testicular atrophy that may be due to changes in the immune micro-environment or abnormal repair of DNA damage.