Mitochondrial remodeling during the peri-implantation stage is the hallmark event essential for normal embryogenesis. Among the changes, enhanced oxidative phosphorylation is critical for supporting high energy demands of postimplantation embryos, but increases mitochondrial oxidative stress, which in turn threatens mitochondrial DNA (mtDNA) stability. However, how mitochondria protect their own histone-lacking mtDNA, during this stage remains unclear. Concurrently, the mitochondrial genome gain DNA methylation by this stage. Its spatiotemporal coincidence with enhanced mitochondrial stress led us to ask if mtDNA methylation has a role in maintaining mitochondrial genome stability. Herein, we report that mitochondrial genome undergoes de novo mtDNA methylation that can protect mtDNA against enhanced oxidative damage during the peri-implantation window. Mitochondrial genome gains extensive mtDNA methylation during transition from blastocysts to postimplantation embryos, thus establishing relatively hypermethylated mtDNA from hypomethylated state in blastocysts. Mechanistic study revealed that DNA methyltransferase 3A (DNMT3A) and DNMT3B enter mitochondria during this process and bind to mtDNA, via their unique mitochondrial targeting sequences. Importantly, loss- and gain-of-function analyses indicated that DNMT3A and DNMT3B are responsible for catalyzing de novo mtDNA methylation, in a synergistic manner. Finally, we proved, in vivo and in vitro, that increased mtDNA methylation functions to protect mitochondrial genome against mtDNA damage induced by increased mitochondrial oxidative stress. Together, we reveal mtDNA methylation dynamics and its underlying mechanism during the critical developmental window. We also provide the functional link between mitochondrial epigenetic remodeling and metabolic changes, which reveals a role for nuclear-mitochondrial crosstalk in establishing mitoepigenetics and maintaining mitochondrial homeostasis.

Significance: The fertility of female mammals depends upon the well-orchestrated progression of oocyte meiosis, the female germ cell division that is essential for haploid oocyte formation. In preovulatory follicles, how LH induces a decrease in CNP and its encoding mRNA Nppc , a prerequisite for oocyte meiotic resumption, remains an outstanding question to understand the mechanism that controls oocyte meiosis. The present study shows that TTP, an mRNA-destabilizing protein, is one of the regulatory components responsible for the LH-induced rapid decrease in Nppc mRNA and oocyte meiotic resumption. This finding not only highlights the importance of posttranscriptional regulation in the follicle periphery to fine-tune oocyte meiosis but also provides an insight into CNP-dependent cGMP homeostasis in other physiological systems.

In vitro embryo production depends on high-quality oocytes. Compared with in vivo matured oocytes, in vitro oocytes undergo precocious meiotic resumption, thus compromising oocyte quality. C-type natriuretic peptide (CNP) is a follicular factor maintaining meiotic arrest. Thus, CNP-pretreatment has been widely used to improve the in vitro maturation (IVM) of oocytes in many species. However, the efficacy of this strategy has remained unsatisfactory in porcine oocytes. Here, by determining the functional concentration and dynamics of CNP in inhibiting spontaneous meiotic resumption, we improved the current IVM system of porcine oocytes. Our results indicate that although the beneficial effect of the CNP pre-IVM strategy is common among species, the detailed method may be largely divergent among them and needs to be redesigned specifically for each one. Focusing on the overlooked role of cumulus cells surrounding the oocytes, we also explore the mechanisms relevant to their beneficial effect. In addition to oocytes per se, the enhanced anti-apoptotic and anti-oxidative gene expression in cumulus cells may contribute considerably to improved oocyte quality. These findings not only emphasize the importance of screening the technical parameters of the CNP pre-IVM strategy for specific species, but also highlight the critical supporting role of cumulus cells in this promising strategy.

Ovulation and granulosa cell luteinization are induced by ovulatory signals, including luteinizing hormone (LH) and human chorionic gonadotropin (hCG). Histone modifications enable rapid, signal-responsive transcriptional reprogramming. However, the effects of LH/hCG-induced histone modification changes on the mural granulosa cells (MGCs) function remain to be fully elucidated. By mining public datasets we integrated transcriptomic and histone-modification profiles of MGCs across the ovulatory interval and tracked LH/hCG-driven gene expression at three time points (0, 4, and 12 h after-hCG). During oocyte maturation, the 4 h LH-surge constitutes a critical window for meiotic resumption, during which many genes display rapid transcriptional changes followed by a return to baseline levels. Early-response genes are enriched for cell locomotion, inflammatory responses, the activation of signaling pathways, and histone modifications. Furthermore, LH/hCG-induced transcriptome remodeling is highly correlated with dynamic gains or losses of H3K4me3 and H3K27ac. Notably, we discovered for the first time that H3K27ac marks super-enhancers (SEs) that regulate LH/hCG-induced transcriptional activation in MGCs. Finally, through complementary in vitro and in vivo pharmacological inhibition, we demonstrate that LH/hCG governs oocyte maturation and ovulation by reshaping the MGC transcriptome via H3K4me3- and H3K27ac-dependent chromatin remodeling. In summary, our study advances the understanding of how gonadotropins regulate MGC function and oocyte maturation through histone-modification-mediated transcriptional control.