Recent studies have demonstrated that mammalian sex determination is governed not only by trans-acting transcription factors but also by cis-acting chromatin modifications. This review summarizes recent advances in understanding how histone and DNA modifications contribute to sex determination, including the precise temporal regulation of the Y-linked testis-determining gene Sry. Key chromatin regulators-such as KDM3A, EHMT1/2, TET2, CBP/p300, KDM6B, CBX2, and CDYL-shape the chromatin landscape to promote male fate. We also highlight the emerging role of iron metabolism in modulating the activity of chromatin-modifying enzymes, thereby linking cellular metabolic state to epigenetic regulation. Together, these findings underscore the critical role of epigenetic fine-tuning in ensuring proper sex determination and may provide novel insights into the etiology of human disorders of sex development (DSDs).
Faithful chromosome segregation in oocytes relies on the precise coordination of core centromeric chromatin, dynamic kinetochore assembly, and spindle interactions. In meiosis I, sister kinetochores are mono-oriented toward the same spindle pole, allowing homologous chromosomes to bi-orient and segregate properly, whereas meiosis II exhibits conventional bi-orientation of sister chromatids. Stepwise assembly of kinetochore subcomplexes, Meikin-mediated mono-orientation, and protection of centromeric cohesion by Shugoshin ensure accurate segregation through meiotic divisions. These same structural and regulatory features, however, are also exploited by selfish centromeres to bias chromosome orientation on the oocyte spindle, leading to biased segregation to the egg and therefore to the next generation in violation of Mendel's Law of Segregation. We will review recent studies that have established a mechanistic framework for both accurate chromosome segregation and non-Mendelian segregation in oocytes, highlighting the central role of meiotic centromeres in female reproduction and evolutionary conflict.
Early mammalian development entails one of the most extensive and physiologically relevant episodes of epigenomic reprogramming. Following fertilization, two highly specialized gametic epigenomes are reset to establish a totipotent zygotic state and are subsequently reorganized to enable lineage specification and pluripotency. A central component of this transition is the dynamic regulation of histone modifications that mark transcriptionally active and inactive chromatin. Studies in mouse embryos have revealed a distinctive sequence of events in which noncanonical, oocyte- and embryo-specific chromatin states are transiently established and then progressively replaced by canonical, somatic-like patterns around zygotic genome activation. However, comparative analyses across mammals demonstrate that several of these features, including broad noncanonical histone domains and Polycomb-mediated imprinting, are not universally conserved and differ substantially in humans and other species. In this chapter, we summarize recent findings on the dynamics of active and inactive histone marks during early embryogenesis, with a focus on principles derived from the mouse model and their limitations. We discuss how histone modifications contribute to transcriptional competence, repression, and lineage priming, how these processes vary across species, and how they together constitute an epigenomic "rebooting" that balances the erasure of parental memory with the preservation of essential regulatory information.
Embryogenesis begins with fertilization, resulting in a totipotent zygote that undergoes a complex series of cellular and molecular events toward the development of a complete organism. During this period, critical events such as zygotic genome activation and cell fate specification occur, characterized by dynamic changes in gene expression programs. Regulation of gene expression is generally explained by chromatin-based mechanisms orchestrated in nuclei. To note, non-chromatin nuclear proteins, referred to as nucleoskeletal proteins, emerge as key players for shaping nuclear and chromatin structures. The nucleoskeleton consists of multiple proteins, and its dynamics are important for establishing cell-type-specific nuclear structure, genome organization, and gene expression patterns. Recent studies have highlighted that nucleoskeletal proteins, particularly lamins and nuclear actin, play diverse and critical roles during early embryogenesis. Furthermore, a growing body of evidence suggests that nuclear architecture itself can actively influence gene expression and embryonic development. In this review, we focus on the nuclear structure of early vertebrate embryos from the viewpoint of the nucleoskeleton. We summarize current findings regarding the dynamics and functions of nucleoskeletal proteins during embryogenesis and further discuss how these constitutional components can affect gene expression and developmental programs.
Spermatogenesis is the process by which spermatozoa are produced from spermatogonia through two rounds of meiotic divisions to form spermatocytes, followed by a series of transformations to form spermatids. Unlike what occurs in oogenesis, during which centrioles are eliminated, centrioles are retained throughout meiosis in spermatocytes and are inherited by spermatids, where they function as basal bodies for the formation of flagella. In spermatocytes, centrioles are duplicated twice independently of pre-meiotic DNA synthesis. In spermatids, centrioles attach to the nucleus and undergo a unique remodeling process. These features are not observed in somatic cells, and the mechanisms that regulate these specialized events are still largely unknown. In this review, we provide an up-to-date overview of the roles of centrioles and the molecular mechanisms that support their functions in spermatogonia maintenance, meiosis in spermatocytes, spermatid transformation into spermatozoa, and the first zygotic division after fertilization.
Germline stem cells (GSCs) in the testis are responsible for producing sperm. In Drosophila, GSCs almost always divide asymmetrically, resulting in one self-renewing GSC and one differentiating cell called the gonialblast (GB), which then develops into sperm. This continuous asymmetric division ensures sperm production throughout the animal's life. During division, some asymmetries are observed. For example, sister chromatids-products of DNA replication that are generally considered identical-are segregated nonrandomly: they are equally segregated into two daughter cells, but one particular chromatid of the sister chromatids is preferentially segregated into the GSC in a strongly biased manner (~85%). Interestingly, only X and Y chromosomes, not autosomes, show such nonrandom sister chromatid segregation (NRSS). Recent studies indicate that NRSS is mediated by an asymmetry in sister chromatids of ribosomal DNA (rDNA), which is unique to the X and Y chromosomes in this species. rDNA is both necessary and sufficient to trigger NRSS. Moreover, NRSS appears to be essential for maintaining germline continuity across generations, mainly by preserving the unstable rDNA copy number. In this review, I will discuss the mechanisms and reasons behind the rDNA-mediated NRSS in Drosophila male GSCs.
Teleosts (teleost fishes), comprising over 30,000 species-roughly half of all living vertebrates-exhibit remarkably diverse patterns of meiotic recombination. Recent genome-wide analyses as well as cytological studies provide new insights built on decades of genetic research. In most teleosts, crossovers are concentrated near chromosome ends, producing strong heterochiasmy largely driven by male meiosis. Despite sharing conserved recombination machinery with mammals, teleosts show strikingly relaxed meiotic checkpoints, allowing meiotic progression and gamete formation even with severe recombination defects. This tolerance likely facilitates hybridization, polyploidy, and clonal reproduction commonly observed in teleosts. The recombination hotspot regulator PRDM9 has diversified dramatically across teleost lineages: some retain the full-length Prdm9α that determines recombination hotspots, whereas most species possess only a truncated Prdm9β and likely rely on PRDM9-independent recombination. Cytological analyses further reveal dynamic synapsis and partial recombination suppression on emerging sex chromosomes. This review summarizes the current understanding of meiotic recombination in teleost fishes, integrating evidence from recombination landscape diversity, molecular pathways, cytological features, PRDM9 evolution, and distinctive phenomena such as sex chromosome differentiation, clonal reproduction, hybridization, polyploidy tolerance, and relaxed checkpoint control. Together, these findings illustrate how the remarkable lineage-specific adaptations of teleosts can serve as a powerful lens for revealing the fundamental significance of meiotic recombination across vertebrates.
Sister chromatid cohesion, established by the cohesin complex, is indispensable for accurate chromosome segregation in both mitosis and meiosis. Whereas mitosis relies predominantly on the canonical cohesin complex composed of SMC1α, SMC3, RAD21, and STAG1/2, meiosis employs specialized variants, including SMC1β, REC8, RAD21L, and STAG3. These meiosis-specific cohesins impart unique structural and regulatory properties essential for gametogenesis. They form the chromosome axis and organize chromatin loops, thereby providing the framework for homolog recognition, synapsis, and crossover control. At centromeres, meiotic cohesins secure proper kinetochore orientation and enable the stepwise release of cohesion across successive divisions. A striking feature of cohesins in oocytes is their extraordinary stability, persisting from fetal stages until adulthood, which underpins female reproductive longevity. Progressive loss of cohesin integrity with age contributes to aneuploidy, infertility, and congenital disorders, highlighting their clinical significance. This review synthesizes recent advances in defining the molecular composition, dynamics, and functions of meiotic cohesins in mammals. We emphasize their multifaceted roles in shaping chromosome architecture, examine mechanisms that link cohesin maintenance to reproductive aging, and outline key unresolved questions that may shed light on both the evolutionary innovations of meiosis and the origins of human reproductive disorders.
Time is an important physical factor controlling all biological phenomena. Within the short period from fertilization to preimplantation, various events occur in a timely manner. A rapid replication of the genome and extensive epigenetic reprogramming occur concomitantly during the transformation from gametes to totipotent zygotes and differentiated cells. In the nuclei, large-scale changes such as genome replication and chromosome segregation progress within a short time. Simultaneously, classical epigenetic information, including histone modifications and DNA methylation patterns, is dynamically remodeled. During this period, the genome, including chromatin architecture and nuclear positioning of gene loci, is under strict control. Recently, single-cell omics analyses and high-throughput chromosome conformation capture (Hi-C) data have provided locus-level resolution of these genomic structures. These global changes inside and outside the cell nuclei occur across several species; however, differences in timing exist because of interspecies variation in developmental speed. In this chapter, we describe the history of research on these spatiotemporal changes inside and outside cell nuclei and present live-cell imaging technology as an important tool for the quantitative analysis of these phenomena.
In metazoans, gametogenesis produces the only cell type capable of transmitting both genetic and epigenetic information to offspring. This process represents one of the most extensive cellular differentiation programs, often originating from germline stem cells (GSCs), as in the female and male Drosophila and C. elegans gonads. These well-defined, unipotent germline lineages provide powerful in vivo models to study epigenetic regulation in multicellular organisms. During gametogenesis, epigenetic mechanisms balance cell differentiation and cellular plasticity. This review summarizes recent findings on how asymmetric sister chromatids are established and segregated during GSC division, the initial step of gametogenesis essential for reproduction in Drosophila and C. elegans. We focus on histones, a major carrier of epigenetic information, and discuss how their inheritance is regulated during GSC asymmetric divisions. Canonical histones and histone variants are dynamically incorporated into chromatin in a cell cycle- and genomic locus-specific manner, and these chromosome-bound epigenetic differences must coordinate with the mitotic machinery to ensure their proper partitioning. Finally, we speculate how these mechanisms may extend beyond the germline, assessing their conservation across species. Understanding these processes provides critical insights into how misregulation contributes to disease and how targeted manipulation could promote tissue homeostasis and regeneration.
Genome mutations in germ cells are fundamental to evolution and genetic diversity, while they also cause developmental abnormalities and genetic diseases, making their study essential in both biology and medicine. Research on germline mutations has advanced from classical genetic approaches, through transgenic model analyses, to next-generation sequencing, contributing to our understanding of germline mutations and their origins and inheritance. In particular, recent advances in sequencing technologies, including duplex sequencing and long-read sequencing, enable highly accurate detection of rare mutations and complex genomic alterations, including structural variants and repeat-associated changes, providing a more comprehensive view of mutational landscapes. Research progress has also been made in transposon regulation and spermatogonial stem cell dynamics, revealing additional layers of genome regulation in the germ line. These advances have greatly expanded our understanding of the germline genome and provide a foundation for future research in genetics, evolution, and reproductive biology.
Male germline development is characterized by a unique perinatal phase in which primordial germ cells (PGCs) differentiate into gonocytes (prospermatogonia) and subsequently give rise to spermatogonia. This chapter outlines the cellular, molecular, and epigenetic programs that orchestrate this transition, establishing the foundation of the lifelong spermatogenic lineage. Gonocytes undergo a transient G0/G1 arrest tightly regulated by retinoic acid (RA) metabolism, CDK inhibition, TGF family signaling, and the RNA-binding protein NANOS2, which integrates somatic cues to suppress meiosis and enforce male fate. Concurrently, gonocytes experience genome-wide de novo DNA methylation driven by NSD1-mediated H3K36me2 deposition and the DNMT3A/DNMT3L/DNMT3C machinery, accompanied by piRNA-directed transposon silencing and paternal imprint establishment. These processes collectively reshape the chromatin landscape and stabilize the male germline epigenome. The subsequent gonocyte-to-spermatogonia transition (GST) involves FGF, GDNF, and RA signaling, as well as dynamic histone demethylation, to generate spermatogonial stem cells (SSCs). Aberrant regulation of these pathways can arrest differentiation and predispose germ cells to transformation, as seen in testicular germ cell tumors (TGCTs), whose precursor cells retain gonocyte-like features. We further discuss the interplay between transposon control, imprinting, and chromatin architecture that underpins gonocyte identity. Emerging single-cell and small-input omics approaches are now redefining this transient developmental state, providing new insight into how epigenetic reprogramming and signaling convergence establish the male germline.
Petroleum-associated environments are among the most chemically complex and biologically extreme systems encountered in the field of industrial biotechnology. Here, microbial activity plays a pivotal role in hydrocarbon biodegradation, reservoir souring, and microbiologically influenced corrosion (MIC). In these systems, proteins constitute the functional interface between microbial metabolism and physicochemical processes affecting infrastructure integrity and environmental impact. This chapter presents an integrated proteomics-based workflow for the characterization of microbial communities inhabiting oil pipeline sludges, with particular emphasis on sample preparation strategies tailored to hydrocarbon-rich, metal-laden, and saline matrices. Optimized phenol-based extraction, electrochemical in vitro corrosion assays, two-dimensional gel electrophoresis, and high-resolution mass spectrometry are combined with metagenomic information to enable robust identification and functional interpretation of proteins involved in redox metabolism, biofilm formation, extracellular electron transfer, sulfur and nitrogen cycling, and stress adaptation. The approach is illustrated through a biocorrosion case study of marine pipeline sludge, revealing key enzymatic systems, including oxidoreductases, hydrolases, cytochromes, ABC transporters, and biofilm-associated structural proteins that mediate metal dissolution and microbial energy conservation. By integrating proteomics with electrochemical measurements and systems-level analysis, this chapter highlights how tailored sample preparation and functional protein profiling can overcome the limitations of culture-dependent methods, providing mechanistic insight into complex petroleum microbiomes. These advances establish proteomics as a critical tool for monitoring, predicting, and ultimately mitigating biocorrosion, as well as for guiding the development of biotechnology-based strategies in the oil and gas industry.
Urine is an ideal biological fluid due to the highly metabolomic and proteomic information it provides and its easy collection in large amounts. Urinary biomarkers reported for different types of diseases included the urological tract and systemic diseases. Although the most common approach in omics is single-omics studies, combining multi-omics biomarkers such as metabolomics, proteomics, transcriptomics, and genomics can improve diagnostic accuracy and provide deeper insights into disease mechanisms than single biomarkers. The gold standard technique for bioanalysis is liquid chromatography coupled to mass spectrometry (LC-MS/MS) due to its high sensitivity, specificity, and selectivity for the analysis of metabolites and proteins in complex biological samples. One of the most important aspects in metabolomics and proteomics is sample extraction and preparation before the analysis. Different types of metabolites and protein extraction methods can be used effectively for urine samples, including protein precipitation, liquid-liquid extraction, and solid-phase extraction. However, in the multi-omics approach integrating metabolomics and proteomics, sample preparation could be either individual for each or simultaneous for both from a single sample. In this chapter, we discuss aspects of LC-MS-based metabolomics and proteomics sample preparation, as well as their integration for a multi-omics approach. In clinical practice, the reported sample preparation methods for bladder cancer metabolomics and proteomics were also discussed.
Biomolecular interactions involving proteins, nucleic acids, and small molecules constitute the molecular foundation of cellular regulation, signaling, and therapeutic intervention. Advances in mass spectrometry-based proteomics have enabled the systematic characterization of these interactions at unprecedented depth, sensitivity, and structural resolution. This chapter provides a comprehensive overview of state-of-the-art proteomics methodologies developed to investigate protein-protein, protein-nucleic acid, and protein-drug interactions, with particular emphasis on experimental design, sample preparation, and data quality control. Targeted and untargeted strategies are discussed, including affinity purification-mass spectrometry, proximity-dependent labeling, cross-linking mass spectrometry, blue native electrophoresis, and size-exclusion chromatography-mass spectrometry for protein-protein interactions; affinity capture, EMSA-MS, chromatin immunoprecipitation-mass spectrometry, CRISPR-based locus-specific enrichment, and CLIP-based approaches for protein-nucleic acid complexes; and chemoproteomics, thermal proteome profiling, and label-free structural proteomics for protein-drug interaction analysis. The chapter further highlights recent technological innovations, computational tools, and integrative multi-omics strategies that enhance interaction mapping across biological scales. By critically evaluating the strengths, limitations, and appropriate applications of each methodology, this work aims to provide practical guidance for researchers seeking to design robust interactomics experiments and to interpret complex molecular networks in both basic and translational research contexts.
A range of mass spectrometry-based proteomic approaches is now employed to characterize the surface proteome-surfaceome-of pathogenic microorganisms, including fungi capable of causing infections in humans. Nevertheless, the preparation of surface-enriched samples remains technically challenging because of the persistent risk of intracellular protein contamination. Cell surface shaving represents a focused sample treatment approach in which proteolytic enzymes act on intact cells to release surface-exposed peptides, thereby enriching this fraction for high-resolution liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis. Optimizing sample preparation for fungal surface proteomics is critical, as reducing sample complexity, enriching surface-exposed components, and ensuring LC-MS/MS compatibility must be achieved while avoiding matrix interferences, partial lysis, and biases toward only trypsin-accessible epitopes. These methodological challenges, as well as strategies for validating sample-treatment selectivity with appropriate controls, are discussed in this chapter.
This chapter summarizes the current knowledge on the practical, methodological, and interpretative aspects of applying metaproteomics in water biotechnology. We outline the full metaproteomic workflow-from sampling and protein extraction to LC-MS/MS acquisition, database construction, quantitative analysis, and bioinformatic interpretation-and emphasize critical considerations specific to complex matrices such as EPS-rich biofilms, granular sludge, and low-biomass drinking water. Case studies illustrate how metaproteomics can clarify mechanisms of micropollutant degradation, nitrogen-transforming pathways, biofilm functional architecture, and microbial resilience under operational stress. Recent advances in data-independent acquisition, metagenome-informed databases, and integrative multi-omics are shown to substantially improve depth, reproducibility, and functional resolution. Finally, we discuss emerging applications in wastewater-based epidemiology, where metaproteomics complement nucleic-acid-based surveillance by enabling the detection of large biomolecule biomarkers of population health and industrial activity. Although metaproteomics is already being applied across a wide range of water cycle contexts and is producing promising, robust results, several challenges, including limitations in analytical chemistry, database completeness, and bioinformatics workflows, continue to hinder its broader implementation. Continued technical research and innovation are therefore essential to fully unlock its potential in water biotechnology.
