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.

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/.

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.

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.

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.

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.

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.

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.

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.

Mismatch Repair Protein Msh6Tt Is Necessary for Nuclear Division and Gametogenesis in Tetrahymena thermophila.

DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in E. coli or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. Tetrahymena undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6Tt localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6Tt only localized in MICs and newly developing MACs. MSH6Tt knockout led to aberrant nuclear division during vegetative growth. The MSH6TtKO mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. MSH6Tt knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6Tt interacted with Msh2Tt and MMR-independent factors. Downregulation of MSH2Tt expression affected the stability of Msh6Tt. In addition, MSH6Tt knockout led to the upregulated expression of several MSH6Tt homologs at different developmental stages. Msh6Tt is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in Tetrahymena.

Plasmodium microtubule-binding protein EB1 is critical for partitioning of nuclei in male gametogenesis.

Sexual reproduction of the malaria parasites is critical for their transmission to a mosquito vector. Several signaling molecules, such as kinases and phosphatases, are known to regulate this process. We previously demonstrated that Plasmodium falciparum (Pf) Ca2+-dependent protein kinase 4 (CDPK4) and serine/arginine-rich protein kinase 1 (SRPK1) are critical for axoneme formation during male gametogenesis, with genetic deletion of either gene causing a complete block in parasite transmission to the mosquito. A comparative phospho-proteome analysis of Pfcdpk4- and RNA-seq analysis of Pfsrpk1- gametocytes showed that these kinases regulate similar biological processes linked to both microtubule (MT) dynamics and cell motility. One of these proteins was a nuclear MT-associated End Binding protein 1 (EB1), which was hypophosphorylated in Pfcdpk4- gametocytes. To study the functional relevance of EB1, we created gene deletion parasites for EB1. We further demonstrate that Pfeb1- parasites like WT NF54 parasites proliferate normally as asexuals and undergo gametocytogenesis and gametogenesis. Strikingly, these parasites suffer a severe defect in nuclear segregation and partitioning of nuclei into emerging microgametes. Further genetic crosses utilizing male- and female-sterile parasites revealed that Pfeb1- parasites only suffer a male fertility defect. Overall, our study reveals an essential function for PfEB1 in male gamete nuclear segregation and suggests a potential therapeutic avenue in the design of transmission-blocking drugs to prevent malaria transmission from humans to mosquito. IMPORTANCE Gametogenesis and subsequent gamete fusion are central to successful transmission of the malaria parasites to a female Anopheles mosquito vector and completion of the sexual phase of the parasite life cycle. Male gametogenesis involves the formation of axonemes inside male gametes from male gametocytes via active cytoskeleton remodeling. The tubulin and tubulin-binding proteins are, thus, attractive anti-malarial drug targets. In the present study, we demonstrate that a microtubule-binding protein PfEB1 is essential for male gamete fertility, specifically for the inheritance of nuclei from activated male gametocytes. Targeting PfEB1 function may provide new avenues into designing interventions to prevent malaria transmission and disease spread.

The transcriptional regulator Ume6 is a major driver of early gene expression during gametogenesis.

The process of gametogenesis is orchestrated by a dynamic gene expression program, where a vital subset constitutes the early meiotic genes. In budding yeast, the transcription factor Ume6 represses early meiotic gene expression during mitotic growth. However, during the transition from mitotic to meiotic cell fate, early meiotic genes are activated in response to the transcriptional regulator Ime1 through its interaction with Ume6. While it is known that binding of Ime1 to Ume6 promotes early meiotic gene expression, the mechanism of early meiotic gene activation remains elusive. Two competing models have been proposed whereby Ime1 either forms an activator complex with Ume6 or promotes Ume6 degradation. Here, we resolve this controversy. First, we identify the set of genes that are directly regulated by Ume6, including UME6 itself. While Ume6 protein levels increase in response to Ime1, Ume6 degradation occurs much later in meiosis. Importantly, we found that depletion of Ume6 shortly before meiotic entry is detrimental to early meiotic gene activation and gamete formation, whereas tethering of Ume6 to a heterologous activation domain is sufficient to trigger early meiotic gene expression and produce viable gametes in the absence of Ime1. We conclude that Ime1 and Ume6 form an activator complex. While Ume6 is indispensable for early meiotic gene expression, Ime1 primarily serves as a transactivator for Ume6.

Genome-wide identification and transcriptome-based expression profiling of E2 gene family: Implication for potential roles in gonad development of Crassostrea gigas.

In this study, we investigated the role of E2 ubiquitin conjugating enzymes (E2) in the Pacific oyster Crassostrea gigas, with a focus on their involvement in gonad development. We identified 34 E2 genes clustered into nine subgroups and 24 subfamilies. The gene structure and intron-exon location were conserved within the same subfamily, but motif variation suggested functional diversity. Tissue transcriptome analyses revealed that most E2 genes were broadly expressed, with UBE2CL showing specific expression in the female gonad. Expression profiling of E2 genes during early embryo-larvae development stages suggested that five E2 genes were highly expressed in early embryo development, indicating their involvement in cell division processes. Furthermore, by profiling the expression of E2 genes in different gonadal developmental stages, we observed a gradual increase in expression for four genes during gametogenesis, with significantly higher expression in the female gonad at the maturation stage. Similarly, five E2 genes displayed elevated expression levels in the male gonad at the maturation stage, indicating their crucial roles in gonadal development and gametogenesis. Our study provides valuable insights into the potential functions of the E2 gene family in C. gigas, shedding light on the molecular mechanisms underlying gonad development in oysters.

From in vivo to in vitro: exploring the key molecular and cellular aspects of human female gametogenesis.

Human oogenesis is a highly complex and not yet fully understood process due to ethical and technological barriers that limit studies in the field. In this context, replicating female gametogenesis in vitro would not only provide a solution for some infertility problems, but also be an excellent study model to better understand the biological mechanisms that determine the formation of the female germline. In this review, we explore the main cellular and molecular aspects involved in human oogenesis and folliculogenesis in vivo, from the specification of primordial germ cells (PGCs) to the formation of the mature oocyte. We also sought to describe the important bidirectional relationship between the germ cell and the follicular somatic cells. Finally, we address the main advances and different methodologies used in the search for obtaining cells of the female germline in vitro.

RBM46 is essential for gametogenesis and functions in post-transcriptional roles affecting meiotic cohesin subunits.

RBM46 is a germ cell-specific RNA-binding protein required for gametogenesis, but the targets and molecular functions of RBM46 remain unknown. Here, we demonstrate that RBM46 binds at specific motifs in the 3'UTRs of mRNAs encoding multiple meiotic cohesin subunits and show that RBM46 is required for normal synaptonemal complex formation during meiosis initiation. Using a recently reported, high-resolution technique known as LACE-seq and working with low-input cells, we profiled the targets of RBM46 at single-nucleotide resolution in leptotene and zygotene stage gametes. We found that RBM46 preferentially binds target mRNAs containing GCCUAU/GUUCGA motifs in their 3'UTRs regions. In Rbm46 knockout mice, the RBM46-target cohesin subunits displayed unaltered mRNA levels but had reduced translation, resulting in the failed assembly of axial elements, synapsis disruption, and meiotic arrest. Our study thus provides mechanistic insights into the molecular functions of RBM46 in gametogenesis and illustrates the power of LACE-seq for investigations of RNA-binding protein functions when working with low-abundance input materials.

Differential phosphorylation of Clr4SUV39H by Cdk1 accompanies a histone H3 methylation switch that is essential for gametogenesis.

Methylation of histone H3 at lysine 9 (H3K9) is a hallmark of heterochromatin that plays crucial roles in gene silencing, genome stability, and chromosome segregation. In Schizosaccharomyces pombe, Clr4 mediates both di- and tri-methylation of H3K9. Although H3K9 methylation has been intensely studied in mitotic cells, its role during sexual differentiation remains unclear. Here, we map H3K9 methylation genome-wide during meiosis and show that constitutive heterochromatin temporarily loses H3K9me2 and becomes H3K9me3 when cells commit to meiosis. Cells lacking the ability to tri-methylate H3K9 exhibit meiotic chromosome segregation defects. Finally, the H3K9 methylation switch is accompanied by differential phosphorylation of Clr4 by the cyclin-dependent kinase Cdk1. Our results suggest that a conserved master regulator of the cell cycle controls the specificity of an H3K9 methyltransferase to prevent ectopic H3K9 methylation and to ensure faithful gametogenesis.

RBM46 is essential for gametogenesis and functions in post-transcriptional roles affecting meiotic cohesin subunits

RBM46 is a germ cell-specific RNA-binding protein required for gametogenesis, but the targets and molecular functions of RBM46 remain unknown. Here, we demonstrate that RBM46 binds at specific motifs in the 3'UTRs of mRNAs encoding multiple meiotic cohesin subunits and show that RBM46 is required for normal synaptonemal complex formation during meiosis initiation. Using a recently reported, high-resolution technique known as LACE-seq and working with low-input cells, we profiled the targets of RBM46 at single-nucleotide resolution in leptotene and zygotene stage gametes. We found that RBM46 preferentially binds target mRNAs containing GCCUAU/GUUCGA motifs in their 3'UTRs regions. In Rbm46 knockout mice, the RBM46-target cohesin subunits displayed unaltered mRNA levels but had reduced translation, resulting in the failed assembly of axial elements, synapsis disruption, and meiotic arrest. Our study thus provides mechanistic insights into the molecular functions of RBM46 in gametogenesis and illustrates the power of LACE-seq for investigations of RNA-binding protein functions when working with low-abundance input materials.

Transcriptome Analysis in a Scleractinian Coral, Acropora tenuis, during the Spawning Season With Reference to the Gonadal Condition.

Synchronous spawning is a striking feature of coral. Although it is important for reproductive success, corals reallocate energy for reproduction to growth when they are damaged by external stimuli. To assess the transcriptome before and after spawning in the scleractinian coral Acropora tenuis, we tagged three colonies (one bleached and two unbleached) in the field around Sesoko Island (Okinawa, Japan) in November 2016, sampled them monthly from May to July 2017, and performed RNA sequencing (RNA-Seq) analysis. Histological analysis revealed that the previously bleached colony possessed gametes in June, by which time the other two colonies had already spawned. In RNA-Seq analyses, multi-dimensional scaling based on gene expression similarity among the samples reflected the differences between colonies and between months except for the sample of a non-spawned colony in May, which was similar to the samples in June. The similarity of the non-spawned colony sample in May to the samples in June was also shown in hierarchical clustering based on the expression patterns of the genes that were differentially expressed between months in the spawned colonies. These results suggest that non-spawning was already decided in May, and that the physiological condition in a non-spawned colony in May was advanced to June. RNA-Seq analysis also showed that genes related to gametogenesis and those related to apoptosis were upregulated before and after spawning, respectively.

Pyp25α is required for male gametocyte exflagellation.

Malaria, a mosquito-borne infectious disease, is caused by the unicellular apicomplexan protozoa of the genus Plasmodium. For malaria parasite transmission, the essential sexual stage includes production of gametocytes through gametocytogenesis in vertebrate hosts and formation of gametes from gametocytes through gametogenesis in mosquito vectors. Whereas each female gametocyte forms a single immotile macrogamete, a male gametocyte produces eight flagella-like microgametes in a process called exflagellation. We identified a conserved protein named as Py05543 (Pyp25α), required for male gametocyte exflagellation in Plasmodium yoelii, which is the ortholog of PFL1770c (PF3D7_1236600). Interestingly, PF3D7_1236600 was previously phenotypically screened to be gametocyte-essential genes during gametocytogenesis of Plasmodium falciparum, using piggyBac transposon-mediated insertional mutagenesis. In this study, using CRISPR/Cas9-mediated genome editing, the Pyp25α¯ (KO) parasite line was successfully established. We found that the KO parasites proliferated asexually in mouse blood normally. In addition, compared with that of the parental parasites, the KO parasites displayed similar levels of gametocytes formation. Unexpectedly, the KO parasites showed considerable deficiency in exflagellation of male gametes, by observing exflagellation centre formation. Taken together, our data suggested that Pyp25α gene, the ortholog of PF3D7_1236600, was nonessential for the growth of asexual parasites, required for male gametocyte exflagellation in P. yoelii.