Evolutionary success in mammals depends on coordinated neurological and reproductive functions, likely mediated by shared genes and molecular pathways between the brain and germ cells. However, direct evidence 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 identify a set of conserved genes shared between neurological and reproductive systems, implying a potential genetic network underlying evolutionary conservation of molecular functions across these systems.

Epigenetic mechanisms profoundly regulate gene expression, developmental trajectories, and phenotypic variation, extending biological influence beyond DNA sequence alone. A growing body of evidence suggests that environmental exposures, including pollutants, drugs, stress, and diet, can induce germline and early embryonic epimutations that alter developmental programs with lasting consequences for neurodevelopmental and cognitive outcomes. However, the fields most relevant to these processes have largely developed independently. These include germline epigenetics, early embryonic patterning, neurodevelopment and cognitive regulation, and intergenerational or transgenerational inheritance. Each field has its own conceptual frameworks and mechanistic models. This fragmentation obscures the biological reality that these systems are tightly interconnected: environmentally induced epigenetic perturbations in gametes can reshape the epigenetic landscape of the early embryo, influence lineage allocation during gastrulation, and ultimately modify the molecular architecture of the developing central nervous system. A systems-biology perspective capable of linking germline epimutations and early embryonic epigenetic instability to later neurodevelopmental and cognitive phenotypes and their potential inheritance is therefore required. This review synthesizes current evidence across these traditionally isolated domains and proposes a coherent mechanistic framework linking germ cell epimutations and early embryonic epigenetic instability to the emergence of neurodevelopmental and cognitive phenotypes. By bridging these conceptual gaps, we aim to establish a cohesive foundation for understanding how early epigenetic disruptions generate long-lasting and in some cases heritable effects on brain development and cognitive function.

The recent rise in global food insecurity has renewed scientific interest in understanding the long-term health consequences of early-life nutritional deprivation. This study critically evaluates the experimental designs and methodological approaches of key publications examining the epigenetic and phenotypic effects of the Dutch and Chinese famines. Specifically, these studies were assessed for sample size, control group selection, relevance of tissue sampling, timing of famine exposure, and the quality of statistical reporting. Research on both famines has centered on prenatal exposure and subsequent health outcomes, providing important insights into how in utero nutritional deprivation may lead to long-lasting epigenetic modifications. These changes have been linked to elevated risks for metabolic, cardiovascular, and neuropsychiatric disorders. Despite these contributions, many studies exhibited notable limitations, including small sample sizes, questionable accuracy in reporting health outcomes, and issues with the selection of control groups. Such methodological shortcomings may have led to the misinterpretation of some findings. Ongoing and recent famines in regions such as Sudan, Somalia, and Gaza—driven by conflict and environmental disasters, including droughts and floods—represent some of the most pressing humanitarian crises of our time. Lessons from studies of the 20th-century Dutch and Chinese famines can inform the design of future research on the biological and intergenerational consequences of famine and trauma. Improved study designs will enhance the ability to generate reliable evidence and guide global health strategies for populations at risk of transgenerational effects from nutritional deprivation.

Epigenetic reprogramming during gametogenesis and early embryogenesis involves genome-wide erasure and re-establishment of DNA methylation, ensuring developmental plasticity. However, specific genomic regions, such as imprinted loci and transposable elements, can escape this reprogramming, allowing environmentally induced epigenetic marks to persist and potentially transmit across generations. This review summarizes current advances in intergenerational and transgenerational epigenetic inheritance in mammals, with emphasis on nutritional influences. Seminal studies from the Dutch famine cohorts and the viable yellow agouti (Avy) mouse model established that maternal diet could remodel the epigenome and induce long-term phenotypic effects. Extending these findings to livestock, our recent studies demonstrate that both maternal and paternal methionine supplementation alter DNA methylation and influence growth and fertility traits in sheep. Notably, some epigenetic marks and phenotypic effects persist through the F4 generation, providing rare evidence of stable transgenerational epigenetic inheritance in a mammalian livestock model. These findings highlight nutrition as a powerful modulator of the germline and embryonic epigenome. Understanding how dietary factors shape heritable epigenetic variation offers new opportunities to improve animal health, fertility, and productivity, while providing fundamental insights into the mechanisms and evolutionary relevance of epigenetic inheritance in mammals.

Abstract Neural tube closure relies on tightly coordinated morphogenetic programs integrating convergent extension, apical constriction of the neuroepithelium, and precise cell-cell interactions across germ layers. Disruption of these processes results in myelomeningocele, a severe complex congenital defect with lifelong multisystem consequences whose genetic and epigenetic determinants remain poorly defined. Using a sheep population with naturally occurring myelomeningocele, we quantified substantial heritability (0.42–0.68) and generated the first integrated multi-omics, multi-tissue atlas of this condition in any mammalian species. Genetic, transcriptomic, and whole-genome DNA methylation profiling across ectoderm- and mesoderm-derived tissues revealed shared and lineage-specific perturbations converging on cell-adhesion, cytoskeletal, migratory, inflammatory, and folate-responsive pathways. Chromosome 24 emerged as a multi-omics hotspot enriched for differentially expressed genes, differentially methylated regions, and candidate regulatory loci of GWAS signals, overlapping with human neurological and embryonic development trajectories. Cross-tissue network analyses highlighted coordinated disruption of neurulation-critical gene modules, establishing sheep as a robust translational model for mechanistic dissection of neural tube defect biology.

Sexual selection theory provides a unifying framework for understanding how cognitive traits, reproductive strategies, and molecular mechanisms coevolve to maximize fitness. Across species, evidence shows that cognitive performance predicts reproductive success through both male-male competition and female mate choice, with individuals displaying superior problem-solving, social dominance, or courtship skills achieving greater mating success. Sexual selection also favors alternative strategies, including sexual mimicry and socially induced sex change, which allow individuals to navigate competitive mating environments and optimize reproductive output. At a mechanistic level, the close evolutionary coupling of cognition and reproduction is reflected in shared cellular, molecular, and genetic architectures between the brain and reproductive tissues, particularly sperm. Conserved genes and pathways involved in signaling, metabolism, epigenetic regulation, and stress responses link neural function to gamete performance across species. We hypothesized that natural selection on cognitive or reproductive traits drives coordinated evolution of shared genetic and molecular pathways between the brain and reproductive tissues, resulting in conserved mechanisms that support both behavioral and fertilization success. To test this hypothesis, we investigated the extent to which the brain and reproductive tissues share conserved genes and molecular pathways, and whether these shared mechanisms provide evidence for coordinated evolution between cognitive and reproductive functions.

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.

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 (EV) released by cells contain mRNAs, microRNAs, long noncoding RNAs, lipids, and proteins, playing crucial roles in cell-cell communication. Although full-length mRNA transcripts have been documented in EV secreted by cancer cells, there are no reports on full transcripts secreted by embryos. Our study aimed to identify EV mRNAs in the culture medium of bovine embryos and investigate their roles in embryo-maternal communication. Following the isolation of EV 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 1 gene was downregulated (false discovery rate <0.01) following DPPA3 transfection, many with known functions in pregnancy recognition. Proteomic analysis revealed 28 differentially expressed proteins, with 17 upregulated and 11 downregulated. Two proteins, ISG15 and MX1, overlapped with the differentially expressed mRNAs. To mimic the natural transfer of EV from embryos to endometrial cells, we performed coculture with d-8 blastocysts or supplemented the cells with embryo-conditioned culture medium. DPPA3 presence was detected in endometrial cells exposed to embryo-conditioned medium after just 30 min. Overall, our study highlights the significant role of EV in cell-cell communication through mRNA signaling from the embryo to the mother.

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.

Understanding differences in chromatin state and changes in gene regulatory landscape of placode (Pc) and dermal condensate are crucial for decoding hair follicle (HF) morphogenesis programs. To identify cell-type-specific chromatin accessibility patterns in the developing HF, we integrated chromatin accessibility and transcriptome profiles at single-cell resolution during the murine HF induction stage. We applied unbiased analyses to identify seven major HF cell types and reclustered dermal (Der) and epithelium (Epi) subtypes to trace their cell fate specification. Our analysis showed that gene regulation in Der and Epi lineages is largely determined by cis-regulatory elements that direct gene expression in response to specific developmental cues. The chromatin accessibility of Twist2, Enpp2, Dkk1, and Sox2 varied from fibroblasts (Fb) to pre-DC lineage, while that of Edar, Lhx2, and Wnt10b varied from Epi to Pc lineage. Cell-type-specific enrichment of transcription factor binding motifs implicated Twist2 and Nfatc4 as key regulators in Fb to pre-DC fate specification, and Fos, Bach1, and Klf1 in Epi to Pc niche fate specification. Additionally, alignment of cell-type-specific peaks to super-enhancer databases identified key regulatory elements in both lineages. We identified and validated the critical cis-regulatory elements in pre-DC and Pc fate specifications through embryonic dorsal skin culture in vitro, suggesting that these elements may regulate critical genes essential for HF induction. Overall, our results provide a foundation for a comprehensive analysis of gene regulatory programs that initiate HF development, offering insights into the molecular mechanism of HF morphogenesis and clinical treatments of alopecia by skin grafts.

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.

Internal and external factors can change an individual's phenotype. A significant external threat to humans and livestock is environmental heat load, a combination of high ambient temperatures and humidity. A heat stress response occurs when an endothermal animal is exposed to a heat load that challenges its’ thermoregulation capacity. With the ongoing climate change trends, the incidence of chronically elevated temperatures causing heat stress is expected to rise, posing an even greater risk to the health and survival of all species. Heat stress is generally related to adverse effects on food intake, health, and performance in mammal livestock species and humans. Evidence from epidemiological and experimental studies of humans and livestock demonstrated that exposing pregnant females to heat stress affects the phenotype of the newborn in various ways. For instance, in utero heat stress is related to lower body weight at birth and changes in metabolic and immune functions in the newborn. In cows, the effects of heat stress on the performance of the offspring last for three or four generations, suggesting intergenerational effects. The molecular mechanism orchestrating these effects of heat stress may be epigenetic regulation, as various epigenetic mechanisms control genome reprogramming. Epigenetic modifications are attached to DNA and histone proteins and can influence how specific genes are expressed, resulting in phenotypic changes. Epigenetic modifications can be triggered in response to environmental heat stress without altering the DNA sequence. Heat stress insults during critical periods of organ development (i.e., fetal exposure) can trigger epigenetic modifications that impact health and productivity across generations. Thus, epigenetic changes caused by extreme temperatures can be passed down to the offspring if the mother is exposed to the insult during pregnancy. Understanding the phenotypic and molecular consequences of maternal heat stress, including the carry-over lingering effects on the resulting progeny, is necessary to develop effective mitigation strategies and gain translational knowledge about the fundamental processes leading to intergenerational and transgenerational inheritance. This review examines the phenotypic and molecular evidence of how maternal exposure to extreme heat can affect future generations in several species, including humans, swine, sheep, goats, and cattle. The current knowledge of the molecular mechanisms involved in intergenerational and transgenerational epigenetic inheritance will also be presented and discussed.

Abstract The publication of the first complete, haploid telomere-to-telomere (T2T) human genome revealed new insights into the structure and function of the heretofore “invisible” parts of the genome including centromeres, tandem repeat arrays, and segmental duplications. Refinement of T2T processes now enables comparative analyses of complete genomes across entire clades to gain a broader understanding of the evolution of chromosome structure and function. The human T2T project involved a unique ad hoc effort involving many researchers and laboratories, serving as a model for collaborative open science. Subsequent generation and analysis of diploid, near T2T assemblies for multiple species represents a substantial increase in scale and would be daunting for any single laboratory. Efforts focused on the primate lineage continue to employ the successful open collaboration strategy and are revealing details of chromosomal evolution, species-specific gene content, and genomic adaptations, which may be general or lineage-specific features. The suborder Ruminantia has a rich history within the field of chromosome biology and includes a broad range of species at varying evolutionary distances with separation of tens of millions of years to subspecies that are still able to interbreed. We propose an open collaborative effort dubbed the “Ruminant T2T Consortium” (RT2T) to generate complete diploid assemblies for species in the Artiodactyla order, focusing on suborder Ruminantia. Here we present the initial near T2T assemblies of cattle, gaur, domestic goat, bighorn sheep, and domestic sheep, and describe the motivation, goals, and proposed comparative analyses to examine chromosomal evolution in the context of natural selection and domestication of species for use as livestock.

A common goal of the dairy industry is to shorten the calving interval to reap several benefits associated with improved fertility. Early pregnancy detection is crucial to shorten this interval, allowing for prompt reinsemination of cows that failed to conceive after the first service. Currently, the industry lacks a method to accurately predict pregnancy within the first 3 wk. The polypeptide cytokine IFN-tau (IFNT) is the primary signal for maternal recognition of pregnancy in ruminants. As IFNT is released from the early conceptus, it initiates a cascade of effects, including upregulation of IFN-stimulated genes (ISG). Expression of ISG can be detected in the peripheral blood. The present study aimed to characterize peripheral transcriptomic changes, including the ISG, as early as d 7 after embryo transfer. A total of 170 Holstein heifers received in vitro-produced embryos. Whole blood was collected from these heifers within 24 h of the embryo transfer (d 0), d 7, and d 14 after embryo transfer. The heifers were divided into 2 groups, pregnant and nonpregnant, based on pregnancy diagnosis on d 28 via ultrasound. Total RNA was extracted from the peripheral blood of pregnant and nonpregnant heifers, pooled and sequenced. Expression analysis on d 7 heifers resulted in 13 significantly differentially expressed genes mostly related to innate immunity. Differential expression analysis comparing pregnant heifers on d 0 to the same heifers on d 14 showed 51 significantly differentially expressed genes. Eight genes were further quantified through reverse-transcription quantitative real-time PCR for biological validation. On d 7 after embryo transfer, mRNA transcriptions of EDN1 , CXCL3 , CCL4 , and IL1A were significantly upregulated in pregnant heifers (n = 14) compared with nonpregnant heifers (n = 14), with respective fold changes of 8.10, 18.12, 29.60, and 29.97. Although on d 14 after embryo transfer, mRNA transcriptions of ISG15 , MX2 , OASY1 , and IFI6 were significantly upregulated in the blood of pregnant heifers (n = 14) compared with the same heifers on d 0, with respective fold changes of 5.09, 2.59, 3.89, and 3.08. These findings demonstrate that several immunerelated genes and ISG are activated during the first 2 wk after embryo transfer, which may explain how the maternal immune system accommodates the allogenic conceptus. To further investigate the diagnostic potentials of these genes, future studies are warranted to analyze the specificity and sensitivity of these biomarkers to predict early pregnancy.

Telomere-to-telomere (T2T) assemblies reveal new insights into the structure and function of the previously ‘invisible’ parts of the genome and allow comparative analyses of complete genomes across entire clades. We present here an open collaborative effort, termed the ‘Ruminant T2T Consortium’ (RT2T), that aims to generate complete diploid assemblies for numerous species of the Artiodactyla suborder Ruminantia to examine chromosomal evolution in the context of natural selection and domestication of species used as livestock. Here we describe an open collaborative effort termed the ‘Ruminant T2T Consortium’. It aims to generate complete diploid assemblies for many species of ruminants to examine chromosomal evolution in the context of natural selection and domestication.

While transgenerational epigenetic inheritance has been extensively documented in plants, nematodes, and fruit flies, its existence in mammals remains controversial. Several factors have contributed to this debate, including the lack of a clear distinction between intergenerational and transgenerational epigenetic inheritance (TEI), the inconsistency of some studies, the potential confounding effects of in-utero vs. epigenetic factors, and, most importantly, the biological challenge of epigenetic reprogramming. Two waves of epigenetic reprogramming occur: in the primordial germ cells and the developing embryo after fertilization, characterized by global erasure of DNA methylation and remodelling of histone modifications. Consequently, TEI can only occur if specific genetic regions evade this reprogramming and persist through embryonic development. These challenges have revived the long-standing debate about the possibility of inheriting acquired traits, which has been strongly contested since the Lamarckian and Darwinian eras. As a result, coupled with the absence of universally accepted criteria for transgenerational epigenetic studies, a vast body of literature has emerged claiming evidence of TEI. Therefore, the goal of this study is to advocate for establishing fundamental criteria that must be met for a study to qualify as evidence of TEI. We identified five criteria based on the consensus of studies that critically evaluated TEI. To assess whether published original research papers adhere to these criteria, we examined 80 studies that either claimed or were cited as supporting TEI. The findings of this analysis underscore the widespread confusion in this field and highlight the urgent need for a unified scientific consensus on TEI requirements.

Environmental effects on gene expression and offspring development can be mediated by epigenetic modifications. It is well established that maternal diet influences DNA methylation patterns and phenotypes in the offspring; however, the epigenetic effects of paternal diet on developing offspring warrants further investigation. Here, we examined how a prepubertal methionine-enriched paternal diet affected sperm DNA methylation and its subsequent effects on embryo gene expression. Three treatment and three control rams were bred to seven ewes, and blastocysts were flushed for RNA extraction. Semen was collected from all rams and submitted for reduced representation bisulfite sequencing analysis. In total, 166 differentially methylated cytosines were identified in the sperm from treatment versus control rams. Nine genes were found to be differentially expressed in embryos produced from treatment versus control rams, and seven differentially methylated cytosines in the sperm were found to be highly correlated with gene expression in the embryos. Our results demonstrate that sperm methylation differences induced by diet may influence fetal programming.