FilamentID reveals the composition and function of metabolic enzyme polymers during gametogenesis.

Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.

An axonemal intron splicing program sustains Plasmodium male development.

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.

GametesOmics: A Comprehensive Multi-omics Database for Exploring the Gametogenesis in Humans and Mice.

Gametogenesis plays an important role in the reproduction and evolution of species. The transcriptomic and epigenetic alterations in this process can influence the reproductive capacity, fertilization, and embryonic development. The rapidly increasing single-cell studies have provided valuable multi-omics resources. However, data from different layers and sequencing platforms have not been uniformed and integrated, which greatly limits their use for exploring the molecular mechanisms that underlie oogenesis and spermatogenesis. Here, we develop GametesOmics, a comprehensive database that integrates the data of gene expression, DNA methylation, and chromatin accessibility during oogenesis and spermatogenesis in humans and mice. GametesOmics provides a user-friendly website and various tools, including Search and Advanced Search for querying the expression and epigenetic modification(s) of each gene; Tools with Differentially expressed gene (DEG) analysis for identifying DEGs, Correlation analysis for demonstrating the genetic and epigenetic changes, Visualization for displaying single-cell clusters and screening marker genes as well as master transcription factors (TFs), and MethylView for studying the genomic distribution of epigenetic modifications. GametesOmics also provides Genome Browser and Ortholog for tracking and comparing gene expression, DNA methylation, and chromatin accessibility between humans and mice. GametesOmics offers a comprehensive resource for biologists and clinicians to decipher the cell fate transition in germ cell development, and can be accessed at http://gametesomics.cn/.

TENT5-mediated polyadenylation of mRNAs encoding secreted proteins is essential for gametogenesis in mice.

Cytoplasmic polyadenylation plays a vital role in gametogenesis; however, the participating enzymes and substrates in mammals remain unclear. Using knockout and knock-in mouse models, we describe the essential role of four TENT5 poly(A) polymerases in mouse fertility and gametogenesis. TENT5B and TENT5C play crucial yet redundant roles in oogenesis, with the double knockout of both genes leading to oocyte degeneration. Additionally, TENT5B-GFP knock-in females display a gain-of-function infertility effect, with multiple chromosomal aberrations in ovulated oocytes. TENT5C and TENT5D both regulate different stages of spermatogenesis, as shown by the sterility in males following the knockout of either gene. Finally, Tent5a knockout substantially lowers fertility, although the underlying mechanism is not directly related to gametogenesis. Through direct RNA sequencing, we discovered that TENT5s polyadenylate mRNAs encoding endoplasmic reticulum-targeted proteins essential for gametogenesis. Sequence motif analysis and reporter mRNA assays reveal that the presence of an endoplasmic reticulum-leader sequence represents the primary determinant of TENT5-mediated regulation.

Conserved genes regulating human sex differentiation, gametogenesis and fertilization.

The study of the functional genome in mice and humans has been instrumental for describing the conserved molecular mechanisms regulating human reproductive biology, and for defining the etiologies of monogenic fertility disorders. Infertility is a reproductive disorder that includes various conditions affecting a couple's ability to achieve a healthy pregnancy. Recent advances in next-generation sequencing and CRISPR/Cas-mediated genome editing technologies have facilitated the identification and characterization of genes and mechanisms that, if affected, lead to infertility. We report established genes that regulate conserved functions in fundamental reproductive processes (e.g., sex determination, gametogenesis, and fertilization). We only cover genes the deletion of which yields comparable fertility phenotypes in both rodents and humans. In the case of newly-discovered genes, we report the studies demonstrating shared cellular and fertility phenotypes resulting from loss-of-function mutations in both species. Finally, we introduce new model systems for the study of human reproductive biology and highlight the importance of studying human consanguineous populations to discover novel monogenic causes of infertility. The rapid and continuous screening and identification of putative genetic defects coupled with an efficient functional characterization in animal models can reveal novel mechanisms of gene function in human reproductive tissues.

Medaka Terb1 Mutant Displays Defects of Synaptonemal Complex Formation and Sexual Difference in Gametogenesis.

Formation of the synaptonemal complex (SC) is a prerequisite for proper recombination and chromosomal segregation during meiotic prophase I. One mechanism that ensures SC formation is chromosomal movement, which is driven by the force derived from cytoskeletal motors. Here, we report the phenotype of medaka mutants lacking the telomere repeat binding bouquet formation protein 1 (TERB1), which, in combination with the SUN/KASH protein, mediates chromosomal movement by connecting telomeres and cytoskeletal motors. Mutations in the terb1 gene exhibit defects in SC formation in medaka. Although SC formation was initiated, as seen by the punctate lateral elements and fragmented transverse filaments, it was not completed in the terb1 mutant meiocytes. The mutant phenotype further revealed that the introduction of double strand breaks was independent of synapsis completion. In association with these phenotypes, meiocytes in both the ovaries and testes exhibited an aberrant arrangement of homologous chromosomes. Interestingly, although oogenesis halted at the zygotene-like stage in terb1 mutant, testes continued to produce sperm-like cells with aberrant DNA content. This indicates that the mechanism of meiotic checkpoint is sexually different in medaka, similar to the mammalian checkpoint in which oogenesis proceeds while spermatogenesis is arrested. Moreover, our results suggest that spermatogenesis is mechanistically dissociable from meiosis.

The Pacific oyster reproduction is affected by early-life exposure to environmental pesticide mixture: A multigenerational study.

Pesticides threat marine organisms worldwide. Among them, the Pacific oyster is a bivalve mollusc model in marine ecotoxicology. A large body of literature already stated on the multiple-scale effects pesticides can trigger in the Pacific oyster, throughout its life cycle and in a delayed manner. In particular, reproductive toxicity is of major concern because of its influence on population dynamics. However, past studies mostly investigated pesticide reprotoxicity as a direct effect of exposure during gametogenesis or directly on gametes and little is known about the influence of an early embryo exposure on the breed capacity. Therefore, we studied delayed and multigenerational consequences through gametogenesis features (i.e. sex ratio, glycogen content, gene expression) and reproductive success in two consecutive oyster generations (F0 and F1) exposed to an environmentally-relevant pesticide mixture (sum nominal concentration: 2.85 µg.L-1) during embryo-larval development (0-48 h post fertilization, hpf). In the first generation, glycogen content increased in exposed individuals and the expression of some gametogenesis target genes was modified. The reproductive success measured 48 hpf was higher in exposed individuals. A multigenerational influence was observed in the second generation, with feminisation, acceleration of gametogenesis processes and the sex-specific modification of glycogen metabolism in individuals from exposed parents. This study is the first to highlight the delayed effects on reproduction induced by an early exposure to pesticides, and its multigenerational implications in the Pacific oyster. It suggests that environmental pesticide contamination can have impacts on the recruitment and the dynamics of natural oyster populations exposed during their embryo-larval phase.

Gametes of semelparous salmon are repeatedly produced by surrogate rainbow trout.

Most Pacific salmon species grow in the ocean, return to their native rivers to reproduce, and then die (semelparous type). However, rainbow trout survive after spawning and reproduce repeatedly until the end of their lives (iteroparous type). Little is known about how germline stem cells behave during gametogenesis in the two types of Pacific salmon. In this study, we show that all germline stem cells disappear after the first gametogenesis in Chinook and Kokanee salmon, whereas germline stem cells are maintained in rainbow trout. However, the germline stem cells of Chinook and Kokanee salmon transplanted into rainbow trout survive even after their spawning seasons and supply salmon gametes for multiple years. These results indicate that the behavior of the germline stem cells is mainly regulated by the somatic environment.

The Cryptosporidium signaling kinase CDPK5 plays an important role in male gametogenesis and parasite virulence.

The protozoan parasite Cryptosporidium is a leading cause of diarrhea in young children. The parasite's life cycle involves a coordinated and timely progression from asexual to sexual stages, leading to the formation of the transmissible oocyst. Underlying molecular signaling mechanisms orchestrating sexual development are not known. Here, we describe the function of a signaling kinase in Cryptosporidium male gametogenesis. We reveal the expression of Cryptosporidium parvum calcium-dependent protein kinase 5 (CDPK5) during male gamete development and its important role in the egress of mature gametes. Genetic ablation of this kinase results in viable parasites, indicating that this gene is dispensable for parasite survival. Interestingly, cdpk5 deletion decreases parasite virulence and impacts oocyst shedding in immunocompromised mice. Using phosphoproteomics, we identify possible CDPK5 substrates and biological processes regulated by this kinase. Collectively, these findings illuminate parasite cell biology by revealing a mechanism controlling male gamete production and a potential target to block disease transmission.

A cyclical switch of gametogenic pathways in hybrids depends on the ploidy level.

The cellular and molecular mechanisms governing sexual reproduction are conserved across eukaryotes. Nevertheless, hybridization can disrupt these mechanisms, leading to asexual reproduction, often accompanied by polyploidy. In this study, we investigate how ploidy level and ratio of parental genomes in hybrids affect their reproductive mode. We analyze the gametogenesis of sexual species and their diploid and triploid hybrids from the freshwater fish family Cobitidae, using newly developed cytogenetic markers. We find that diploid hybrid females possess oogonia and oocytes with original (diploid) and duplicated (tetraploid) ploidy. Diploid oocytes cannot progress beyond pachytene due to aberrant pairing. However, tetraploid oocytes, which emerge after premeiotic genome endoreplication, exhibit normal pairing and result in diploid gametes. Triploid hybrid females possess diploid, triploid, and haploid oogonia and oocytes. Triploid and haploid oocytes cannot progress beyond pachytene checkpoint due to aberrant chromosome pairing, while diploid oocytes have normal pairing in meiosis, resulting in haploid gametes. Diploid oocytes emerge after premeiotic elimination of a single-copied genome. Triploid hybrid males are sterile due to aberrant pairing and the failure of chromosomal segregation during meiotic divisions. Thus, changes in ploidy and genome dosage may lead to cyclical alteration of gametogenic pathways in hybrids.

Gene expression profiles of Japanese precious coral Corallium japonicum during gametogenesis.

Background: Corallium japonicum, a prized resource in Japan, plays a vital role in traditional arts and fishing industries. Because of diminished stock due to overexploitation, ongoing efforts are focused on restoration through transplantation. This study aimed to enhance our understanding of the reproductive biology of these valuable corals and find more efficient methods for sex determination, which may significantly contribute to conservation initiatives. Methods: We used 12 three-month aquarium reared C. japonicum colony fragments, conducted histological analysis for maturity and sex verification, and performed transcriptome analysis via de novo assembly and mapping using the C. rubrum transcriptome to explore gene expression differences between female and male C. japonicum. Results: Our histological observations enabled sex identification in 33% of incompletely mature samples. However, the sex of the remaining 67% of samples, classified as immature, could not be identified. RNA-seq yielded approximately 21-31 million short reads from 12 samples. De novo assembly yielded 404,439 highly expressed transcripts. Among them, 855 showed significant differential expression, with 786 differentially expressed transcripts between females and males. Heatmap analysis highlighted 283 female-specific and 525 male-specific upregulated transcripts. Transcriptome assembly mapped to C. rubrum yielded 28,092 contigs, leading to the identification of 190 highly differentially expressed genes, with 113 upregulated exclusively in females and 70 upregulated exclusively in males. Blastp analysis provided putative protein annotations for 83 female and 72 male transcripts. Annotation analysis revealed that female biological processes were related to oocyte proliferation and reproduction, whereas those in males were associated with cell adhesion. Discussion: Transcriptome analysis revealed sex-specific gene upregulation in incompletely mature C. japonicum and shared transcripts with C. rubrum, providing insight into its gene expression patterns. This study highlights the importance of using both de novo and reference-based assembly methods. Functional enrichment analysis showed that females exhibited enrichment in cell proliferation and reproduction pathways, while males exhibited enrichment in cell adhesion pathways. To the best of our knowledge, this is the first report on the gene expressions of each sex during the spawning season. Our findings offer valuable insights into the physiological ecology of incompletely mature red Japanese precious corals and suggest a method for identifying sex using various genes expressed in female and male individuals. In the future, techniques such as transplantation, artificial fertilization, and larval rearing may involve sex determination methods based on differences in gene expression to help conserve precious coral resources and ecosystems.

DNA methylation remodeling and the functional implication during male gametogenesis in rice.

BACKGROUND: Epigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However, the timing and functional significance of the remodeling during plant gametogenesis remain obscure. RESULTS: Here we show that DNA methylation remodeling starts after male meiosis in rice, with non-CG methylation, particularly at CHG sites, being first enhanced in the microspore and subsequently decreased in sperm. Functional analysis of rice CHG methyltransferase genes CMT3a and CMT3b indicates that CMT3a functions as the major CHG methyltransferase in rice meiocyte, while CMT3b is responsible for the increase of CHG methylation in microspore. The function of the two histone demethylases JMJ706 and JMJ707 that remove H3K9me2 may contribute to the decreased CHG methylation in sperm. During male gametogenesis CMT3a mainly silences TE and TE-related genes while CMT3b is required for repression of genes encoding factors involved in transcriptional and translational activities. In addition, CMT3b functions to repress zygotic gene expression in egg and participates in establishing the zygotic epigenome upon fertilization. CONCLUSION: Collectively, the results indicate that DNA methylation is dynamically remodeled during male gametogenesis, distinguish the function of CMT3a and CMT3b in sex cells, and underpin the functional significance of DNA methylation remodeling during rice reproduction.

Ready for renascence in mosquito: The regulation of gene expression in Plasmodium sexual development.

Malaria remains one of the most perilous vector-borne infectious diseases for humans globally. Sexual gametocyte represents the exclusive stage at which malaria parasites are transmitted from the vertebrate to the Anopheles host. The feasible and effective approach to prevent malaria transmission is by addressing the sexual developmental processes, that is, gametocytogenesis and gametogenesis. Thus, this review will comprehensively cover advances in the regulation of gene expression surrounding the transmissible stages, including epigenetic, transcriptional, and post-transcriptional control.

Sexually dimorphic dynamics of the microtubule network in medaka (Oryzias latipes) germ cells.

Gametogenesis is the process through which germ cells differentiate into sexually dimorphic gametes, eggs and sperm. In the teleost fish medaka (Oryzias latipes), a germ cell-intrinsic sex determinant, foxl3, triggers germline feminization by activating two genetic pathways that regulate folliculogenesis and meiosis. Here, we identified a pathway involving a dome-shaped microtubule structure that may be the basis of oocyte polarity. This structure was first established in primordial germ cells in both sexes, but was maintained only during oogenesis and was destabilized in differentiating spermatogonia under the influence of Sertoli cells expressing dmrt1. Although foxl3 was dispensable for this pathway, dazl was involved in the persistence of the microtubule dome at the time of gonocyte development. In addition, disruption of the microtubule dome caused dispersal of bucky ball RNA, suggesting the structure may be prerequisite for the Balbiani body. Collectively, the present findings provide mechanistic insight into the establishment of sex-specific polarity through the formation of a microtubule structure in germ cells, as well as clarifying the genetic pathways implementing oocyte-specific characteristics.

Sexual conflict drive in the rapid evolution of new gametogenesis genes.

The evolutionary forces underlying the rapid evolution in sequences and functions of new genes remain a mystery. Adaptation by natural selection explains the evolution of some new genes. However, many new genes perform sex-biased functions that have rapidly evolved over short evolutionary time scales, suggesting that new gene evolution may often be driven by conflicting selective pressures on males and females. It is well established that such sexual conflict (SC) plays a central role in maintaining phenotypic and genetic variation within populations, but the role of SC in driving new gene evolution remains essentially unknown. This review explores the connections between SC and new gene evolution through discussions of the concept of SC, the phenotypic and genetic signatures of SC in evolving populations, and the molecular mechanisms by which SC could drive the evolution of new genes. We synthesize recent work in this area with a discussion of the case of Apollo and Artemis, two extremely young genes (<200,000 years) in Drosophila melanogaster, which offered the first empirical insights into the evolutionary process by which SC could drive the evolution of new genes. These new duplicate genes exhibit the hallmarks of sexually antagonistic selection: rapid DNA and protein sequence evolution, essential sex-specific functions in gametogenesis, and complementary sex-biased expression patterns. Importantly, Apollo is essential for male fitness but detrimental to female fitness, while Artemis is essential for female fitness but detrimental to male fitness. These sexually antagonistic fitness effects and complementary changes to expression, sequence, and function suggest that these duplicates were selected for mitigating SC, but that SC has not been fully resolved. Finally, we propose Sexual Conflict Drive as a self-driven model to interpret the rapid evolution of new genes, explain the potential for SC and sexually antagonistic selection to contribute to long-term evolution, and suggest its utility for understanding the rapid evolution of new genes in gametogenesis.

Dietary steroids promote body weight growth and induce gametogenesis by increasing the expressions of genes related to cell proliferation of sea cucumber (Apostichopus japonicus).

Steroids play a vital role in animal survival, promoting growth and development when administered appropriate concentration exogenously. However, it remains unclear whether steroids can induce gonadal development and the underlying mechanism. This study assessed sea cucumber weights post-culturing, employing paraffin sections and RNA sequencing (RNA-seq) to explore gonadal changes and gene expression in response to exogenous steroid addition. Testosterone and cholesterol, dissolved in absolute ethanol, were incorporated into sea cucumber diets. After 30 days, testosterone and cholesterol significantly increased sea cucumber weights, with the total weight of experimental groups surpassing the control. The testosterone-fed group exhibited significantly higher eviscerated weight than the control group. In addition, dietary steroids influenced gonad morphology and upregulated genes related to cell proliferation，such as RPL35, PC, eLF-1, MPC2, ADCY10 and CYP2C18. Thees upregulated differentially expressed genes were significantly enriched in the organic system, metabolism, genetic information and environmental information categories. These findings imply that steroids may contribute to the growth and the process of genetic information translation and protein synthesis essential for gonadal development and gametogenesis.

MBD2 couples DNA methylation to transposable element silencing during male gametogenesis.

DNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood1-5. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2 and MBD4 and show that MBD2 acts as a TE repressor during male gametogenesis. MBD2 bound chromatin regions containing high levels of CG methylation, and MBD2 was capable of silencing the FWA gene when tethered to its promoter. MBD2 loss caused activation at a small subset of TEs in the vegetative cell of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. TE activation in mbd2 became more significant in the mbd5 mbd6 and adcp1 mutant backgrounds, suggesting that MBD2 acts redundantly with other silencing pathways to repress TEs. Overall, our study identifies MBD2 as a methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis.

Gene regulation during meiosis.

Meiosis is essential for gamete production in all sexually reproducing organisms. It entails two successive cell divisions without DNA replication, producing haploid cells from diploid ones. This process involves complex morphological and molecular differentiation that varies across species and between sexes. Specialized genomic events like meiotic recombination and chromosome segregation are tightly regulated, including preparation for post-meiotic development. Research in model organisms, notably yeast, has shed light on the genetic and molecular aspects of meiosis and its regulation. Although mammalian meiosis research faces challenges, particularly in replicating gametogenesis in vitro, advances in genetic and genomic technologies are providing mechanistic insights. Here we review the genetics and molecular biology of meiotic gene expression control, focusing on mammals.

Making human eggs in a dish: are we close?

In the space of 50 years, we have seen incredible achievements in human reproductive medicine. With these leaps forward, it is no wonder that there is a major interest in women's reproductive health research, including extension of reproductive lifespan. Substantial effort is currently being made to address this challenge, including from the commercial sector. In vitro gametogenesis (IVG) in mice is a spectacular breakthrough and has the potential to offer hope to women with intractable infertility. However, with such lofty goals, some reflection may be called for: mastering all of the techniques required for complete and safe IVG in women is likely to be extraordinarily difficult.