Melatonin supplementation in the culture medium rescues impaired glucose metabolism in IVF mice offspring.

Increasing evidence suggests that in vitro fertilization (IVF) may be associated with an increased risk of developing obesity and metabolic diseases later in life in the offspring. Notably, the addition of melatonin to culture medium may improve embryo development and prevent cardiovascular dysfunction in IVF adult mice. This study aimed to determine if melatonin supplementation in the culture medium can reverse impaired glucose metabolism in IVF mice offspring and the underlying mechanisms. Blastocysts used for transfer were generated by natural mating (control group) or IVF with or without melatonin (10-6  M) supplementation (mIVF and IVF group, respectively) in clinical-grade culture media. Here, we first report that IVF decreased hepatic expression of Fbxl7, which was associated with impaired glucose metabolism in mice offspring. Melatonin addition reversed the phenotype by up-regulating the expression of hepatic Fbxl7. In vitro experiments showed that Fbxl7 enhanced the insulin signaling pathway by degrading RhoA through ubiquitination and was up-regulated by transcription factor Foxa2. Specific knockout of Fbxl7 in the liver of adult mice, through tail intravenous injection of recombinant adeno-associated virus, impaired glucose tolerance, while overexpression of hepatic Fbxl7 significantly improved glucose tolerance in adult IVF mice. Thus, the data suggest that Fbxl7 plays an important role in maintaining glucose metabolism of mice, and melatonin supplementation in the culture medium may rescue the long-term risk of metabolic diseases in IVF offspring.

Distinct H3K9me3 and DNA methylation modifications during mouse spermatogenesis.

DNA methylation and histone modifications critically regulate the expression of many genes and repeat regions during spermatogenesis. However, the molecular details of these processes in male germ cells remain to be addressed. Here, using isolated murine sperm cells, ultra-low-input native ChIP-Seq (ULI-NChIP-Seq), and whole genome bisulfite sequencing (WGBS), we investigated genome-wide DNA methylation patterns and histone 3 Lys-9 trimethylation (H3K9me3) modifications during mouse spermatogenesis. We found that DNA methylation and H3K9me3 have distinct sequence preferences and dynamics in promoters and repeat elements during spermatogenesis. H3K9me3 modifications in histones at gene promoters were highly enriched in round spermatids. H3K9me3 modification on long terminal repeats (LTRs) and long interspersed nuclear elements (LINEs) was involved in silencing active transcription from these regions in conjunction with reestablishment of DNA methylation. Furthermore, H3K9me3 remodeling on the X chromosome was involved in meiotic sex chromosome inactivation and in partial transcriptional reactivation of sex chromosomes in spermatids. Our findings also revealed the DNA methylation patterns and H3K9me3 modification profiles of paternal and maternal germline imprinting control regions (gICRs) during spermatogenesis. Taken together, our results provide a genome-wide map of H3K9me3 modifications during mouse spermatogenesis that may be helpful for understanding male reproductive disorders.

Identification and rescue of a novel TUBB8 mutation that causes the first mitotic division defects and infertility.

PURPOSE: Tubulin beta eight class VIII (TUBB8) is essential for oogenesis, fertilization, and pre-implantation embryo development in human. Although TUBB8 mutations were recently discovered in meiosis-arrested oocytes of infertile females, there is no effective therapy for this gene mutation caused infertility. Our study aims to further reveal the infertility-causing gene mutations in the patient's family and to explore whether the infertility could be rescued by optimizing the conditions of embryo culture and finally achieve the purpose of making the patient pregnant. METHODS: Whole-exome sequence analysis and Sanger sequencing were performed on patients' family members to screen and identify candidate mutant genes. Construction of plasmids, in vitro transcription, microinjection of disease-causing gene cRNA, and immunofluorescence staining were used to recapitulate the infertility phenotype observed in patients and to understand the pathogenic principles. Simultaneously, overexpression of mutant and wild-type cRNA of the candidate gene in mouse oocytes at either germinal vesicle (GV) or metaphase II (MII) stage was performed in the rescue experiment. RESULTS: We first identified a novel heritable TUBB8 mutation (c.1041C>A: p.N347K) in the coding region which specifically affects the first mitosis and causes the developmental arrest of early embryos in a three-generation family. We further demonstrated that TUBB8 mutation could lead to abnormal spindle assemble. And moreover, additional expression of wild-type TUBB8 cRNA in the mouse oocytes in which the mutant TUBB8 were expressed can successfully rescue the developmental defects of resulting embryo and produce full-term offspring. CONCLUSIONS: Our study not only defines a novel mutation of TUBB8 causing the early cleavage arrest of embryos, but also provides an important basis for treating such female infertility in the future.

Dosage effects of ZP2 and ZP3 heterozygous mutations cause human infertility.

The zona pellucida (ZP) is an extracellular matrix universally surrounding mammalian eggs, which is essential for oogenesis, fertilization, and pre-implantation embryo development. Here, we identified two novel heritable mutations of ZP2 and ZP3, both occurring in an infertile female patient with ZP-abnormal eggs. Mouse models with the same mutations were generated by CRISPR/Cas9 gene editing system, and oocytes obtained from female mice with either single heterozygous mutation showed approximately half of the normal ZP thickness compared to wild-type oocytes. Importantly, oocytes with both heterozygous mutations showed a much thinner or even missing ZP that could not avoid polyspermy fertilization, following the patient's pedigree. Further analysis confirmed that precursor proteins produced from either mutated ZP2 or ZP3 could not anchor to oocyte membranes. From these, we conclude that ZP mutations have dosage effects which can cause female infertility in humans. Finally, this patient was treated by intracytoplasmic sperm injection (ICSI) with an improved culture system and successfully delivered a healthy baby.

Human-caused increases in organic carbon burial in plateau lakes: The response to warming effect.

The patterns of organic carbon sequestration in lakes, along with their temporal dynamics, have profound implications for assessing the strength of terrestrial carbon sinks and the global carbon budget. The complexity of fluctuations in organic carbon burial in freshwater lake basins, along with the intricate interactions among various controlling factors over time, remains challenging to comprehend. By utilizing data on the organic carbon burial of sedimentary cores from twelve plateau lakes in a gradient of urbanization, this study employed a rigorous methodology to quantify the factors and origins that contribute to lacustrine carbon sequestration. The findings indicate that the rate of Total Organic Carbon (TOC) accumulation in lakes in highly urbanized areas has significantly surpassed that in areas with minimal urbanization since 1985. This trend of divergence has persisted for more than four decades. During the period from 1958 to 2008, soil nutrient characteristics (29.576 %) and human impact (16.684 %) were the major factors regulating the organic carbon burial in plateau lakes. Human pressures indirectly impact carbon sequestration through earth-surface processes in the lake basin, causing carbon burial to lag behind environmental indicators (e.g., δ13C and C/N) by approximately 5 years. Meanwhile, the carbon sequestration efficiency of plateau lakes shows a positive feedback to climatic warming with intensified urbanization, primarily regulated through the impacts on lake basin environments. The results will further enhance our understanding of the response of the lake ecosystem carbon cycle to anthropogenic influences.

RNA m6A modification regulates cell fate transition between pluripotent stem cells and 2-cell-like cells.

N6-methyladenosine (m6A) exerts essential roles in early embryos, especially in the maternal-to-zygotic transition stage. However, the landscape and roles of RNA m6A modification during the transition between pluripotent stem cells and 2-cell-like (2C-like) cells remain elusive. Here, we utilised ultralow-input RNA m6A immunoprecipitation to depict the dynamic picture of transcriptome-wide m6A modifications during 2C-like transitions. We found that RNA m6A modification was preferentially enriched in zygotic genome activation (ZGA) transcripts and MERVL with high expression levels in 2C-like cells. During the exit of the 2C-like state, m6A facilitated the silencing of ZGA genes and MERVL. Notably, inhibition of m6A methyltransferase METTL3 and m6A reader protein IGF2BP2 is capable of significantly delaying 2C-like state exit and expanding 2C-like cells population. Together, our study reveals the critical roles of RNA m6A modification in the transition between 2C-like and pluripotent states, facilitating the study of totipotency and cell fate decision in the future.

Immune rebalancing at the maternal-fetal interface of maternal SARS-CoV-2 infection during early pregnancy.

The current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) remains a threat to pregnant women. However, the impact of early pregnancy SARS-CoV-2 infection on the maternal-fetal interface remains poorly understood. Here, we present a comprehensive analysis of single-cell transcriptomics and metabolomics in placental samples infected with SARS-CoV-2 during early pregnancy. Compared to control placentas, SARS-CoV-2 infection elicited immune responses at the maternal-fetal interface and induced metabolic alterations in amino acid and phospholipid profiles during the initial weeks post-infection. However, subsequent immune cell activation and heightened immune tolerance in trophoblast cells established a novel dynamic equilibrium that mitigated the impact on the maternal-fetal interface. Notably, the immune response and metabolic alterations at the maternal-fetal interface exhibited a gradual decline during the second trimester. Our study underscores the adaptive immune tolerance mechanisms and establishment of immunological balance during the first two trimesters following maternal SARS-CoV-2 infection.

Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells.

Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.

[Jinshuibao capsule combined losartan potassium intervened early renal damage of hypertension patients of yin and yang deficiency: a clinical research].

OBJECTIVE: To observe the effects of Jinshuibao Capsule (JC) combined losartan potassium on some indices of early renal damage of hypertension patients of yin and yang deficiency syndrome (YYDS), such as levels of serum cystatin C (Cys C), beta2-microglobulin (beta2-MG), hypersensitive C-reactive protein (hs-CRP), uric acid (UA), blood pressure, blood lipids, and fasting blood glucose (FBG), and to explore their protective effects on early renal damage of hypertension patients and on the metabolisms of blood lipids and blood glucose. METHODS: Totally 106 hypertension patients of YYDS were randomly assigned to two groups, 53 patients in the control group (treated by losartan potassium) and 53 patients in the treatment group (treated by JC + losartan potassium). The treatment lasted for 16 weeks. The serum changes of UA, Cys C, beta2-MG, hs-CRP, blood lipids [including total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C)], and FBG levels were measured to evaluate the renal protective effects and to assess their effect on the metabolisms of blood lipids and blood glucose. RESULTS: Compared with before treatment in the same group, the systolic blood pressure (SBP) decreased in the two groups after treatment, showing statistical difference (P < 0.05, P < 0.01), but there was no statistical difference between the two groups (P > 0.05). The diastolic blood pressure (DBP) was not obviously declined in the two groups after treatment, showing no statistical difference. Compared with before treatment in the same group, the LDL-C level decreased obviously after treatment in the control group. But there was no obvious change in FBG, TC, HDL-C, and TG in the control group, showing no statistical difference when compared with before treatment (P < 0.05). The FBG, TC, and LDL-C obviously decreased in the treatment group more obviously after treatment than before treatment, showing statistical difference (P < 0.05, P < 0.01). There was no statistical difference when compared with the control group after treatment (P > 0.05). Compared with before treatment in the same group, the levels of UA, Cys C, beta2-MG, and hs-CRP all decreased in the two groups, showing statistical difference (P < 0.05, P < 0.01). The SCr level decreased in the treatment group more obviously after treatment than before treatment, showing statistical difference (P < 0.05). Compared with the control group after treatment, the levels of Cys C, beta2-MG, and hs-CRP decreased more obviously after treatment in the treatment group, showing statistical difference (P < 0.05). CONCLUSIONS: JC combined losartan potassium showed better effects in treating early renal damage of hypertension patients of YYDS. They could protect and stabilize the renal functions more effectively. JC could regulate blood lipids and blood glucose.

Inhibition of Wnt activity improves peri-implantation development of somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells into totipotency. Although pre-implantation development of SCNT embryos has greatly improved, most SCNT blastocysts are still arrested at the peri-implantation stage, and the underlying mechanism remains elusive. Here, we develop a 3D in vitro culture system for SCNT peri-implantation embryos and discover that persistent Wnt signals block the naïve-to-primed pluripotency transition of epiblasts with aberrant H3K27me3 occupancy, which in turn leads to defects in epiblast transformation events and subsequent implantation failure. Strikingly, manipulating Wnt signals can attenuate the pluripotency transition and H3K27me3 deposition defects in epiblasts and achieve up to a 9-fold increase in cloning efficiency. Finally, single-cell RNA-seq analysis reveals that Wnt inhibition markedly enhances the lineage developmental trajectories of SCNT blastocysts during peri-implantation development. Overall, these findings reveal diminished potentials of SCNT blastocysts for lineage specification and validate a critical peri-implantation barrier for SCNT embryos.

Remodeling of H3K9me3 during the pluripotent to totipotent-like state transition.

Multiple chromatin modifiers associated with H3K9me3 play important roles in the transition from embryonic stem cells to 2-cell (2C)-like cells. However, it remains elusive how H3K9me3 is remodeled and its association with totipotency. Here, we integrated transcriptome and H3K9me3 profiles to conduct a detailed comparison of 2C embryos and 2C-like cells. Globally, H3K9me3 is highly preserved and H3K9me3 dynamics within the gene locus is not associated with gene expression change during 2C-like transition. Promoter-deposited H3K9me3 plays non-repressive roles in the activation of genes during 2C-like transition. In contrast, transposable elements, residing in the nearby regions of up-regulated genes, undergo extensive elimination of H3K9me3 and are tended to be induced in 2C-like transitions. Furthermore, a large fraction of trophoblast stem cell-specific enhancers undergo loss of H3K9me3 exclusively in MERVL+/Zscan4+ cells. Our study therefore reveals the unique H3K9me3 profiles of 2C-like cells, facilitating the further exploration of totipotency.

Resetting histone modifications during human prenatal germline development.

Histone modifications play critical roles in regulating gene expression and present dynamic changes during early embryo development. However, how they are reprogrammed during human prenatal germline development has not yet been elucidated. Here, we map the genome-wide profiles of three key histone modifications in human primordial germ cells (hPGCs) from weeks 8 to 23 of gestation for the first time by performing ULI-NChIP-seq. Notably, H3K4me3 exhibits a canonical promoter-enriched pattern, though with relatively lower enrichment, and is positively correlated with gene expression in globally hypomethylated hPGCs. In addition, H3K27me3 presents very low enrichment but plays an important role in not only dynamically governing specific bivalent promoters but also impeding complete X chromosome reactivation in female hPGCs. Given the activation effects of both global DNA demethylation and H3K4me3 signals, repressive H3K9me3 and H3K27me3 marks are jointly responsible for the paradoxical regulation of demethylation-resistant regions in hPGCs. Collectively, our results provide a unique roadmap of three core histone modifications during hPGC development, which helps to elucidate the architecture of germ cell reprogramming in an extremely hypomethylated DNA environment.

Bilineage embryo-like structure from EPS cells can produce live mice with tetraploid trophectoderm.

Self-organized blastoids from extended pluripotent stem (EPS) cells possess enormous potential for investigating postimplantation embryo development and related diseases. However, the limited ability of postimplantation development of EPS-blastoids hinders its further application. In this study, single-cell transcriptomic analysis indicated that the "trophectoderm (TE)-like structure" of EPS-blastoids was primarily composed of primitive endoderm (PrE)-related cells instead of TE-related cells. We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure. Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation. Furthermore, we demonstrated that blastocyst-like structures reconstituted by combining the EPS-derived bilineage embryo-like structure (BLES) with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses. In summary, our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.

BMP4 preserves the developmental potential of mESCs through Ube2s- and Chmp4b-mediated chromosomal stability safeguarding.

Chemically defined medium is widely used for culturing mouse embryonic stem cells (mESCs), in which N2B27 works as a substitution for serum, and GSK3ß and MEK inhibitors (2i) help to promote ground-state pluripotency. However, recent studies suggested that MEKi might cause irreversible defects that compromise the developmental potential of mESCs. Here, we demonstrated the deficient bone morphogenetic protein (BMP) signal in the chemically defined condition is one of the main causes for the impaired pluripotency. Mechanistically, activating the BMP signal pathway by BMP4 could safeguard the chromosomal integrity and proliferation capacity of mESCs through regulating downstream targets Ube2s and Chmp4b. More importantly, BMP4 promotes a distinct in vivo developmental potential and a long-term pluripotency preservation. Besides, the pluripotent improvements driven by BMP4 are superior to those by attenuating MEK suppression. Taken together, our study shows appropriate activation of BMP signal is essential for regulating functional pluripotency and reveals that BMP4 should be applied in the serum-free culture system.

Aberrant H3K4me3 modification of epiblast genes of extraembryonic tissue causes placental defects and implantation failure in mouse IVF embryos.

Assisted reproductive technology has been widely applied in the treatment of human infertility. However, accumulating evidence indicates that in vitro fertilization (IVF) is associated with a low pregnancy rate, placental defects, and metabolic diseases in offspring. Here, we find that IVF manipulation notably disrupts extraembryonic tissue-specific gene expression, and 334 epiblast (Epi)-specific genes and 24 Epi-specific transcription factors are abnormally expressed in extraembryonic ectoderm (ExE) of IVF embryos at embryonic day 7.5. Combined histone modification analysis reveals that aberrant H3K4me3 modification at the Epi active promoters results in increased expression of these genes in ExE. Importantly, we demonstrate that knockdown of the H3K4me3-recruited regulator Kmt2e, which is highly expressed in IVF embryos, greatly improves the development of IVF embryos and reduces abnormal gene expression in ExE. Our study therefore identifies that abnormal H3K4me3 modification in extraembryonic tissue is a major cause of implantation failure and abnormal placental development of IVF embryos.

Unique Patterns of H3K4me3 and H3K27me3 in 2-Cell-like Embryonic Stem Cells.

A small subgroup of embryonic stem cells (ESCs) exhibit molecular features similar to those of two-cell embryos (2C). However, it remains elusive whether 2C-like cells and 2C embryos share similar epigenetic features. Here, we map the genome-wide profiles of histone H3K4me3 and H3K27me3 in 2C-like cells. We found that the majority of genes in 2C-like cells inherit their histone status from ESCs. Among the genes showing a switch in their histone methylation status during 2C-like transitions, only a small number acquire 2C-embryo epigenetic signatures. In contrast, broad H3K4me3 domains display extensive loss in 2C-like cells. Most of the differentially expressed genes display decreased H3K4me3 and H3K27me3 levels in 2C-like cells, whereas de novo H3K4me3 deposition is closely linked with the expression levels of upregulated 2C-specific genes. Taken together, our study reveals the unique epigenetic profiles of 2C-like cells, facilitating the further exploration of totipotency in the future.

Dcaf11 activates Zscan4-mediated alternative telomere lengthening in early embryos and embryonic stem cells.

Telomeres play vital roles in ensuring chromosome stability and are thus closely linked with the onset of aging and human disease. Telomeres undergo extensive lengthening during early embryogenesis. However, the detailed molecular mechanism of telomere resetting in early embryos remains unknown. Here, we show that Dcaf11 (Ddb1- and Cul4-associated factor 11) participates in telomere elongation in early embryos and 2-cell-like embryonic stem cells (ESCs). The deletion of Dcaf11 in embryos and ESCs leads to reduced telomere sister-chromatid exchange (T-SCE) and impairs telomere lengthening. Importantly, Dcaf11-deficient mice exhibit gradual telomere erosion with successive generations, and hematopoietic stem cell (HSC) activity is also greatly compromised. Mechanistically, Dcaf11 targets Kap1 (KRAB-associated protein 1) for ubiquitination-mediated degradation, leading to the activation of Zscan4 downstream enhancer and the removal of heterochromatic H3K9me3 at telomere/subtelomere regions. Our study therefore demonstrates that Dcaf11 plays important roles in telomere elongation in early embryos and ESCs through activating Zscan4.

Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation.

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.

Genome transfer for the prevention of female infertility caused by maternal gene mutation.

Poor oocyte quality is associated with early embryo developmental arrest and infertility. Maternal gene plays crucial roles in the regulation of oocyte maturation, and its mutation is a common cause of female infertility. However, how to improve oocyte quality and develop effective therapy for maternal gene mutation remains elusive. Here, we use Zar1 as an example to assess the feasibility of genome transfer to cure maternal gene mutation-caused female infertility. We first discover that cytoplasmic deficiency primarily leads to Zar1-null embryo developmental arrest by disturbing maternal transcript degradation and minor zygotic genome activation (ZGA) during the maternal-zygotic transition. We next perform genome transfer at the oocyte (spindle transfer or polar body transfer) and zygote (early pronuclear transfer or late pronuclear transfer) stages to validate the feasibility of preventing Zar1 mutation-caused infertility. We finally demonstrate that genome transfer either at the oocyte or at the early pronuclear stage can support normal preimplantation embryo development and produce live offspring. Moreover, those pups grow to adulthood and show normal fertility. Therefore, our findings provide an effective basis of therapies for the treatment of female infertility caused by maternal gene mutation.

Nuclear Exosome Targeting Complex Core Factor Zcchc8 Regulates the Degradation of LINE1 RNA in Early Embryos and Embryonic Stem Cells.

The nuclear exosome targeting (NEXT) complex is responsible for specific nuclear RNA degradation in mammalian cells. However, its function in development remains unknown. Here, we find that the depletion of a central factor of the NEXT complex, Zcchc8, in mouse results in developmental defects, a shortened lifespan, and infertility. We find that Zcchc8-deficient embryonic stem cells (ESCs) exhibit proliferation abnormalities and reduced developmental potencies. Importantly, the transcripts of retrotransposon element LINE1 are found to be targeted by Zcchc8 and degraded by a Zcchc8-mediated mechanism. We further find that sustained expression of higher levels of LINE1 RNA is detected in maternal Zcchc8-depleted oocytes and embryos. Zcchc8-depleted oocytes show higher chromatin accessibility and developmental defects in both meiotic maturation and embryogenesis after fertilization. Collectively, our study defines Zcchc8-mediated RNA degradation as an important post-transcription regulation of LINE1 transcripts in early embryos and ESCs, which play vital roles in the pluripotency and early development.