Despite two extensive reprogramming events during early embryogenesis and gametogenesis, epigenetic information can be passed to the next generations, which constitutes the transgenerational epigenetic inheritance of phenotypes. Considering its utmost importance, there have been few studies focused on the transgenerational effects of dietary interventions, such as methionine supplementation, in livestock. Using whole-genome bisulfite sequencing, we implemented a single-base resolution differential methylation analysis for the F3 and F4 descendants of control vs. methionine-supplemented F0 twin-pair rams. Based on the results of our previous study on F0, F1, and F2 generations, we compared current results of 2981 and 1726 differentially methylated cytosines (DMCs), as well as 798 and 553 unique differentially methylated genes (DMGs), in F3 and F4, respectively. We identified 41 DMGs that exhibited transgenerational epigenetic inheritance (TEI-DMGs) across four generations and 11 TEI-DMGs across five generations. Finally, we estimated the effect size of F0 diet group on F3 and F4 growth and fertility-related phenotypes, providing evidence for transgenerational effects of diet group accompanying inherited differentially methylated genes. Here, for the first time using gene-level and phenotypic data, we demonstrate that a moderate dietary intervention can exert long-lasting transgenerational effects on offspring phenotypes extending beyond the F2 generation in sheep.

Seminal plasma (SP) primarily serves as the transport medium for sperm; however, protein and miRNA expression in SP have been found to be indicative of reproductive function and have been used to identify changes in spermatogenesis. It is also postulated that SP proteins and miRNAs play a role in direct communication with the female reproductive tract to alter uterine function and drive physiological changes that increase pregnancy success. Importantly, SP has been shown to be susceptible to paternal diet, indicating another method in which altered fetal programming may occur. Most work regarding the role of SP in pregnancy establishment as well as dietary contributions to SP composition has been conducted in mice and humans; however, the effects of diet on SP composition in livestock species warrants further investigation. Therefore, this study aims to investigate how paternal diet can alter the proteome and miRNA composition of SP from sheep to better understand their potential roles in male fertility and fetal programming. Here, we examined how a prepubertal methionine-enriched diet affected the miRNA and protein compositions of SP in Polypay rams. Comparative proteomics revealed that a total of 28 SP proteins were significantly increased, and 32 were decreased in abundance (P < 0.05, fold change > 1.5) in response to methionine supplementation. Additionally, 138 significant miRNAs were identified in the SP from treatment versus control rams, with 80 upregulated and 58 downregulated (P < 0.05, fold change > 2). Given that miRNAs are known regulators of mRNA expression, we performed a functional enrichment analysis of our differentially expressed miRNAs and found that 79 miRNAs target 37 proteins exhibiting differential abundance. Gene Ontology analysis revealed that targeted proteins were enriched in biological processes, including reproduction, fertilization, and embryo development. Overall, these results demonstrate that the SP composition of rams is susceptible to a methionine-enriched diet and may impact male fertility and offspring development.

Environmental factors can influence gene expression and developmental outcomes through epigenetic modifications. Although maternal diet influences offspring DNA methylation and phenotypes, its effects on the oocyte and the resulting embryonic epigenome remain poorly understood. Here, we investigated the effect of maternal methionine supplementation on DNA methylation patterns in oocytes and embryos in Polypay sheep. Whole-genome bisulfite sequencing (WGBS) was performed on oocytes collected from 16 twin ewe pairs (8 methionine-treated and 8 controls). These ewes were later bred to control rams, and embryos were flushed for WGBS as well. In oocytes, 2,056 differentially methylated cytosines (DMCs) were identified. Additionally, 17 mitochondrial DMCs were identified, with 12 hypermethylated and 5 hypomethylated. In embryos, 113 DMCs were identified. Mitochondrial DNA analysis revealed 22 hypermethylated DMCs. To assess the inheritance of methyl marks, we compared DMCs between oocytes and embryos. While no direct overlaps were found in nuclear DNA, 3 CpGs exhibited opposite methylation trends - hypomethylated in oocytes but hypermethylated in embryos. In contrast, 5 mitochondrial DMCs overlapped between oocytes and embryos. To functionally assess the role of differentially methylated genes, we performed siRNA-mediated knockdown of 2 embryo DMC-associated genes: SCRIB and CERS3. Knockdown of SCRIB led to a 16.4% average decrease in blastocyst formation rate (p = 0.001), while CERS3 knockdown resulted in a 9.5% decrease (p = 0.005). These results demonstrate that maternal methionine supplementation alters nuclear and mitochondrial DNA methylation in oocytes and embryos, and that affected genes may play critical roles in early embryonic development, contributing to fetal programming.

Extracellular vesicles (EVs) released by cells contain mRNAs, miRNAs, lncRNAs, lipids, and proteins, playing crucial roles in cell-cell communication. While full-length mRNA transcripts have been documented in EVs secreted by cancer cells, there are no reports on full transcripts secreted by embryos. Our study aimed to identify EV mRNAs in the culture media of bovine embryos and investigate their roles in embryo-maternal communication. Following the isolation of EVs from in vitro fertilization media samples and RNA sequencing, we identified a full mRNA transcript of DPPA3, known to play an essential role in embryo development. To examine the role of DPPA3 in embryo-maternal communication, an in vitro transcribed mRNA of DPPA3 was transfected into bovine endometrial epithelial cells. Transfected and control cells were subsequently analyzed with RNA sequencing and proteomics to assess the effects of DPPA3 on gene expression. A total of 24 genes were found to be upregulated, and one gene was downregulated (FDR <0.01) following DPPA3 transfection, many with known functions in pregnancy recognition. Proteomic analysis revealed 28 differentially expressed proteins, with 19 upregulated and 15 downregulated. Two proteins, ISG15 and MX1, overlapped with the differentially expressed mRNAs. To mimic the natural transfer of EVs from embryos to endometrial cells, we performed coculture with day-8 blastocysts or supplemented the cells with embryo-conditioned culture media. DPPA3 presence was detected in endometrial cells exposed to embryo-conditioned media after just 30 min. Overall, our study highlights the significant role of EVs in cell-cell communication through mRNA signaling from the embryo to the mother.

Evolutionary success in mammals requires coordinated regulation of cognitive functions and reproductive capacity. Such coordination must involve shared genes and molecular pathways between the brain and germ cells, yet direct evidence linking cognition to reproduction across species remains limited. Here, proteomic and transcriptomic analyses were performed experimentally in Ovis aries and Rattus norvegicus, while transcriptomic datasets from Mus musculus, Macaca mulatta, and Homo sapiens were analyzed in silico. We identified 8,464 protein-coding genes shared between the brain and sperm/testis and conserved across five species. In rats, 8,444 of these genes were also shared between the brain and the ovary. Functional annotation classified 3,890 genes as associated with both neurological and reproductive functions, and 1,752 as uncharacterized in these contexts, highlighting candidates for future studies on reproductive and neurological disorders. These findings reveal a deeply conserved genetic network linking neurological and reproductive systems, underscoring the evolutionary interplay that supports mammalian fitness.

Background/Objectives: Parental environmental factors can shape developmental outcomes through epigenetic mechanisms that regulate gene expression. While maternal dietary effects on offspring epigenetics have been well characterized, the impact of paternal diet on embryonic DNA methylation remains poorly understood. Here, we investigated the effect of paternal methionine supplementation on DNA methylation patterns in preimplantation embryos in Polypay sheep. Methods: Four yearling rams (two control and two methionine-supplemented) were bred to twelve ewes following estrus synchronization and superovulation. Embryos were collected after natural mating and analyzed using whole-genome bisulfite sequencing (WGBS). Results: A total of 842 differentially methylated cytosines (DMCs) were identified in embryos derived from methionine-supplemented sires compared to controls, with 835 hypermethylated and 7 hypomethylated. The majority of DMCs were located in intergenic regions, with minimal representation in exonic regions. To assess overlap between parental dietary effects, DMCs identified in this study were compared with those previously reported in embryos derived from methionine-supplemented dams. Eight hypermethylated DMCs were shared between the two datasets, while no hypomethylated DMCs overlapped. To evaluate the functional relevance of differentially methylated genes, we performed siRNA-mediated knockdown of SSU72, a gene associated with multiple DMCs. Knockdown of SSU72 resulted in an average 18% decrease in blastocyst formation rate (p < 0.001). Conclusions: These results demonstrate that paternal methionine supplementation alters embryonic DNA methylation patterns and that affected genes may play critical roles in early embryonic development, contributing to fetal programming.