Gene mutations impede oocyte maturation, fertilization, and early embryonic development.

Reproductive diseases are a long-standing problem and have become more common in the world. Currently, 15% of the world's population suffers from infertility, and half of them are women. Maturation of oocytes, successful fertilization, and high-quality embryos are prerequisites for pregnancy. With the development of assisted reproductive technology and advanced genetic assays, we have found that infertility in many young female patients is caused by mutations in various developmental regulators. These pathogenic factors may result in impediment of oocyte maturation, failure of fertilization or early embryonic development arrest. In this review, we categorize these clinically-identified, mutated genetic factors by their molecular characteristics: nuclear factors (PALT2, TRIP13, WEE2, TBPL2, REC114, MEI1 and CDC20), cytoplasmic factors (TLE6, PADI6, NLRP2/5, FBXO43, MOS and BTG4), a factor unique to primates (TUBB8), cell membrane factor (PANX1), and zona pellucida factors (ZP1-3). We compared discrepancies observed in phenotypes between human and mouse models to provide clues for clinical diagnosis and treatment of related reproductive diseases.

H3K36me2 methyltransferase NSD2 orchestrates epigenetic reprogramming during spermatogenesis.

Spermatogenesis is precisely controlled by sophisticated gene expression programs and is driven by epigenetic reprogramming, including histone modification alterations and histone-to-protamine transition. Nuclear receptor binding SET domain protein 2 (Nsd2) is the predominant histone methyltransferase catalyzing H3K36me2 and its role in male germ cell development remains elusive. Here, we report that NSD2 protein is abundant in spermatogenic cells. Conditional loss of Nsd2 in postnatal germ cells impaired fertility owing to apoptosis of spermatocytes and aberrant spermiogenesis. Nsd2 deficiency results in dysregulation of thousands of genes and remarkable reduction of both H3K36me2 and H3K36me3 in spermatogenic cells, with H3K36me2 occupancy correlating positively with expression of germline genes. Nsd2 deficiency leads to H4K16ac elevation in spermatogenic cells, probably through interaction between NSD2 and PSMA8, which regulates acetylated histone degradation. We further reveal that Nsd2 deficiency impairs EP300-induced H4K5/8ac, recognized by BRDT to mediate the eviction of histones. Accordingly, histones are largely retained in Nsd2-deficient spermatozoa. In addition, Nsd2 deficiency enhances expression of protamine genes, leading to increased protamine proteins in Nsd2-deficient spermatozoa. Our findings thus reveal a previously unappreciated role of the Nsd2-dependent chromatin remodeling during spermatogenesis and provide clues to the molecular mechanisms in epigenetic abnormalities impacting male reproductive health.

CRISPR/Cas9 technology: applications in oocytes and early embryos.

CRISPR/Cas9, a highly versatile genome-editing tool, has garnered significant attention in recent years. Despite the unique characteristics of oocytes and early embryos compared to other cell types, this technology has been increasing used in mammalian reproduction. In this comprehensive review, we elucidate the fundamental principles of CRISPR/Cas9-related methodologies and explore their wide-ranging applications in deciphering molecular intricacies during oocyte and early embryo development as well as in addressing associated diseases. However, it is imperative to acknowledge the limitations inherent to these technologies, including the potential for off-target effects, as well as the ethical concerns surrounding the manipulation of human embryos. Thus, a judicious and thoughtful approach is warranted. Regardless of these challenges, CRISPR/Cas9 technology undeniably represents a formidable tool for genome and epigenome manipulation within oocytes and early embryos. Continuous refinements in this field are poised to fortify its future prospects and applications.

dCas9 techniques for transcriptional repression in mammalian cells: Progress, applications and challenges.

Innovative loss-of-function techniques developed in recent years have made it much easier to target specific genomic loci at transcriptional levels. CRISPR interference (CRISPRi) has been proven to be the most effective and specific tool to knock down any gene of interest in mammalian cells. The catalytically deactivated Cas9 (dCas9) can be fused with transcription repressors to downregulate gene expression specified by sgRNA complementary to target genomic sequence. Although CRISPRi has huge potential for gene knockdown, there is still a lack of systematic guidelines for efficient and widespread use. Here we describe the working mechanism and development of CRISPRi, designing principles of sgRNA, delivery methods and applications in mammalian cells in detail. Finally, we propose possible solutions and future directions with regard to current challenges.

Ppan is essential for preimplantation development in mice†.

PETER PAN (PPAN), located to nucleoli and mitochondria, is a member of the Brix domain protein family, involved in rRNA processing through its rRNA binding motif and mitochondrial apoptosis by protecting mitochondria structure and suppressing basal autophagic flux. Ppan is important for cell proliferation and viability, and mutation of Ppan in Drosophila caused larval lethality and oogenesis failure. Yet, its role in mammalian reproduction remains unclear. In this study, we explored the function of Ppan in oocyte maturation and early embryogenesis using conditional knockout mouse model. Deficiency of maternal Ppan significantly downregulated the expression level of 5.8S rRNA, 18S rRNA, and 28S rRNA, though it had no effect on oocyte maturation or preimplantation embryo development. However, depletion of both maternal and zygotic Ppan blocked embryonic development at morula stage. Similar phenotype was obtained when only zygotic Ppan was depleted. We further identified no DNA binding activity of PPAN in mouse embryonic stem cells, and depletion of Ppan had minimum impact on transcriptome but decreased expression of 5.8S rRNA, 18S rRNA, and 28S rRNA nevertheless. Our findings demonstrate that Ppan is indispensable for early embryogenesis in mice.

Regulatory roles of alternative splicing at Ezh2 gene in mouse oocytes.

BACKGROUND: Enhancer of zeste homologue 2 (EZH2), the core member of polycomb repressive complex 2 (PRC2), has multiple splicing modes and performs various physiological functions. However, function and mechanism of alternative splicing at Ezh2 exon 3 in reproduction are unknown. METHODS: We generated Ezh2Long and Ezh2Short mouse models with different point mutations at the Ezh2 exon 3 alternative splicing site, and each mutant mouse model expressed either the long or the short isoform of Ezh2. We examined mutant mouse fertility and oocyte development to assess the function of Ezh2 alternative splicing at exon 3 in the reproductive system. RESULTS: We found that Ezh2Long female mice had normal fertility. However, Ezh2Short female mice had significantly decreased fertility and obstructed oogenesis, with compromised mitochondrial function in Ezh2Short oocytes. Interestingly, increased EZH2 protein abundance and accumulated H3K27me3 were observed in Ezh2Short oocytes. CONCLUSIONS: Our results demonstrate that correct Ezh2 alternative splicing at exon 3 is important for mouse oogenesis.

Exposure of mouse oocytes to N,N-dimethylformamide impairs mitochondrial functions and reduces oocyte quality.

N,N-dimethylformamide (DMF) is a widely-used solvent for the synthesis of synthetic fibers such as polyacrylonitrile fiber, and can also be used to make medicine. Although this organic solvent has multipurpose applications, its biological toxicity cannot be ignored and its impact on mammalian reproduction remains largely unexplored. Our study found that DMF exposure inhibited oocyte maturation and fertilization ability. Transcriptomic analysis indicated that DMF exposure changed the expression of genes and transposable elements in oocytes. Subcellular structure examination found that DMF exposure caused mitochondrial dysfunction, abnormal aggregation of mitochondria and decreased mitochondrial membrane potential in mouse oocytes. Its exposure also caused abnormal distribution of Golgi apparatus and endoplasmic reticulum which formed large number of clusters. In addition, oxidative stress occurs in oocytes exposed to DMF, which was manifested by an increase in the level of reactive oxygen species. We found that DMF exposure induced disordered spindle and chromosomes abnormality. Meanwhile, we examined various histone modification levels in oocytes exposed to DMF and found that DMF exposure reduced H3K9me3, H3K9ac, H3K27ac, and H4K16ac levels in mouse oocytes. Moreover, DMF-treated oocytes failed to form pronuclei after fusion with normal sperm. Collectively, DMF exposure caused mitochondrial damage, oxidative stress, spindle assembly and chromosome arrangement disorder, leading to oocyte maturation arrest and fertilization failure.

Evaluating the impact of COVID-19 on male reproduction.

Invasion or damage of the male reproductive system is one of the reported outcomes of viral infection. Current studies have documented that SARS-CoV-2, which causes COVID-19, can damage the male reproductive system in large part by inflammatory damage caused by a cytokine storm. However, whether SARS-CoV-2 can infect the human testis directly and enter semen is controversial. Other adverse effects of SARS-CoV-2 on male reproduction are also of concern and require comprehensive evaluation. Here, we analyze the invasiveness of SARS-CoV-2 in the testis and examine reported mechanisms by which SARS-CoV-2 interferes with male reproduction. Long-term implications of SARS-CoV-2 infection on male reproduction are also discussed. It should be emphasized that although COVID-19 may induce testicular damage, a substantial decrease in male reproductive capacity awaits clinical evidence. We propose that there is an urgent need to track male COVID-19 patients during their recovery. The development of suitable experimental models, including human reproductive organoids, will be valuable to further investigate the viral impact on reproduction for current and future pandemics.

Suggestions on cleavage embryo and blastocyst vitrification/transfer based on expression profile of ACE2 and TMPRSS2 in current COVID-19 pandemic.

An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is leading to an unprecedented worldwide health crisis. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2. Our objectives are to analysis the expression profile of ACE2 and TMPRSS2 in human spermatogenic cells, follicle cells, and preimplantation embryos, thereby providing mechanistic insights into viral entry and viral impact on reproduction. We found that ACE2 is mainly expressed during gametogenesis in spermatogonia and oocytes of antral follicles, granulosa cells of antral follicles and pre-ovulatory follicles, while TMPRSS2 almost has no expression in spermatogenic cells, oocytes or granulosa cells. In preimplantation embryos, ACE2 is expressed in early embryos before eight-cell stage, and trophectoderm of late blastocysts, while TMPRSS2 initiates its robust expression in late blastocyst stage. ACE2 and TMPRSS2 only show significant co-expression in trophectoderm of late blastocysts in all above cell types. We speculate that trophectoderm of late blastocysts is susceptible to SARS-CoV-2, and that the chance of SARS-CoV-2 being passed on to offspring through gametes is very low. Therefore, we propose that fertility preservation for COVID-19 patients is relatively safe and rational. We also recommend embryo cryopreservation and embryo transfer into healthy recipient mother at cleavage stage instead of blastocyst stage. Moreover, we unexpectedly found that co-expression pattern of ACE2 and TMPRSS2 in oocytes and preimplantation embryos in human, rhesus monkey and mouse are totally different, so animal models have significant limitations for evaluating transmission risk of SARS-CoV-2 in reproduction.

H3.3 kinetics predicts chromatin compaction status of parental genomes in early embryos.

BACKGROUND: After fertilization, the fusion of gametes results in the formation of totipotent zygote. During sperm-egg fusion, maternal factors participate in parental chromatin remodeling. H3.3 is a histone H3 variant that plays essential roles in mouse embryogenesis. METHODS: Here, we used transgenic early embryos expressing H3.3-eGFP or H2B-mCherry to elucidate changes of histone mobility. RESULTS: We used FRAP analysis to identify that maternally stored H3.3 has a more significant change than H2B during maternal-to-embryonic transition. We also found that H3.3 mobile fraction, which may be regulated by de novo H3.3 incorporation, reflects chromatin compaction of parental genomes in GV oocytes and early embryos. CONCLUSIONS: Our results show that H3.3 kinetics in GV oocytes and early embryos is highly correlated with chromatin compaction status of parental genomes, indicating critical roles of H3.3 in higher-order chromatin organization.

Transcription factor OTX2 silences the expression of cleavage embryo genes and transposable elements.

Upon mammalian fertilization, zygotic genome activation (ZGA) and activation of transposable elements (TEs) occur in early embryos to establish totipotency and support embryogenesis. However, the molecular mechanisms controlling the expression of these genes in mammals remain poorly understood. The 2-cell-like population of mouse embryonic stem cells (mESCs) mimics cleavage-stage embryos with transient Dux activation. In this study, we demonstrated that deficiency of the transcription factor OTX2 stimulates the expression of ZGA genes in mESCs. Further analysis revealed that OTX2 is incorporated at the Dux locus with corepressors for transcriptional inhibition. We also found that OTX2 associates with TEs and silences the subtypes of TEs. Therefore, OTX2 protein plays an important role in ZGA and TE expression in mESCs to orchestrate the transcriptional network.

Cleavage-embryo genes and transposable elements are regulated by histone variant H2A.X.

During mammalian preimplantation development, stimulation of zygotic genome activation (ZGA) and transposable elements (TEs) shapes totipotency profiling. A rare mouse embryonic stem cells (mESCs) subpopulation is capable of transiently entering a state resembling 2-cell stage embryos, with subtypes of TEs expressed and ZGA genes transiently activated. In this study, we found that deletion of H2A.X in mESCs led to a significant upregulation of ZGA genes and misregulated TEs. ChIP-seq analysis indicated a direct association of H2A.X at the Dux locus for silencing the Dux gene and its downstream ZGA genes in mESCs. We also demonstrated that histone variant H2A.X is highly enriched in human cleavage embryos when ZGA genes and TEs are active. Therefore, we propose that H2A.X plays an important role in regulating ZGA genes and TEs to establish totipotency.

Diagnostic value of loop-mediated isothermal amplification assay for hand, foot, and mouth disease.

BACKGROUND: Nowadays, hand, foot, and mouth disease (HFMD) has a significant negative impact on children's health, especially in the Asia-Pacific region. Loop-mediated isothermal amplification assay (LAMP) is a highly efficient and convenient novel tool. However, its diagnostic accuracy for HFMD is still not clear. Therefore, we conducted a meta-analysis in order to evaluate the potential of LAMP assay for the diagnosis of HFMD, in which the reference standard was polymerase chain reaction (PCR). METHODS: A protocol was predetermined (CRD42020212882) in PROSPERO. We retrieved seven databases including PubMed for relevant studies published before October 2020. Articles were included if they compared the diagnostic efficiency of LAMP with PCR for HFMD through detecting clinical samples which was more than 15. Statistical analysis was performed by STATA 15.1 software. Risk of bias and applicability were assessed using Quality Assessment of Diagnostic Accuracy Studies. No funding was used for the study. RESULTS: A total of 18 retrospective studies including 2495 samples from China were finally included. Reference standards of them included RT-PCR and non-RT-PCR. The merged sensitivity and specificity with 95% confidence interval (95% CI) were 1.00 (0.97-1.00) and 0.97 (0.88-0.99), respectively. The pooled PLR, NLR, and DOR with 95% CI were 11.17 (5.91-21.11), 0.05 (0.03-0.09), and 538.12 (183.17-1580.83), respectively. The AUC of SROC was 1.00 (95% CI: 0.99-1.00). CONCLUSION: In conclusion, our research revealed high sensitivity and specificity of LAMP in diagnosing HFMD. However, more high-quality research is required to prove this conclusion.

H3.3 impedes zygotic transcriptional program activated by Dux.

During development, fertilization triggers totipotency establishment, featured by zygotic genome activation/embryonic genome activation (ZGA/EGA). Mouse embryonic stem cells (mESCs) occasionally cycle through a two-cell (2C)-like status with activated expression of Dux and its targeted ZGA genes. Here, we demonstrate that deficiency of histone variant H3.3 dramatically stimulates expression of ZGA genes in mESCs. Our analysis revealed that H3.3 directly associates with Dux locus and inhibits Dux expression, therefore it is an important upstream regulator of Dux. Our finding is further supported by transcriptome change in early mouse embryos with H3.3 knockdown. We suggest that proper H3.3 level in early embryos is important to orchestrate ZGA activity for totipotency establishment.

Histone H3 methylation orchestrates transcriptional program in mouse spermatogenic cell line.

Changes in histone modifications always correlate with altered transcriptional activities of genes. Recent studies have shown that the mutation of certain lysine residues to methionine in the histone variant H3.3 can act as a valuable tool to reduce specific H3 methylation levels. In our study, we used the mouse spermatogenic cell line GC-2 as a model to generate cells stably expressing H3.3 K4, H3.3 K9, H3.3 K27, and H3.3 K36M. The expression of these H3.3 K-to-M mutants influenced the expression of different subsets of genes, and a total of 891 differentially expressed genes were identified through global gene expression profiling. Moreover, the H3.3 K-to-M transgenes, especially H3.3 K36M, impacted the expression of endogenous retrovirus ERVK. This study gives a global view of how different H3 modifications regulate transcriptomes in spermatogenic cell lines, and identifies potential targets of H3 modifications in male germ line.

PML4 facilitates erythroid differentiation by enhancing the transcriptional activity of GATA-1.

Promyelocytic leukemia protein (PML) has been implicated as a participant in multiple cellular processes including senescence, apoptosis, proliferation, and differentiation. Studies of PML function in hematopoietic differentiation previously focused principally on its myeloid activities and also indicated that PML is involved in erythroid colony formation. However, the exact role that PML plays in erythropoiesis is essentially unknown. In this report, we found that PML4, a specific PML isoform expressed in erythroid cells, promotes endogenous erythroid genes expression in K562 and primary human erythroid cells. We show that the PML4 effect is GATA binding protein 1 (GATA-1) dependent using GATA-1 knockout/rescued G1E/G1E-ER4 cells. PML4, but not other detected PML isoforms, directly interacts with GATA-1 and can recruit it into PML nuclear bodies. Furthermore, PML4 facilitates GATA-1 trans-activation activity in an interaction-dependent manner. Finally, we present evidence that PML4 enhances GATA-1 occupancy within the globin gene cluster and stimulates cooperation between GATA-1 and its coactivator p300. These results demonstrate that PML4 is an important regulator of GATA-1 and participates in erythroid differention by enhancing GATA-1 trans-activation activity.

SIRT1 deacetylates SATB1 to facilitate MAR HS2-MAR ε interaction and promote ε-globin expression.

The higher order chromatin structure has recently been revealed as a critical new layer of gene transcriptional control. Changes in higher order chromatin structures were shown to correlate with the availability of transcriptional factors and/or MAR (matrix attachment region) binding proteins, which tether genomic DNA to the nuclear matrix. How posttranslational modification to these protein organizers may affect higher order chromatin structure still pending experimental investigation. The type III histone deacetylase silent mating type information regulator 2, S. cerevisiae, homolog 1 (SIRT1) participates in many physiological processes through targeting both histone and transcriptional factors. We show that MAR binding protein SATB1, which mediates chromatin looping in cytokine, MHC-I and ß-globin gene loci, as a new type of SIRT1 substrate. SIRT1 expression increased accompanying erythroid differentiation and the strengthening of ß-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the ß-globin locus, MAR(HS2) and MAR(ε). We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation. Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.

Surfeit locus protein 4 modulates endoplasmic reticulum function and maintains oocyte quality.

Surfeit locus protein 4 is a cargo receptor mediating cargo transport from the endoplasmic reticulum lumen to the Golgi apparatus. Loss of Surf4 gene led to embryonic lethality in mice. However, the role of Surf4 during oocyte development remains unknown. In this study, we generated the mouse model with oocyte-specific knockout of Surf4 gene. We found that adult mice with deletion of Surf4 showed normal folliculogenesis, ovulation and fertility. However, loss of Surf4 slightly impaired oocyte quality, thus led to partial oocyte meiotic arrest and reduced ratio of blastocyst formation. Consistent with this, the distribution of endoplasmic reticulum was disturbed in Surf4-deficient oocytes in mice. These results demonstrated that although Surf4 is dispensable for female mouse fertility, Surf4 modulates endoplasmic reticulum arrangement and participates in regulation of developmental competence of oocytes.

Deficiency of nucleosome-destabilizing factor GLYR1 dampens spermatogenesis in mice.

Aberrant sperm morphology hinders sperm motility and causes male subfertility. Spermatogenesis, a complex process in male germ cell development, necessitates precise regulation of numerous developmental genes. However, the regulatory pathways involved in this process remain partially understood. We have observed the widespread expression of Glyr1, the gene encoding a nucleosome-destabilizing factor, in mouse testicular cells. Our study demonstrates that mice experiencing Glyr1 depletion in spermatogenic cells exhibit subfertility characterized by a diminished count and motility of spermatozoa. Furthermore, the rate of sperm malformation significantly increases in the absence of Glyr1, with a predominant occurrence of head and neck malformation in spermatozoa within the cauda epididymis. Additionally, a reduction in spermatocyte numbers across different meiotic stages is observed, accompanied by diminished histone acetylation in spermatogenic cells upon Glyr1 depletion. Our findings underscore the crucial roles of Glyr1 in mouse spermiogenesis and unveil novel insights into the etiology of male reproductive diseases.

The landscape of transcriptional profiles in human oocytes with different chromatin configurations.

With increasingly used assisted reproductive technology (ART), the acquisition of high-quality oocytes and early embryos has become the focus of much attention. Studies in mice have found that the transition of chromatin conformation from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) is essential for oocyte maturation and early embryo development, and similar chromatin transition also exists in human oocytes. In this study, we collected human NSN and SN oocytes and investigated their transcriptome. The analysis of differentially expressed genes showed that epigenetic functions, cyclin-dependent kinases and transposable elements may play important roles in chromatin transition during human oocyte maturation. Our findings provide new insights into the molecular mechanism of NSN-to-SN transition of human oocyte and obtained new clues for improvement of oocyte in vitro maturation technique.