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.

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.

Chromatin Modifier EP400 Regulates Oocyte Quality and Zygotic Genome Activation in Mice.

Epigenetic modifiers that accumulate in oocytes, play a crucial role in steering the developmental program of cleavage embryos and initiating life. However, the identification of key maternal epigenetic regulators remains elusive. In the findings, the essential role of maternal Ep400, a chaperone for H3.3, in oocyte quality and early embryo development in mice is highlighted. Depletion of Ep400 in oocytes resulted in a decline in oocyte quality and abnormalities in fertilization. Preimplantation embryos lacking maternal Ep400 exhibited reduced major zygotic genome activation (ZGA) and experienced developmental arrest at the 2-to-4-cell stage. The study shows that EP400 forms protein complex with NFYA, occupies promoters of major ZGA genes, modulates H3.3 distribution between euchromatin and heterochromatin, promotes transcription elongation, activates the expression of genes regulating mitochondrial functions, and facilitates the expression of rate-limiting enzymes of the TCA cycle. This intricate process driven by Ep400 ensures the proper execution of the developmental program, emphasizing its critical role in maternal-to-embryonic transition.

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.

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.

Regulation of cleavage embryo genes upon DRP1 inhibition in mouse embryonic stem cells.

Dynamic-related protein 1 (DRP1) is a key protein of mitochondrial fission. In this study, we found that inhibition of activity of DRP1 led to increased levels of cleavage embryo genes in mouse embryonic stem cells (mESCs), which might reflect a transient totipotency status derived from pluripotency. This result indicates that DRP1 inhibition in mESCs leads to a tendency to obtain a new expression profile similar to that of the 2C-like state. Meanwhile, we also noticed that the glycolysis/gluconeogenesis pathway and its related enzymes were significantly downregulated, and the key glycolytic enzymes were also downregulated in various 2C-like cells. Moreover, when DRP1 activity was inhibited from the late zygote when cleavage embryo genes started to express, development of early embryos was inhibited, and these cleavage embryo genes failed to be efficiently silenced at the late 2-cell (2C) stage. Taken together, our result shows that DRP1 plays an important role in silencing cleavage embryo genes for totipotency-to-pluripotency transition.

H3K4 Methylation Promotes Expression of Mitochondrial Dynamics Regulators to Ensure Oocyte Quality in Mice.

Significantly decreased H3K4 methylation in oocytes from aged mice indicates the important roles of H3K4 methylation in female reproduction. However, how H3K4 methylation regulates oocyte development remains largely unexplored. In this study, it is demonstrated that oocyte-specific expression of dominant negative mutant H3.3-K4M led to a decrease of the level of H3K4 methylation in mouse oocytes, resulting in reduced transcriptional activity and increased DNA methylation in oocytes, disturbed oocyte developmental potency, and fertility of female mice. The impaired expression of genes regulating mitochondrial functions in H3.3-K4M oocytes, accompanied by mitochondrial abnormalities, is further noticed. Moreover, early embryos from H3.3-K4M oocytes show developmental arrest and reduced zygotic genome activation. Collectively, these results show that H3K4 methylation in oocytes is critical to orchestrating gene expression profile, driving the oocyte developmental program, and ensuring oocyte quality. This study also improves understanding of how histone modifications regulate organelle dynamics in oocytes.

Transcriptome and DNA methylome analysis of peripheral blood samples reveals incomplete restoration and transposable element activation after 3-months recovery of COVID-19.

Comprehensive analyses showed that SARS-CoV-2 infection caused COVID-19 and induced strong immune responses and sometimes severe illnesses. However, cellular features of recovered patients and long-term health consequences remain largely unexplored. In this study, we collected peripheral blood samples from nine recovered COVID-19 patients (median age of 36 years old) from Hubei province, China, 3 months after discharge as well as 5 age- and gender-matched healthy controls; and carried out RNA-seq and whole-genome bisulfite sequencing to identify hallmarks of recovered COVID-19 patients. Our analyses showed significant changes both in transcript abundance and DNA methylation of genes and transposable elements (TEs) in recovered COVID-19 patients. We identified 425 upregulated genes, 214 downregulated genes, and 18,516 differentially methylated regions (DMRs) in total. Aberrantly expressed genes and DMRs were found to be associated with immune responses and other related biological processes, implicating prolonged overreaction of the immune system in response to SARS-CoV-2 infection. Notably, a significant amount of TEs was aberrantly activated and their activation was positively correlated with COVID-19 severity. Moreover, differentially methylated TEs may regulate adjacent gene expression as regulatory elements. Those identified transcriptomic and epigenomic signatures define and drive the features of recovered COVID-19 patients, helping determine the risks of long COVID-19, and guiding clinical intervention.

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.

Insm2 deficiency results in female infertility by disturbing steroid pathway and decreasing ovarian reserve in mice.

The number and quality of oocytes in the ovarian reserve are related to fertility and reproductive lifespan in mammals. Some transcription factors have been demonstrated to determine oogenesis. The insulinoma-associated 2 (Insm2) gene is a member of the Snail transcriptional repressor superfamily. Recent studies have demonstrated Insm2 plays an essential role for insulin secretion and glucose intolerance in mice, but the functions of Insm2 in reproduction remain elusive. Here, by examination of Insm2 knockout mice, we found Insm2 was essential for female fertility. Loss of Insm2 resulted in female infertility with major defects in primordial follicle pool, ovarian folliculogenesis and ovulation. Transcriptomic profiling of ovaries suggests that loss of Insm2 caused defects in oocyte meiosis and steroid synthesis. Both oocyte- and granulosa cell-expressed genes were dysregulated, including Foxo1 and other known genes involved in primary ovarian insufficiency. Together, these studies show that Insm2 is required for oocyte development and their communication with ovarian somatic cells.

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.

Effect and Mechanism of lncRNA CERS6-AS1 on the Biological Behavior of Prostate Cancer Cell.

Objective: To investigate the effect of long noncoding RNA (lncRNA) CERS6 antisense RNA1 (CERS6-AS1) on the biological behavior of prostate cancer cells DU145 and its mechanism. Methods: RT-PCR was used to detect the relative level of CERS6-AS1 and miR-16-5p in prostate cancer tissues, adjacent tissues, prostate cancer cells DU145, and human normal prostate epithelial cells RWPE-1. DU145 cells were divided into control group, si-CERS6-AS1 group, si-NC group, miR-16-5p mimic group, miR-NC group, and si-CERS6-AS1+miR-16-5p inhibitor group. And CCK-8 method and colony formation test was applied to detect cell proliferation ability, flow cytometry was selected to calculate cell apoptosis, and scratch healing test was employed to assess cell migration ability. Western blot was determined to detect high mobility protein A2 (HMGA2) expression. RT-PCR and dual-luciferase reporter experiments were used to analyze the targeting relationship among CERS6-AS1, miR-16-5p, and HMGA2. Results: Compared with the adjacent tissues, the relative level of CERS6-AS1 in prostate cancer tissue was increased (P < 0.05), and the relative level of miR-16-5p was decreased (P < 0.05). Compared with RWPE-1 cells, the relative level of CERS6-AS1 in DU145 cells was increased (P < 0.05), and the relative level of miR-16-5p was decreased (P < 0.05). Compared with the control group and the si-NC group, the HMGA2 protein expression, the colony formation number, and the scratch healing rate of DU145 cells in the si-CERS6-AS1 group and the miR-16-5p mimic group were reduced (P < 0.05), and the relative level of miR-16-5p and the proliferation inhibition rate and apoptosis were increased (P < 0.05). miR-16-5p is specifically bound to CERS6-AS1 and HMGA2, respectively. Compared with the si-CERS6-AS1 group, the HMGA2 protein expression, the colony formation number, and the scratch healing rate of DU145 cells in the si-CERS6-AS1+miR-16-5p inhibitor group were increased (P < 0.05), and the cell proliferation inhibition rate and apoptosis rate were reduced (P < 0.05). Conclusion: Silencing CERS6-AS1 can inhibit the proliferation and migration of prostate cancer cell DU145 and induce cell apoptosis, the mechanism is related to the regulation of the miR-16-5p/HMGA2 axis.

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.

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.

Aberrant Expression of Mitochondrial SAM Transporter SLC25A26 Impairs Oocyte Maturation and Early Development in Mice.

The immature germinal vesicle (GV) oocytes proceed through metaphase I (MI) division, extrude the first polar body, and become mature metaphase II (MII) oocytes for fertilization which is followed by preimplantation and postimplantation development until birth. Slc25a26 is the gene encoding S-adenosylmethionine carrier (SAMC), a member of the mitochondrial carrier family. Its major function is to catalyze the uptake of S-adenosylmethionine (SAM) from cytosol into mitochondria, which is the only known mitochondrial SAM transporter. In the present study, we demonstrated that excessive SLC25A26 accumulation in mouse oocytes mimicked naturally aged oocytes and resulted in lower oocyte quality with decreased maturation rate and increased reactive oxygen species (ROS) by impairing mitochondrial function. Increased level of Slc25a26 gene impacted gene expression in mouse oocytes such as mt-Cytb which regulates mitochondrial respiratory chain. Furthermore, increased level of Slc25a26 gene in fertilized oocytes slightly compromised blastocyst formation, and Slc25a26 knockout mice displayed embryonic lethality around 10.5 dpc. Taken together, our results showed that Slc25a26 gene plays a critical role in oocyte maturation and early mouse development.

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.

Lactate Activates Germline and Cleavage Embryo Genes in Mouse Embryonic Stem Cells.

Lactate was recently found to mediate histone lysine lactylation and facilitate polarization of M1 macrophages, indicating its role in metabolic regulation of gene expression. During somatic cell reprogramming, lactate promotes histone lactylation of pluripotency genes and improves reprogramming efficiency. However, the function of lactate in cell fate control in embryonic stem cells (ESCs) remains elusive. In this study, we revealed that lactate supplementation activated germline genes in mouse ESCs. Lactate also induced global upregulation of cleavage embryo genes, such as members of the Zscan4 gene family. Further exploration demonstrated that lactate stimulated H3K18 lactylation accumulation on germline and cleavage embryo genes, which in turn promoted transcriptional elongation. Our findings indicated that lactate supplementation expanded the transcriptional network in mouse ESCs.

Inhibition of DRP1 Impedes Zygotic Genome Activation and Preimplantation Development in Mice.

Mitochondrion plays an indispensable role during preimplantation embryo development. Dynamic-related protein 1 (DRP1) is critical for mitochondrial fission and controls oocyte maturation. However, its role in preimplantation embryo development is still lacking. In this study, we demonstrate that inhibition of DRP1 activity by mitochondrial division inhibitor-1, a small molecule reported to specifically inhibit DRP1 activity, can cause severe developmental arrest of preimplantation embryos in a dose-dependent manner in mice. Meanwhile, DRP1 inhibition resulted in mitochondrial dysfunction including decreased mitochondrial activity, loss of mitochondrial membrane potential, reduced mitochondrial copy number and inadequate ATP by disrupting both expression and activity of DRP1 and mitochondrial complex assembly, leading to excessive ROS production, severe DNA damage and cell cycle arrest at 2-cell embryo stage. Furthermore, reduced transcriptional and translational activity and altered histone modifications in DRP1-inhibited embryos contributed to impeded zygotic genome activation, which prevented early embryos from efficient development beyond 2-cell embryo stage. These results show that DRP1 inhibition has potential cytotoxic effects on mammalian reproduction, and DRP1 inhibitor should be used with caution when it is applied to treat diseases. Additionally, this study improves our understanding of the crosstalk between mitochondrial metabolism and zygotic genome activation.

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.

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.