1. Introduction
The process of oocyte maturation is a unique form of cell division that sets it apart from mitosis. It represents the reactivation and culmination of the initial meiotic division [
1]. Oocytes remain in a state of arrest at the germinal vesicle (GV) stage for many years. Upon stimulation by a surge of luteinizing hormone (LH), there is an occurrence of germinal vesicle breakdown (GVBD) with a gradual compaction of chromosomes [
2]. Once chromosomal condensation is complete, oocytes progress into metaphase I (MI), where chromosomes align along the metaphase plate, and spindle microtubules attach to kinetochores, laying the foundation for the subsequent segregation of chromosomes during anaphase I [
3]. Following the expulsion of the first polar body, oocytes reach a state of arrest at metaphase II (MII) until fertilization occurs. Any aberrations in this intricate process may result in meiotic arrest and, consequently, fertilization failure. Considering the absence of transcriptional activity during oocyte maturation, the significance of post-translational modifications (PTMs) becomes evident in facilitating the successful completion of oocyte maturation [
4]. Thus, PTMs play critical roles during oocyte maturation.
Following fertilization, the zygote initiates the expression of genes specific to embryonic development, and this expression pattern undergoes dynamic changes throughout the preimplantation development phase. Maternal mRNAs and proteins that originate during oogenesis exert control over virtually all aspects of the initial embryonic development, even though the transcription of the zygotic genome remains quiescent [
5]. These proteins are primarily activated at the moment of fertilization and during the maternal-to-zygotic transition. Zygotic genome activation is predominantly observed at the 2-cell stage in mice, while in other species, it occurs at the 4-cell and 8-cell stages [
6]. Because the zygotic genome remains dormant during the early stages of embryogenesis, the significance of post-translational modifications (PTMs) becomes particularly evident. Recent research has demonstrated that PTMs play a crucial role in eliminating maternal mRNAs and degrading proteins [
7]. Deviations in PTMs during both oocyte maturation and the early stages of embryo development may lead to complications such as implantation failure and fetal abnormalities [
8,
9].
Numerous post-translational modifications are involved in the processes of oocyte growth and maturation, encompassing acetylation, methylation, phosphorylation, ubiquitination, and SUMOylation of a variety of proteins. These modifications play roles of varying significance in the regulation of chromatin structure and gene expression [
10,
11,
12]. Recent investigations have unveiled a novel function of lactate in orchestrating the shift from inflammatory to reparative macrophages and instigating the expression of homeostatic genes through histone lysine lactylation, thereby upholding immune equilibrium [
13,
14]. While previous studies have revealed the role of Tfap2a in regulating mouse oocyte maturation and its effects on histone acetylation and lactylation levels [
15], the specific functions and dynamic changes of lactylation during oocyte maturation and early embryo development processes remain to be explored in depth. Currently, there is a lack of systematic studies investigating the roles of histone lactylation in regulating these critical biological processes. Moreover, Yang et al. demonstrated the presence of histone lactylation marks in mouse oocytes and preimplantation embryos, as well as the potential impact of hypoxic conditions on lactylation levels and embryo development [
16]; a comprehensive understanding of the regulatory roles of lactylation during these critical developmental stages is still lacking.
Therefore, in this study, we first examined the expression and distribution of lactylated proteins in various mouse tissues to confirm that lactylation is a prevalent post-translational modification. We then focused on investigating the expression patterns and dynamic changes in histone lactylation during the maturation of mouse oocytes and the early embryo development of using immunofluorescence staining with pan-lactylation and site-specific antibodies. Additionally, we supplemented exogenous lactate to oocytes and embryos to explore the consequences of modulating histone lactylation levels on oocyte maturation and embryo development processes. Furthermore, we analyzed the impact of enhanced lactylation on the transcriptome of MII oocytes and 2-cell embryos.
3. Discussion
Lactylation is a novel post-translational modification occurring intracellularly that can influence protein structure, function, and interactions [
18]. Lactylated proteins play important roles in many physiological and pathological processes, including energy metabolism, cell cycle, gene expression, stress response, neurodegenerative diseases, and cancer [
18]. In this study, we examined lactylation levels in different mouse tissues, and specifically investigated whether histone lactylation occurs and its dynamic changes during mouse oocyte maturation and early embryo development. First of all, our study revealed that lactylated proteins are widely present across different mouse tissues with tissue- and cell-specific distribution patterns, indicating lactylation as a common post-translational modification involved in diverse physiological processes. Then, we found that in the ovary, lactylated proteins were mainly localized in oocytes, stromal and granulosa cells, with more localization in the oocyte cytoplasm, which may be associated with oocyte growth and maturation. In uterine tissues, lactylated proteins were predominantly localized in the serosa, myometrium, and endometrium, with greater localization in the endometrium, which may relate to endometrial maintenance and blood supply during pregnancy. In testicular tissues, lactylated proteins were primarily detected in spermatogenic, spermatogonial, and testicular interstitial cells, with enriched localization in interstitial cells, which may correlate with their functions such as androgen production and regulation of spermatogenesis. During spermatogenesis, lactate is mainly produced by Sertoli cells through LDH-catalyzed glycolysis and secreted into seminiferous tubules to provide energy for spermatogenic cells [
19]. The activity of LDH and lactate levels in Sertoli cells vary with the stages of the spermatogenic cycle. Decreased lactate levels in Sertoli cells can affect energy metabolism, morphology, quality, and fertilization capacity of spermatogenic cells [
20]. Lactylated proteins can regulate intracellular metabolic activities and thus influence energy production and utilization.
Consistent with previous studies showing pan histone lactylation and H3K23la but not H3K18la detected from condensed chromosomes when oocytes reach MII [
16], our results confirmed that histone lactylation levels were highest at the GV stage, sharply decreased at GVBD, and rebounded slightly at MII during mouse oocyte maturation, indicating dynamic changes throughout this process. This pattern is analogous to the dynamics of acetylation, methylation, phosphorylation, and other modifications that regulate DNA binding ability, chromatin remodeling, transcription factor access, and chromosome condensation during meiotic division of oocytes [
4,
21,
22]. Moreover, our examination of specific lysine residues revealed H3K9la, H3K14la, H4K8la, and H4K12la fluorescence was strongest at GV but diminished at GVBD and MII, while H4K5la peaked at GVBD, suggesting lactylation at different sites may confer distinct functions during maturation. Analogous to our findings on lactylation dynamics, studies report changes in histone acetylation patterns like H3K18 acetylation increasing from GV to GVBD, peaking at GVBD, deacetylating at MI, and reacetylating at MII in buffalo oocytes [
23]. Similarly, methylation patterns like H3K9me2 (a heterochromatin marker) tend to decrease in aged mouse GV oocytes compared to young ones [
24], which could impact chromatin states and transcription. These findings highlight the intricate interplay of histone modifications like lactylation, acetylation, and methylation in orchestrating the epigenetic landscape and transcriptional programming governing oocyte meiotic maturation, with lactylation potentially complementing other modifications in this intricate regulatory system.
Lactate is an end product of glycolysis that is reused for gluconeogenesis in the liver. Recent studies have revealed a novel function of lactate in being utilized for histone lysine lactylation, which promotes the transition of macrophages from inflammatory to reparative phenotypes and activates homeostatic gene expression to maintain immune homeostasis [
13,
14]. The discovery of lactoyl-CoA (lactyl-CoA) in mammalian cells has indicated that lactoyl-CoA generated from glucose metabolism may constitute a potential biochemical link between lactate and histone lactylation in vivo. Our study also confirmed that total fluorescence intensities of GV and MII oocytes increased with escalating concentrations of exogenous sodium lactate, indicating sodium lactate enhances histone lactylation levels in oocytes. In GV oocytes, no significant differences in lactylation were observed at the H3K9la, H3K14la, H3K56la, and H4K5la sites between 0 and 10 mM sodium lactate, suggesting these sites are either insensitive to exogenous lactate or the effects are counterbalanced by other factors. In contrast, significant differences were detected at the H3K18la, H4K8la, H4K12la, and H4K16la sites between 0 and 10 mM sodium lactate in GV oocytes. Unlike GV oocytes, only the H3K14la and H4K12la sites showed markedly increased lactylation under 10 mM versus 0 mM sodium lactate in MII oocytes, while the other six sites remained unchanged. H3K14 and H4K12 are considered as transcription activating epigenetic markers that can increase chromatin accessibility for the binding of transcription factors and recruitment of RNA polymerases [
25,
26]. Therefore, we postulate sodium lactate may activate beneficial genes for oocyte maturation and development by elevating lactylation levels specifically at the H3K14la and H4K12la sites. Moreover, our study found that although sodium lactate at different concentrations exerted no significant effects on GVBD in mouse oocytes, 10 mM sodium lactate substantially improved oocyte maturation rates. This indicates that enhancing histone lactylation by sodium lactate does not markedly regulate resumption of meiosis in GV oocytes. Lactate can act as both an energy substrate and signaling molecule; hence, the effects on oocyte maturation may also involve modulation of energy metabolism and signal transduction. Our transcriptomic data suggest that altering lactylation levels affects oxidative phosphorylation in mouse oocytes, which in turn impacts reactive oxygen species levels and oocyte quality. Collectively, these results demonstrate sodium lactate may regulate histone lactylation at specific sites in oocytes, thereby influencing chromatin states, gene expression, and oocyte maturation in a site-specific manner.
In early mouse embryos, lactate is an important metabolic intermediate. Studies found that from zygotes to the 2-cell stage, mouse embryos rely heavily on glycolysis-derived lactate as the energy source, and thus have high demands for lactate. At this stage, embryos exist in a reductive metabolic state where lactate helps maintain low pH and high reduction potential, which facilitates zygotic genome activation and erasure of epigenetic modifications. After the morula stage, glucose utilization increases in embryos as they begin employing the tricarboxylic acid cycle and oxidative phosphorylation for ATP production [
27]. Hence, lactate levels gradually decline while TCA cycle intermediates like α-ketoglutarate rise in embryos. Our data showed that 2-cell development rates were comparable across the five sodium lactate concentrations, indicating minimal impacts of sodium lactate at this stage. For 8-cell, morula, and blastocyst stages, development rates progressively increased from 0 to 10 mM sodium lactate and peaked at 10 mM, demonstrating appropriate sodium lactate levels can promote embryo cell division and differentiation to enhance developmental competence. This is likely attributed to elevated histone lactylation by sodium lactate, which in turn activates related signaling pathways and promotes gene expression and protein synthesis. In contrast, development rates declined from 10 to 30 mM sodium lactate, reaching the lowest level at 30 mM. This implies excessive sodium lactate inhibits embryo cell division and differentiation to impair developmental potential, either through drastic histone hyper-lactylation, which is detrimental for early embryos, or increased osmotic pressure, causing cell shrinkage.
Moreover, our research revealed that among the eight lactylation sites, H3K14la, H4K5la, and H4K12la fluorescence was detectable at all embryonic stages examined and displayed distinct changing patterns during development, suggesting differential regulatory roles of lactylation at these sites across stages. These changes may be closely related to the epigenetic reprogramming before and after zygotic genome activation, and they may participate in regulating the expression of the zygotic genome. Lactylation may alter the conformation and affinity of histone proteins, influencing the transcriptional activity of specific genes, thereby guiding the gene expression program during this critical developmental stage. Additionally, overall histone lactylation levels showed an increasing trend in 2-cell embryos, with escalating sodium lactate, especially with markedly enhanced signals at the H3K9la, H3K14la, H4K5la, H4K8la, H4K12la, and H4K16la sites. Transcriptomic data indicated that enhancing embryo lactylation altered glycolysis, which directly impacts endogenous lactate production and thus lactylation levels. As lactylation is an emerging epigenetic modification, it may play important roles in biological processes such as energy balance, oxidative stress response, and cell cycle regulation, which warrant further in-depth research. Collectively, these findings suggest sodium lactate may modulate histone lactylation at different sites to regulate the epigenetic state and expression of critical genes or transcription factors during early embryogenesis.
It is important to acknowledge some limitations of our study. First, while we observed dynamic changes in histone lactylation during oocyte maturation and embryo development, the specific mechanisms underlying the regulation of lactylation levels at different stages remain to be elucidated. Second, our study primarily focused on histone lactylation, but the potential crosstalk between lactylation and other epigenetic modifications, such as acetylation and methylation, warrants further investigation. The important message from our study is that histone lactylation plays important regulatory roles during oocyte maturation and early embryogenesis, and modulating lactylation levels can influence these processes. Our findings provide new insights into the epigenetic regulation of oocyte and embryo development, potentially leading to strategies for improving oocyte maturation rates and embryo quality. Future research should aim to elucidate the specific mechanisms by which lactylation regulates gene expression and cellular processes during oocyte maturation and embryogenesis.
4. Materials and Methods
4.1. Animals and Treatment
We obtained ICR mice (10 weeks old) for oocyte and embryo collection from SLAC Experimental Animal Co. Ltd. (Shanghai, China) and housed them in a specific pathogen-free facility. The mice were kept at a temperature of 22 ± 2 °C under a 12 h light/dark cycle and provided with unlimited access to food and water. A one-week adaptation period was allowed before commencing the experiments. All animal procedures strictly adhered to the guidelines of the Institutional Animal Care and Use Committee of Hainan University. Euthanasia of the mice was carried out after anesthesia, and various organs, including the heart, liver, spleen, lungs, kidneys, stomach, uterus, testes, muscles, ovaries, and intestine, were harvested for further analysis.
For in vivo collection of oocytes and embryos, the procedures were performed as previously described [
16]. Ovaries were removed from mice, and GV oocytes were isolated by gently puncturing small antral follicles (200–300 μm) under a stereomicroscope. Embryos were obtained by flushing the fallopian tubes and uterine horns with M2 medium. Embryos at the 2-cell, 4-cell, morula, and blastocyst stages were collected at 42–44, 51–53, 84–86, and 92–94 h post-hCG injection, respectively.
To investigate the influence of sodium lactate on enhancing histone lactylation during oocyte maturation, the base medium was Opti-MEM supplemented with 4 mg of BSA. Sodium lactate solution was added with various concentrations (0 mM, 5 mM, 10 mM, 20 mM). To ensure adequate sodium lactate uptake, oocytes were initially cultured with 3-isobutyl-1-methylxanthine (IBMX, phosphodiesterase inhibitor) and sodium lactate for 24 h, after which they were transferred to the same concentration medium without IBMX for continued culture. GVBD rates were assessed at 2, 4, and 6 h after releasing inhibition, and maturation rates were determined following 14 h of culture.
To investigate the influence of sodium lactate on early embryonic development, the basal culture medium used was KSOM-AA supplemented with 4 mg of BSA and devoid of sodium lactate. Sodium lactate solutions were added in accordance with the required concentrations (0 mM, 5 mM, 10 mM, 20 mM, 30 mM). To maintain consistent osmotic pressure in the KSOM-AA culture medium with varying sodium lactate concentrations and prevent osmotic changes resulting from sodium lactate content variations, NaCl was added based on the amount of sodium lactate introduced. This ensured that the KSOM-AA culture medium with different sodium lactate concentrations possessed identical osmotic pressure.
4.2. Immunofluorescent Staining
The heart, liver, spleen, lungs, kidneys, stomach, uterus, testes, muscles, ovaries, and intestine were fixed and preserved in paraffin after an overnight fixation process. Tissue sections were subjected to incubation with primary antibodies (Pan la, PTM-1401, PTM Bio) at 37 °C for 2 h. Following this, the sections underwent three PBS rinses and were subsequently incubated for 1 h at room temperature with Alexa-labeled secondary antibodies (Invitrogen, Life Technologies) at a 1:500 dilution at 37 °C for 2 h. Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole). Subsequently, the slides were visualized using a fluorescence microscope (Nikon Inc., Melville, NY, USA).
For oocytes and embryos, collected samples were fixed in 4% (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at room temperature for 20 min. Following fixation, samples underwent three washes with 1% BSA/PBS. Samples were permeabilized with 0.5% Triton X-100 and 1% BSA/PBS for 20 min, followed by three rinses with 1% BSA/PBS. The samples were blocked with 0.1% Triton X-100, 1% BSA/PBS for 30 min and incubated with primary antibodies overnight at 4 °C, including Pan la, H3K9la, H3K14la, H3K18la, H3K56la, H4K5la, H4K8la, H4K12la, H4K16la. Oocytes and embryos were incubated with secondary antibodies in the dark for 1 h and subsequently washed with 1% BSA/PBS for 5 min, repeating this process three times. Afterward, the samples were stained with DAPI for 10 min. Finally, the samples were mounted on slides, and immunofluorescence images were captured using a fluorescence microscope.
4.3. Western Blot
The heart, liver, spleen, lungs, kidneys, stomach, uterus, testes, muscles, ovaries, and intestine were collected and subjected to homogenization. Each well of a 12% SDS-PAGE gel received a loading of 30 micrograms of total protein for electrophoretic separation. Subsequently, the proteins were transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Following the blocking of non-specific binding sites, the membranes underwent overnight incubation at 4 °C with anti-Pan la (PTM Bio, 1:1000 dilution) and anti-ACTB antibody (Proteintech Group, 1:2000 dilution). The membranes were then incubated with HRP-conjugated secondary antibodies. Finally, protein bands were visualized using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) and quantified with Quantity One software (Version 4.62, Bio-Rad Laboratories, Hercules, CA, USA).
4.4. RNA-Sequencing and Bioinformatic Analysis
RNA sequencing was carried out by Geekgene Co., Ltd. (Beijing, China). Total RNA was extracted to construct cDNA libraries. Following a quality assessment, the libraries were combined based on their effective concentrations and the desired data output targets. Subsequently, sequencing was performed on the Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) were identified using the criteria of log2(fold change) > 1.2 and p < 0.05. To gain insights into the functional implications of these DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted. The raw sequencing data were deposited in the China National Center for Bioinformation under the accession code PRJCA021062.
4.5. Statistical Analysis
Unless specifically indicated, all data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS version 22 (IBM-SPSS Inc., Chicago, IL, USA). Variables were subjected to one-way analysis of variance (ANOVA), followed by post hoc LSD tests for group-wise comparisons. Statistical significance was considered as p < 0.05.
5. Conclusions
In this study, we have confirmed that lactylated proteins were widely detected in various mouse tissues, such as heart, liver, spleen, lung, kidney, stomach, intestine, muscle, uterus, ovary, and testis, indicating lactylation as a ubiquitous post-translational modification. During mouse oocyte maturation, histone lactylation levels significantly decreased. Supplementation of 10 mM sodium lactate in culture medium elevated both the oocyte maturation rate and histone Kla modification levels, with prominent changes at the H4K8la, H4K12la, and H4K16la sites in GV oocytes, and markedly increased Kla at the H3K14la and H4K12la sites in MII oocytes. It also affected the transcription of molecules involved in oxidative phosphorylation. Additionally, histone lactylation levels changed dynamically with embryo cell division and development during mouse early embryogenesis. Sodium lactate at 10 mM enhanced early embryo development as well as lactylation at the H3K9la, H3K14la, H4K5la, H4K8la, H4K12la, and H4K16la sites, and impacted the transcription of glycolytic molecules. This study helps reveal the roles of lactylation during oocyte maturation and embryo development, providing new insights into improving oocyte maturation rates and embryo quality.