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Abstract BackgroundMorphologic sex differences between males and females typically emerge after the primordial germ cell migration and gonad formation, although sex is determined at fertilization based on chromosome composition. A key debated sexual difference is the embryonic developmental rate, within vitroproduced male embryos often developing faster. However, the molecular mechanisms driving early embryonic sex differences remain unclear. ResultsTo investigate the transcriptional sex difference during early development,in vitroproduced bovine blastocysts were collected and sexed by PCR. A significant male-biased development was observed in expanded blastocysts. Ultra-low input RNA-seq analysis identified 837 DEGs, with 231 upregulated and 606 downregulated in males. Functional enrichment analysis revealed male-biased DEGs were associated with metabolic regulation, whereas female-biased DEGs were related to female gonad development, sex differentiation, inflammatory pathways, and TGF-beta signaling. Comparing X chromosome and autosome expression ratio, we found that female-biased DEGs contributed to the higher X-linked gene dosage, a phenomenon not observed in male embryos. Moreover, we identified the sex-biased transcription factors and RNA-bind proteins, including pluripotent factors such asSOX21andPRDM14, and splicing factorsFMR1andHNRNPH2. Additionally, we revealed 1,555 significantly sex-biased differential alternative splicing (AS), predominantly skipped exons, mapped to 906 genes, with 59 overlapping with DEGs enriched in metabolic and autophagy pathways. By incorporating novel isoforms from long reads sequencing, we identified 1,151 sex-biased differentially expressed isoforms (DEIs) associated with 1,017 genes. Functional analysis showed that female-biased DEIs were involved in the negative regulation of transcriptional activity, while male-biased DEIs were related to energy metabolism. Furthermore, we identified sex-biased differential exon usage inDENND1B, DIS3L2, DOCK11, IL1RAPL2,andZRSR2Y,indicating their sex-specific regulation in early embryo development. ConclusionThis study provided a comprehensive analysis of transcriptome differences between male and female bovine blastocysts, integrating sex-biased gene expression, alternative splicing, and isoform dynamics. Our findings indicate that enriched metabolism processes in male embryos may contribute to the faster developmental pace, providing insights into sex-specific regulatory mechanisms during early embryogenesis. Plain English summaryMale and female early embryos develop at different speeds, with male embryos often developing faster than female embryos. However, the reasons behind these early differences remain unclear. In this study, we examined gene activity in bovine embryos to uncover the biological factors regulating these early sex differences. We collected in vitro-produced bovine blastocysts, examined their sex, and confirmed that male embryos develop faster. By analyzing global gene activity, including alternative splicing, which allows one gene to code for multiple RNA isoforms and proteins, we found distinct gene expression profiles between male and female embryos. Male embryos showed higher activity in genes related to metabolism and cellular functions, while female embryos had increased activity in genes associated with female-specific gonad development and gene expression regulation. We also examined differences in how genes on the X chromosome were expressed. Female embryos had higher X-linked gene expression, which may contribute to sex-specific developmental regulation. Additionally, we identified sex-specific transcription factors and RNA-binding proteins that regulate early embryo development, some of which are known to control pluripotency and gene splicing. Overall, our study provides new insights into how gene activity shapes early sex differences, suggesting that enhanced metabolism in male embryos may be a key driver of their faster developmental rate. HighlightsMale embryos develop faster due to increased gene expression in metabolism pathwaysFemale embryos exhibit higher X-linked gene expression, suggesting X-dosage compensation plays a role in early developmentSex-biased alternative splicing events contribute to embryonic metabolism, autophagy, and transcriptional regulation in embryosSex-biased isoform diversity contributes to distinct developmental regulation in male and female embryosKey pluripotency factors (SOX21, PRDM14) and splicing regulators (FMR1, HNRNPH2) drive sex-specific gene expression 

Summary Pluripotency, the ability of cells to self-renew and differentiate into all the cell types in an animal’s body, is vital for mammalian early development. This study presented a comprehensive comparative transcriptomic analysis of embryonic stem cells across multiple mammalian species, defining their progression through expanded/extended, naïve, formative, and primed pluripotency states. Our findings revealed both conserved and species-specific mechanisms underlying pluripotency regulation. We also emphasized the limitations of existing state-specific markers and their limited cross-species applicability, while identifyingde novopluripotency markers that can inform future research. Despite variability in gene expression dynamics, gene co-expression networks showed remarkable conservation across species. Among pluripotency states, the primed state demonstrated the highest conservation, evidenced by shared markers, preserved gene networks, and stronger selective pressures acting on its genes. These findings provide critical insights into the evolution and regulation of pluripotency, laying a foundation for refining stem cell models to enhance their translational potential in regenerative medicine, agriculture, and conservation biology. 

Abstract Although multiple high-performing epigenetic aging clocks exist, few are based directly on gene expression. Such transcriptomic aging clocks allow us to extract age-associated genes directly. However, most existing transcriptomic clocks model a subset of genes and are limited in their ability to predict novel biomarkers. With the growing popularity of single-cell sequencing, there is a need for robust single-cell transcriptomic aging clocks. Moreover, clocks have yet to be applied to investigate the elusive phenomenon of sex differences in aging. We introduce TimeFlies, a pan-cell-type scRNA-seq aging clock for theDrosophila melanogasterhead. TimeFlies uses deep learning to classify the donor age of cells based on genome-wide gene expression profiles. Using explainability methods, we identified key marker genes contributing to the classification, with lncRNAs showing up as highly enriched among predicted biomarkers. The top biomarker gene across cell types is lncRNA:roX1, a regulator of X chromosome dosage compensation, a pathway previously identified as a top biomarker of aging in the mouse brain. We validated this finding experimentally, showing a decrease in survival probability in the absence of roX1in vivo. Furthermore, we trained sex-specific TimeFlies clocks and noted significant differences in model predictions and explanations between male and female clocks, suggesting that different pathways drive aging in males and females. Graphical Abstract 

ABSTRACT Telomeres, protective caps at the ends of linear chromosomes, are frequently found to shorten with age. Telomere length is commonly measured in wild populations to investigate age‐related changes in somatic integrity and is considered a hallmark of ageing. Despite interest, there is no clear picture regarding sex differences in telomere length or rate of attrition across species. Bats are of considerable interest in studies of ageing and telomeres, owing to their remarkable longevity and the absence of age‐associated telomere attrition observed in some species. Additionally, multiple bat species show evidence of sex differences in longevity. However, few studies of bat telomeres have included both sexes. We collected DNA from wild‐caught males and females of the highly polygynous greater spear‐nosed bat,Phyllostomus hastatus, in which mortality is strongly male‐biased, and measured relative telomere lengths. We found that, while telomeres were shorter in older bats, there was no evidence of shorter telomeres in males. In fact, males tended to have longer telomeres. This runs counter to our prediction of shorter telomeres in the shorter‐lived sex but is not completely unexpected in light of other observations, including that of shorter telomeres in longer lived species. 

ABSTRACT Males of polygynous mammals often do not live as long as females and, in some cases, exhibit evidence of earlier senescence. Patterns of DNA methylation (DNAm) have recently been used to predict chronological age in mammals. Whether DNAm also changes as a consequence of survival and senescence is largely untested in wild animals. In this study, we estimate mortality rates using recaptures of 2700 greater spear‐nosed bats,Phyllostomus hastatus, over 34 years and DNAm profiled for over 300 adult bats. In this species, one male typically controls mating access to a group of unrelated females. Bayesian analysis reveals that mortality risk in males is 1.8 times that of females, and comparison of age‐associated differences in DNAm indicates that DNAm changes 1.4 times faster in males than females. Therefore, even though the age of either sex is predicted by a common set of sites, the methylome of males is more dynamic than that of females. Sites associated with sex differences in the rate of DNAm change are sensitive to androgens and enriched on the X chromosome. Sites that exhibit hypermethylation are enriched in promoters of genes involved in the regulation of metabolic processes. Unexpectedly, subordinate males have higher mortality rates than reproductively dominant males and exhibit faster DNAm change than dominants at dozens of sites. Our results reveal that differences in mortality associated with sex and social status are reflected by changes in DNA methylation, providing novel insights into mechanisms of aging and mortality in this and likely other wild animal populations. 

Abstract The Midwest Aging Consortium (MAC) has emerged as a critical collaborative initiative aimed at advancing our understanding of aging and developing strategies to combat the rising prevalence of age-related diseases. Founded in 2019, MAC brings together researchers from various disciplines and institutions across the Midwestern United States to foster interdisciplinary geroscience research. This report summarizes the highlights of the Fourth Annual Symposium of MAC, which was held at Iowa State University in May 2023. The symposium featured presentations on a wide array of topics, including studies on slow-aging animals, cellular senescence and senotherapeutics, the role of the immune system in aging, metabolic changes in aging, neuronal health in aging, and biomarkers for measuring the aging process. Speakers shared findings from studies involving a variety of animals, ranging from commonly used species such as mice, rats, worms, yeast, and fruit flies, to less-common ones like naked mole-rats, painted turtles, and rotifers. MAC continues to emphasize the importance of supporting emerging researchers and fostering a collaborative environment, positioning itself as a leader in aging research. This symposium not only showcased the current state of aging biology research but also highlighted the consortium’s role in training the next generation of scientists dedicated to improving the healthspan and well-being of the aging population. 

Synopsis Context-dependent allocation of resources drives trade-offs among fitness-related traits and other phenotypes to which those traits are linked. In addition, the amount and type of acquired resources can also affect the phenotypes of other organisms through indirect genetic effects, as exemplified by the maternal provisioning of offspring. Despite a large literature on maternal effects, we lack a comprehensive understanding of the extent to which mothers might affect the phenotypes of their offspring, as well as the various mechanisms by which they do so, particularly with regard to many functional traits that are key determinants of survival and reproduction. Our goals in this paper are to review the various approaches to measuring and understanding maternal effects and to highlight some promising avenues for integration of maternal effects with some other key areas of evolutionary ecology. We focus especially on nutritional geometry; maternal age; and traits proximate to fitness such as whole-organism performance. Finally, we discuss the logistic and practical limits of quantifying these effects in many animal systems and emphasize the value of integrative approaches in understanding the mechanisms underlying maternal influence on offspring phenotypes. 

Abstract Comparative studies of aging are a promising approach to identifying general properties of and processes leading to aging. While to date, many comparative studies of aging in animals have focused on relatively narrow species groups, methodological innovations now allow for studies that include evolutionary distant species. However, comparative studies of aging across a wide range of species that have distinct life histories introduce additional challenges in experimental design. Here, we discuss these challenges, highlight the most pressing problems that need to be solved, and provide suggestions based on current approaches to successfully carry out comparative aging studies across the animal kingdom. 

Abstract Sex chromosomes play an outsized role in adaptation and speciation, and thus deserve particular attention in evolutionary genomics. In particular, fusions between sex chromosomes and autosomes can produce neo‐sex chromosomes, which offer important insights into the evolutionary dynamics of sex chromosomes. Here, we investigate the evolutionary origin of the previously reportedDanausneo‐sex chromosome within the tribe Danaini. We assembled and annotated genomes ofTirumala septentrionis(subtribe Danaina),Ideopsis similis(Amaurina),Idea leuconoe(Euploeina) andLycorea halia(Itunina) and identified their Z‐linked scaffolds. We found that theDanausneo‐sex chromosome resulting from the fusion between a Z chromosome and an autosome corresponding to theMelitaea cinxiachromosome (McChr) 21 arose in a common ancestor of Danaina, Amaurina and Euploina. We also identified two additional fusions as the W chromosome further fused with the synteny block McChr31 inI. similisand independent fusion occurred between ancestral Z chromosome and McChr12 inL. halia. We further tested a possible role of sexually antagonistic selection in sex chromosome turnover by analysing the genomic distribution of sex‐biased genes inI. leuconoeandL. halia. The autosomes corresponding to McChr21 and McChr31 involved in the fusions are significantly enriched in female‐ and male‐biased genes, respectively, which could have hypothetically facilitated fixation of the neo‐sex chromosomes. This suggests a role of sexual antagonism in sex chromosome turnover in Lepidoptera. The neo‐Z chromosomes of bothI. leuconoeandL. haliaappear fully compensated in somatic tissues, but the extent of dosage compensation for the ancestral Z varies across tissues and species. 

Sex-biased longevity is observed across a wide range of animal taxa, including bats, for reasons not well understood. Patterns of cytosine methylation vary predictably with age in many organisms, offering a valuable means to investigate differences in patterns of aging at the molecular level. We tested sex differences in cytosine methylation across 14 bat species and compared patterns of age-associated variation. Sex differences were overrepresented on the X chromosome, showing a strong pattern of female hypermethylation within promoter regions. Sex and age-associated differences in methylation were non-randomly distributed with respect to proximity to putative sex hormone receptor binding sites, with sites hypermethylated in males and females tending to be underrepresented near androgen and estrogen receptor binding sites, respectively. Across species, we observed the relative steepness of male versus female slopes of age-associated variation was associated with the strength of precopulatory sexual selection, with especially strong trends towards male-biased age-associated slopes in two harem-polygynous species that exhibit female-biased longevity. Our results offer insights into how patterns of methylation differ across sexes and ages, and raise intriguing questions for future research, such as whether sex differences in molecular aging reflect sex-biased longevity, for which records in bats are sparse.