Histone lysine lactylation (Kla) is a novel epigenetic modification that plays a crucial role in cellular processes driven by glycolysis and lactate production. However, the mechanisms of histone lactylation and its interaction with m6A RNA methylation during early embryonic development remain underexplored. This study systematically investigated the effects of oxygen levels-atmospheric oxygen (atmosO; 20% O) and physiological oxygen (physO; 5% O)-on hallmark events during early embryonic development, revealing that lactylation modification regulates early goat embryonic development through the m6A methyltransferase-like 3 (METTL3). We observed that physO conditions significantly promote embryonic development, with higher expression levels of METTL3, global histone lactylation, and histone H3 lysine 18 lactylation (H3K18la) compared to atmosO exposure. Furthermore, the addition of lactate dehydrogenase inhibitors led to a decrease in global lactylation, which was accompanied by a significant reduction in METTL3 expression. Sequencing analysis of the METTL3 knockdown embryo revealed that the differentially expressed genes (DEGs) were primarily enriched in the ribosome, oxidative phosphorylation, thermogenesis, RNA degradation, and RNA polymerase pathways. These findings provide novel insights into the epigenetic regulatory mechanisms of histone lactylation during early embryonic development in livestock, highlighting potential molecular targets and strategies to enhance mammalian in vitro embryo production techniques.

Post-translational modifications of histone H3 on lysine 9, specifically acetylation (H3K9ac) and tri-methylation (H3K9me3), play a critical role in regulating chromatin accessibility. However, the role of these modifications in lineage segregation in the mammalian blastocyst remains poorly understood. We demonstrate that di- and tri-methylation marks, H3K9me2 and H3K9me3, decrease during cavitation and expansion of the rabbit blastocyst. Notably, H3K9me3 levels are particularly low in inner cell mass cells at the onset of blastocyst formation but increase again just before gastrulation. Conversely, H3K9ac is abundant in early blastocyst stages but decreases during the transition from the inner cell mass to the epiblast. These distinct distribution patterns correlate with high expression levels of methyltransferases (EHMT1, EHMT2, SETDB1) and deacetylases (HDAC1, HDAC2, HDAC5) in expanding blastocysts. Functionally, inhibiting H3K9me2/3 through an EHMT1/2 inhibitor disrupts primitive endoderm segregation, whereas enhancing histone acetylation (including H3K9ac) using a class I HDAC inhibitor promotes epiblast expansion at the expense of the primitive endoderm. These modifications impact the expression of genes associated with pluripotency and lineage determination, underscoring the importance of H3K9 modifications in embryonic cell fate decisions.

It remains poorly understood about the regulation of gene and transposon transcription during human early embryogenesis. Here, we report that broad H3K27ac domains are genome-widely distributed in human 2-cell and 4-cell embryos and transit into typical peaks in the 8-cell embryos. The broad H3K27ac domains in early embryos before zygotic genome activation (ZGA) are also observed in mouse. It suggests that broad H3K27ac domains play conserved functions before ZGA in mammals. Intriguingly, a large portion of broad H3K27ac domains overlap with broad H3K4me3 domains. Further investigation reveals that histone deacetylases are required for the removal or transition of broad H3K27ac domains and ZGA. After ZGA, the number of typical H3K27ac peaks is dynamic, which is associated with the stage-specific gene expression. Furthermore, P300 is important for the establishment of H3K27ac peaks and the expression of associated genes in early embryos after ZGA. Our data also indicate that H3K27ac marks active transposons in early embryos. Interestingly, H3K27ac and H3K18ac signals rather than H3K9ac signals are enriched at ERVK elements in mouse embryos after ZGA. It suggests that different types of histone acetylations exert distinct roles in the activation of transposons. In summary, H3K27ac modification undergoes extensive reprogramming during early embryo development in mammals, which is associated with the expression of genes and transposons.

BACKGROUND:Previous studies have shown that the vitrification of metaphase II (MII) oocytes significantly represses their developmental potential. Abnormally increased oxidative stress is the probable factor; however, the underlying mechanism remains unclear. The walnut-derived peptide TW-7 was initially isolated and purified from walnut protein hydrolysate. Accumulating evidences implied that TW-7 was a powerful antioxidant, while its prospective application in oocyte cryopreservation has not been reported. RESULT:Here, we found that parthenogenetic activation (PA) zygotes derived from vitrified MII oocytes showed elevated ROS level and delayed progression of pronucleus formation. Addition of 25 μmol/L TW-7 in warming, recovery, PA, and embryo culture medium could alleviate oxidative stress in PA zygotes from vitrified mouse MII oocytes, furtherly increase proteins related to histone lactylation such as LDHA, LDHB, and EP300 and finally improve histone lactylation in PA zygotes. The elevated histone lactylation facilitated the expression of minor zygotic genome activation (ZGA) genes and preimplantation embryo development. CONCLUSIONS:Our findings revealed the mechanism of oxidative stress inducing repressed development of PA embryos from vitrified mouse MII oocytes and found a potent and easy-obtained short peptide that could significantly rescue the decreased developmental potential of vitrified oocytes, which would potentially contribute to reproductive medicine, animal protection, and breeding.

Numerous post-translational modifications are involved in oocyte maturation and embryo development. Recently, lactylation has emerged as a novel epigenetic modification implicated in the regulation of diverse cellular processes. However, it remains unclear whether lactylation occurs during oocyte maturation and embryo development processes. Herein, the lysine lactylation (Kla) modifications were determined during mouse oocyte maturation and early embryo development by immunofluorescence staining. Exogenous lactate was supplemented to explore the consequences of modulating histone lactylation levels on oocyte maturation and embryo development processes by transcriptomics. Results demonstrated that lactylated proteins are widely present in mice with tissue- and cell-specific distribution. During mouse oocyte maturation, immunofluorescence for H3K9la, H3K14la, H4K8la, and H4K12la was most intense at the germinal vesicle (GV) stage and subsequently weakened or disappeared. Further, supplementing the culture medium with 10 mM sodium lactate elevated both the oocyte maturation rate and the histone Kla levels in GV oocytes, and there were substantial increases in Kla levels in metaphase II (MII) oocytes. It altered the transcription of molecules involved in oxidative phosphorylation. Moreover, histone lactylation levels changed dynamically during mouse early embryogenesis. Sodium lactate at 10 mM enhanced early embryo development and significantly increased lactylation, while impacting glycolytic gene transcription. This study reveals the roles of lactylation during oocyte maturation and embryo development, providing new insights to improving oocyte maturation and embryo quality.

Histone lysine modifications were well-established epigenetic markers, with many types identified and extensively studied. The discovery of histone lysine lactylation had revealed a new form of epigenetic modification. The intensification of this modification was associated with glycolysis and elevated intracellular lactate levels, both of which were closely linked to cellular metabolism. Histone lactylation plays a crucial role in multiple cellular homeostasis, including immune regulation and cancer progression, thereby significantly influencing cell fate. Lactylation can modify both histone and non-histone proteins. This paper provided a comprehensive review of the typical epigenetic effects and lactylation on classical transcription-related lysine sites and summarized the known enzymes involved in histone lactylation and delactylation. Additionally, some discoveries of histone lactylation in tumor biology were also discussed, and some prospects for this field were put forward.

Lactylation is a novel post-translational modification (PTM) involving proteins that is induced by lactate accumulation. Histone lysine lactylation alters chromatin spatial configuration, influencing gene transcription and regulating the expression of associated genes. This modification plays a crucial role as an epigenetic regulatory factor in the progression of various diseases. Glycolytic reprogramming is one of the most extensively studied forms of metabolic reprogramming, recognized as a key hallmark of cancer cells. It is characterized by an increase in glycolysis and the inhibition of the tricarboxylic acid (TCA) cycle, accompanied by significant lactate production and accumulation. The two processes are closely linked by lactate, which interacts in various physiological and pathological processes. On the one hand, lactylation levels generally correlate positively with the extent of glycolytic reprogramming, being directly influenced by the lactate concentration produced during glycolytic reprogramming. On the other hand, lactylation can also regulate glycolytic pathways by affecting the transcription and structural functions of essential glycolytic enzymes. This review comprehensively outlines the mechanisms of lactylation and glycolytic reprogramming and their interactions in tumor progression, immunity, and inflammation, with the aim of elucidating the relationship between glycolytic reprogramming and lactylation.

BACKGROUND:Dynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated. RESULTS:We examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O), gradient oxygen concentration (5% O to 2% O) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate. CONCLUSIONS:We provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.

It is demonstrated that repeated superovulation has deleterious effects on mouse ovaries and cumulus cells. However, little is known about the effects of repeated superovulation on early embryos. Epigenetic reprogramming is an important event in early embryonic development and could be easily disrupted by the environment. Thus, we speculated that multiple superovulations may have adverse effects on histone modifications in the early embryos. Female CD1 mice were randomly divided into four groups: (a) spontaneous estrus cycle (R0); (b) with once superovulation (R1); (c) with three times superovulation at a 7-day interval (R3) and (d) with five times superovulation at a 7-day interval (R5). We found that repeated superovulation remarkably decreased the fertilization rate. With the increase of superovulation times, the rate of early embryo development was decreased. The expression of Oct4, Sox2 and Nanog was also affected by superovulation in blastocysts. The immunofluorescence results showed that the acetylation level of histone 4 at lysine 12 (H4K12ac) was significantly reduced by repeated superovulation in mouse early embryos (P < 0.01). Acetylation level of histone 4 at lysine 16 (H4K16ac) was also significantly reduced in pronuclei and blastocyst along with the increase of superovulation times (P < 0.01). H3K9me2 and H3K27me3 were significantly increased in four-cell embryos and blastocysts. We further found that repeated superovulation treatment increased the mRNA level of histone deacetylases Hdac1, Hdac2 and histone methyltransferase G9a, but decreased the expression level of histone demethylase-encoding genes Kdm6a and Kdm6b in early embryos. In a word, multiple superovulations alter histone modifications in early embryos.

BACKGROUND:Controlled ovarian stimulation is a common skill of assisted reproductive technologies (ARTs). In the clinic, some females would undergo more than one controlled ovarian stimulation cycle. However, few studies have focused on the influence of multi-superovulation on oocytes and offspring. RESULTS:Here, we found that multi-superovulation disrupted the transcriptome of oocytes and that the differentially expressed genes (DEGs) were associated mainly with metabolism and fertilization. The disruption of mRNA degradation via poly (A) size and metabolism might be a reason for the reduced oocyte maturation rate induced by repeated superovulation. Multi-superovulation results in hypo-genomic methylation in oocytes. However, there was an increase in the methylation level of CGIs. The DMRs are not randomly distributed in genome elements. Genes with differentially methylated regions (DMRs) in promoters are enriched in metabolic pathways. With increasing of superovulation cycles, the glucose and insulin tolerance of offspring is also disturbed. CONCLUSIONS:These results suggest that multi-superovulation has adverse effects on oocyte quality and offspring health.

Lactate is present at a high level in the microenvironment of mammalian preimplantation embryos and . However, its role in preimplantation development is unclear. Here, we report that lactate is highly enriched in the nuclei of early embryos when major zygotic genome activation (ZGA) occurs in humans and mice. The inhibition of its production and uptake results in developmental arrest at the 2-cell stage, major ZGA failure, and loss of lactate-derived H3K18lac, which could be rescued by the addition of Lac-CoA and recapitulated by overexpression of H3K18R mutation. By profiling the landscape of H3K18lac during mouse preimplantation development, we show that H3K18lac is enriched on the promoter regions of most major ZGA genes and correlates with their expressions. In humans, H3K18lac is also enriched in ZGA markers and temporally concomitant with their expressions. Taken together, we profile the landscapes of H3K18lac in mouse and human preimplantation embryos, and demonstrate the important role for H3K18lac in major ZGA, showing that a conserved metabolic mechanism underlies preimplantation development of mammalian embryos.

Assisted reproductive technologies are known to alter the developmental environment of gametes and early embryos during the most dynamic period of establishing the epigenome. This may result in the introduction of errors during active DNA methylation reprogramming. Controlled ovarian hyperstimulation, or superovulation, is a ubiquitously used intervention which has been demonstrated to alter the methylation of certain imprinted genes. The objective of this study was to investigate whether ovarian hyperstimulation results in genome-wide DNA methylation changes in mouse early embryos. Ovarian hyperstimulation was induced by treating mice with either low doses (5 IU) or high doses (10 IU) of PMSG and hCG. Natural mating (NM) control mice received no treatment. Zygotes and 8-cell embryos were collected from each group and DNA methylomes were generated by whole-genome bisulfite sequencing. In the NM group, mean CpG methylation levels slightly decreased from zygote to 8-cell stage, whereas a large decrease in mean CpG methylation level was observed in both superovulated groups. A separate analysis of the mean CpG methylation levels within each developmental stage confirmed that significant genome-wide erasure of CpG methylation from the zygote to 8-cell stage only occurred in the superovulation groups. Our results suggest that superovulation alters the genome-wide DNA methylation erasure process in mouse early pre-implantation embryos. It is not clear whether these changes are transient or persistent. Further studies are ongoing to investigate the impact of ovarian hyperstimulation on DNA methylation re-establishment in later stages of embryo development.