[Method of Inducible Knockdown of Essential Genes in OSC Cell Culture of Drosophila melanogaster].

An RNA interference-based method was proposed to achieve an inducible knockdown of genes essential for cell viability. In the method, a genetic cassette in which a copper ion-dependent inducible metallothionein promoter controls expression of a siRNA precursor is inserted into a genomic pre-integrated transgene by CRIPSR/Cas9 technology. The endogenous siRNA source allows the gene knockdown in cell cultures that are refractory to conventional transfection with exogenous siRNA. The efficiency of the method was demonstrated in Drosophila ovarian somatic cell culture (OSC) for two genes that are essential for oogenesis: Cul3, encoding a component of the multiprotein ubiquitin-ligase complex with versatile functions in proteostasis, and cut, encoding a transcription factor regulating differentiation of ovarian follicular cells.

The mRNA dynamics underpinning translational control mechanisms of Drosophila melanogaster oogenesis.

Advances in the study of mRNAs have yielded major new insights into post-transcriptional control of gene expression. Focus on the spatial regulation of mRNAs in highly polarized cells has demonstrated that mRNAs translocate through cells as mRNA:protein granules (mRNPs). These complex self-assemblies containing nuclear and cytoplasmic proteins are fundamental to the coordinated translation throughout cellular development. Initial studies on translational control necessitated fixed tissue, but the last 30 years have sparked innovative live-cell studies in several cell types to deliver a far more nuanced picture of how mRNA-protein dynamics exert translational control. In this review, we weave together the events that underpin mRNA processes and showcase the pivotal studies that revealed how a multitude of protein factors engage with a transcript. We highlight a mRNA's ability to act as a 'super scaffold' to facilitate molecular condensate formation and further moderate translational control. We focus on the Drosophila melanogaster germline due to the extensive post-transcriptional regulation occurring during early oogenesis. The complexity of the spatio-temporal expression of maternal transcripts in egg chambers allows for the exploration of a wide range of mechanisms that are crucial to the life cycle of mRNAs.

foxl2l is a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes. The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system, where most males initially develop juvenile ovaries before committing to male fate. In species with chromosomal sex determination, gonadal somatic cells are recognized as key determinants of germ cell fate. Notably, the loss of germ cells in zebrafish leads to masculinization, implying that germ cells harbor an intrinsic feminization signal. However, the specific signal triggering oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells. Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis, leading to premature spermatogenesis during early gonadal development. Furthermore, while mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis, with a large proportion of germ cells arrested as germline stem cells, highlighting the crucial role of foxl2l in oogenesis. Overall, this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Single-cell transcriptome sequencing reveals that Wolbachia induces gene expression changes in Drosophila ovary cells to favor its own maternal transmission.

Wolbachia is an obligate endosymbiont that is maternally inherited and widely distributed in arthropods and nematodes. It remains in the mature eggs of female hosts over generations through multiple strategies and manipulates the reproduction system of the host to enhance its spreading efficiency. However, the transmission of Wolbachia within the host's ovaries and its effects on ovarian cells during oogenesis, have not been extensively studied. We used single-cell RNA sequencing to comparatively analyze cell-typing and gene expression in Drosophila ovaries infected and uninfected with Wolbachia. Our findings indicate that Wolbachia significantly affects the transcription of host genes involved in the extracellular matrix, cytoskeleton organization, and cytomembrane mobility in multiple cell types, which may make host ovarian cells more conducive for the transmission of Wolbachia from extracellular to intracellular. Moreover, the genes nos and orb, which are related to the synthesis of ribonucleoprotein complexes, are specifically upregulated in early germline cells of ovaries infected with Wolbachia, revealing that Wolbachia can increase the possibility of its localization to the host oocytes by enhancing the binding with host ribonucleoprotein-complex processing bodies (P-bodies). All these findings provide novel insights into the maternal transmission of Wolbachia between host ovarian cells.IMPORTANCEWolbachia, an obligate endosymbiont in arthropods, can manipulate the reproduction system of the host to enhance its maternal transmission and reside in the host's eggs for generations. Herein, we performed single-cell RNA sequencing of ovaries from Drosophila melanogaster and observed the effects of Wolbachia (strain wMel) infection on different cell types to discuss the potential mechanism associated with the transmission and retention of Wolbachia within the ovaries of female hosts. It was found that the transcriptions of multiple genes in the ovary samples infected with Wolbachia are significantly altered, which possibly favors the maternal transmission of Wolbachia. Meanwhile, we also discovered that Wolbachia may flexibly regulate the expression level of specific host genes according to their needs rather than rigidly changing the expression level in one direction to achieve a more suitable living environment in the host's ovarian cells. Our findings contribute to a further understanding of the maternal transmission and possible universal effects of Wolbachia within the host.

Female-specific mechanisms of meiotic initiation and progression in mammalian oocyte development.

Meiosis is regulated in sexually dimorphic manners in mammals. In females, the commitment to and entry into meiosis are coordinated with the developmental program of oocytes. Female germ cells initiate meiosis within a short time window during the fetal period and then undergo meiotic arrest until puberty. However, the genetic mechanisms underlying the orchestration of oocyte development and meiosis to maximize the reproductive lifespan of mammalian females remain largely elusive. While meiotic initiation is regulated by a sexually common mechanism, where meiosis initiator and Stimulated by Retinoic Acid Gene 8 (STRA8) activate the meiotic genes, the female-specific mode of meiotic initiation is mediated by the interaction between retinoblastoma (RB) and STRA8. This review highlights the female-specific mechanisms of meiotic initiation and meiotic prophase progression in the context of oocyte development. Furthermore, the downstream pathway of the RB-STRA8 interaction that may regulate meiotic arrest will be discussed in the context of oocyte development, highlighting a potential genetic link between the female-specific mode of meiotic entry and meiotic arrest.

Single-nucleus atlas of the Artemia female reproductive system suggests germline repression of the Z chromosome.

Our understanding of the molecular pathways that regulate oogenesis and define cellular identity in the Arthropod female reproductive system and the extent of their conservation is currently very limited. This is due to the focus on model systems, including Drosophila and Daphnia, which do not reflect the observed diversity of morphologies, reproductive modes, and sex chromosome systems. We use single-nucleus RNA and ATAC sequencing to produce a comprehensive single nucleus atlas of the adult Artemia franciscana female reproductive system. We map our data to the Fly Cell Atlas single-nucleus dataset of the Drosophila melanogaster ovary, shedding light on the conserved regulatory programs between the two distantly related Arthropod species. We identify the major cell types known to be present in the Artemia ovary, including germ cells, follicle cells, and ovarian muscle cells. Additionally, we use the germ cells to explore gene regulation and expression of the Z chromosome during meiosis, highlighting its unique regulatory dynamics and allowing us to explore the presence of meiotic sex chromosome silencing in this group.

Maternal Spargel/dPGC-1 is critical for embryonic development and influences chorion gene amplification via Cyclin E activity.

The function of spargel/dPGC-1 in Drosophila oogenesis has been unequivocally established. Here, we sought to assess whether Spargel protein or RNA is essential for developmentally competent eggs. The trans-heterozygotic combination of two spargel mutant alleles allowed us to decrease Spargel expression to very low levels. Using this model, we now demonstrated the requirement for Spargel in eggshell patterning and embryonic development, which led us to establish that spargel is a maternal effect gene. Further examination of Spargel's potential mechanism of action in eggshell biogenesis revealed that low levels of Spargel in the adult ovary cause diminished Cyclin E activity, resulting in reduced chorion gene amplification levels, leading to eggshell biogenesis defects. Thus, another novel role for spargel/dPGC-1 is exposed whereby, through Cyclin E activity, this conserved transcriptional coactivator regulates the chorion gene amplification process.

RNAseq analysis of oocyte maturation from the germinal vesicle stage to metaphase II in pig and human.

During maturation oocytes at the germinal vesicle (GV) stage progress to metaphase II (MII). However, during in vitro maturation a proportion often fail to progress. To understand these processes, we employed RNA sequencing to examine the transcriptome profile of these three groups of oocytes from the pig. We compared our findings with similar public oocyte data from humans. The transcriptomes in oocytes that failed to progress was similar to those that did. We found in both species, the most upregulated genes in MII oocytes were associated with chromosome segregation and cell cycle processes, while the most down regulated genes were relevant to ribosomal and mitochondrial pathways. Moreover, those genes involved in chromosome segregation during GV to MII transition were conserved in pig and human. We also compared MII and GV oocyte transcriptomes at the isoform transcript level in both species. Several thousands of genes (including DTNBP1, MAPK1, RAB35, GOLGA7, ATP1A1 and ATP2B1) identified as not different in expression at a gene transcript level were found to have differences in isoform transcript levels. Many of these genes were involved in ATPase-dependent or GTPase-dependent intracellular transport in pig and human, respectively. In conclusion, our study suggests the failure to progress to MII in vitro may not be regulated at the level of the genome and that many genes are differentially regulated at the isoform level, particular those involved ATPase- or GTPase-dependent intracellular transport.

Proteomics profiling reveals lipid metabolism abnormalities during oogenesis in unexplained recurrent pregnancy loss.

Background: Unexplained recurrent pregnancy loss (URPL) is a clinical dilemma in reproductive fields. Its diagnosis is mainly exclusionary after extensive clinical examination, and some of the patients may still face the risk of miscarriage. Methods: We analyzed follicular fluid (FF) from in vitro fertilization (IVF) in eight patients with URPL without endocrine abnormalities or verifiable causes of abortion and eight secondary infertility controls with no history of pregnancy loss who had experienced at least one normal pregnancy and delivery by direct data-independent acquisition (dDIA) quantitative proteomics to identify differentially expressed proteins (DEPs). In this study, bioinformatics analysis was performed using online software including g:profiler, String, and ToppGene. Cytoscape was used to construct the protein-protein interaction (PPI) network, and ELISA was used for validation. Results: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEPs are involved in the biological processes (BP) of complement and coagulation cascades. Apolipoproteins (APOs) are key proteins in the PPI network. ELISA confirmed that APOB was low-expressed in both the FF and peripheral blood of URPL patients. Conclusion: Dysregulation of the immune network intersecting coagulation and inflammatory response is an essential feature of URPL, and this disequilibrium exists as early as the oogenesis stage. Therefore, earlier intervention is necessary to prevent the development of URPL. Moreover, aberrant lipoprotein regulation appears to be a key factor contributing to URPL. The mechanism by which these factors are involved in the complement and coagulation cascade pathways remains to be further investigated, which also provides new candidate targets for URPL treatment.

Decoding molecular features of bovine oocyte fate during antral follicle growth via single-cell multi-omics analysis†.

Antral follicle size is a useful predictive marker of the competency of enclosed oocytes for yielding an embryo following in vitro maturation and fertilization. However, the molecular mechanisms underpinning oocyte developmental potential during bovine antral follicle growth are still unclear. Here, we used a modified single-cell multi-omics approach to analyze the transcriptome, DNA methylome, and chromatin accessibility in parallel for oocytes and cumulus cells collected from bovine antral follicles of different sizes. Transcriptome profiling identified three types of oocytes (small, medium, and large) that underwent different developmental trajectories, with large oocytes exhibiting the largest average follicle size and characteristics resembling metaphase-II oocytes. Differential expression analysis and real-time polymerase chain reaction assay showed that most replication-dependent histone genes were highly expressed in large oocytes. The joint analysis of multi-omics data revealed that the transcription of 20 differentially expressed genes in large oocytes was associated with both DNA methylation and chromatin accessibility. In addition, oocyte-cumulus interaction analysis showed that inflammation, DNA damage, and p53 signaling pathways were active in small oocytes, which had the smallest average follicle sizes. We further confirmed that p53 pathway inhibition in the in vitro maturation experiments using oocytes obtained from small antral follicles could improve the quality of oocytes and increased the blastocyte rate after in vitro fertilization and culture. Our work provides new insights into the intricate orchestration of bovine oocyte fate determination during antral folliculogenesis, which is instrumental for optimizing in vitro maturation techniques to optimize oocyte quality.

ADR-2 regulates fertility and oocyte fate in Caenorhabditis elegans.

RNA-binding proteins (RBPs) play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the adenosine deaminase acting on RNA family of RBPs and the sole adenosine-to-inosine RNA-editing enzyme in Caenorhabditis elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RBPs that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogeneous nuclear ribonucleoprotein family of RBPs. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and RNA editing-independent mechanisms in regulating embryogenesis.

Oocyte-specific EXOC5 expression is required for mouse oogenesis and folliculogenesis.

EXOC5 is a crucial component of a large multi-subunit tethering complex, the exocyst complex, that is required for fusion of secretory vesicles with the plasma membrane. Exoc5 deleted mice die as early embryos. Therefore, to determine the role of EXOC5 in follicular and oocyte development, it was necessary to produce a conditional knockout (cKO), Zp3-Exoc5-cKO, in which Exoc5 was deleted only in oocytes. The first wave of folliculogenesis appeared histologically normal and progressed to the antral stage. However, after IVF with normal sperm, oocytes collected from the first wave (superovulated 21-day-old cKO mice) were shown to be developmentally incompetent. Adult follicular waves did not progress beyond the secondary follicle stage where they underwent apoptosis. Female cKO mice were infertile. Overall, these data suggest that the first wave of folliculogenesis is less sensitive to oocyte-specific loss of Exoc5, but the resulting gametes have reduced developmental competence. In contrast, subsequent waves of folliculogenesis require oocyte-specific Exoc5 for development past the preantral follicle stage. The Zp3-Exoc5-cKO mouse provides a model for disrupting folliculogenesis that also enables the separation between the first and subsequent waves of folliculogenesis.

Expression profiling and function analysis identified new genes regulating cumulus expansion and cumulus cell apoptosis in mouse oocytes.

In brief: Genes expressed in cumulus cells might be used as markers for competent oocytes/embryos. This study identified and validated a new group of cumulus expansion and/or apoptosis-regulating genes, which may be used for selection of quality oocytes/embryos. Abstract: Studies on the mechanisms behind cumulus expansion and cumulus cell (CC) apoptosis are essential for understanding the mechanisms for oocyte maturation. Genes expressed in CCs might be used as markers for competent oocytes and/or embryos. In this study, both in vitro (IVT) and in vivo (IVO) mouse oocyte models with significant difference in cumulus expansion and CC apoptosis were used to identify and validate new genes regulating cumulus expansion and CC apoptosis of mouse oocytes. We first performed mRNA sequencing and bioinformatic analysis using the IVT oocyte model to identify candidate genes. We then analyzed functions of the candidate genes by RNAi or gene overexpression to select the candidate cumulus expansion and CC apoptosis-regulating genes. Finally, we validated the cumulus expansion and CC apoptosis-regulating genes using the IVO oocyte model. The results showed that while Spp1, Sdc1, Ldlr, Ezr and Mmp2 promoted, Bmp2, Angpt2, Edn1, Itgb8, Cxcl10 and Agt inhibited cumulus expansion. Furthermore, Spp1, Sdc1 and Ldlr inhibited CC apoptosis. In conclusion, by using both IVT and IVO oocyte models, we have identified and validated a new group of cumulus expansion and/or apoptosis-regulating genes, which may be used for selection of quality oocytes/embryos and for elucidating the molecular mechanisms behind oocyte maturation.

Transcriptome Dynamics and Cell Dialogs Between Oocytes and Granulosa Cells in Mouse Follicle Development.

The development and maturation of follicles is a sophisticated and multistage process. The dynamic gene expression of oocytes and their surrounding somatic cells and the dialogs between these cells are critical to this process. In this study, we accurately classified the oocyte and follicle development into nine stages and profiled the gene expression of mouse oocytes and their surrounding granulosa cells and cumulus cells. The clustering of the transcriptomes showed the trajectories of two distinct development courses of oocytes and their surrounding somatic cells. Gene expression changes precipitously increased at Type 4 stage and drastically dropped afterward within both oocytes and granulosa cells. Moreover, the number of differentially expressed genes between oocytes and granulosa cells dramatically increased at Type 4 stage, most of which persistently passed on to the later stages. Strikingly, cell communications within and between oocytes and granulosa cells became active from Type 4 stage onward. Cell dialogs connected oocytes and granulosa cells in both unidirectional and bidirectional manners. TGFB2/3, TGFBR2/3, INHBA/B, and ACVR1/1B/2B of TGF-ß signaling pathway functioned in the follicle development. NOTCH signaling pathway regulated the development of granulosa cells. Additionally, many maternally DNA methylation- or H3K27me3-imprinted genes remained active in granulosa cells but silent in oocytes during oogenesis. Collectively, Type 4 stage is the key turning point when significant transcription changes diverge the fate of oocytes and granulosa cells, and the cell dialogs become active to assure follicle development. These findings shed new insights on the transcriptome dynamics and cell dialogs facilitating the development and maturation of oocytes and follicles.

Tolerance thresholds underlie responses to DNA damage during germline development.

Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we found that in P-M hybrid dysgenesis in Drosophila, a sterility syndrome triggered by the P-element DNA transposon, germ cells harbor unexpectedly few new TE insertions despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P-elements or other noncoding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development.

STAT transcription factor is indispensable for oogenesis in silkworm.

Signal Transducer and Activator of Transcription (STAT) proteins represent a critical transcription factor family with multifaceted roles in diverse fundamental eukaryotic processes. In Drosophila, STAT exerts a pivotal regulatory influence on oogenesis, governing the early differentiation of follicular cells and ensuring proper encapsulation of germ-line cells. However, the role of STAT in egg development in silkworms remains unknown. In the present study, using CRISPR/Cas9 technology, we successfully generated a strain of silkworms with targeted deletion of the STAT-L gene, which resulted in significant reproductive abnormalities observed in female moths, including shortened fallopian tubes and reduced egg production. The ovaries dissected from STAT-L knockout silkworms during the pupal stage of silkworm exhibited varying degrees of fusion among egg chambers. Additionally, paraffin sections of prepupal ovaries also revealed evidence of egg chambers fusion. To elucidate the molecular mechanism underlying the role of the STAT-L gene regulation on egg development in silkworm, we performed ovarian transcriptomic analysis following STAT-L knockout. Our findings indicated that STAT-L gene can modulate Notch signaling pathway by down-regulating APH-1 gene expression. These results suggest that STAT-L gene plays a crucial role in normal egg chamber formation in silkworms, potentially through its influence on Notch signaling pathway expression.

Regulation of Oocyte mRNA Metabolism: A Key Determinant of Oocyte Developmental Competence.

The regulation of mRNA transcription and translation is uncoupled during oogenesis. The reason for this uncoupling is two-fold. Chromatin is only accessible to the transcriptional machinery during the growth phase as it condenses prior to resumption of meiosis to ensure faithful segregation of chromosomes during meiotic maturation. Thus, transcription rates are high during this time period in order to produce all of the transcripts needed for meiosis, fertilization, and embryo cleavage until the newly formed embryonic genome becomes transcriptionally active. To ensure appropriate timing of key developmental milestones including chromatin condensation, resumption of meiosis, segregation of chromosomes, and polar body extrusion, the translation of protein from transcripts synthesized during oocyte growth must be temporally regulated. This is achieved by the regulation of mRNA interaction with RNA binding proteins and shortening and lengthening of the poly(A) tail. This chapter details the essential factors that regulate the dynamic changes in mRNA synthesis, storage, translation, and degradation during oocyte growth and maturation.

Meiotic maturation failure in primary ovarian insufficiency: insights from a bovine model.

PURPOSE: Oocytes from women presenting primary ovarian insufficiency (POI) generate viable embryos at a lower rate than non-POI women, but the mechanisms responsible for the lower oocyte quality remain elusive. Due to the scarcity of human oocytes for research, animal models provide a promising way forward. We aimed at investigating the molecular events characterizing final maturation in POI oocytes in a well-defined POI-like bovine model. METHODS: Single-cell RNA-sequencing of bovine control and POI-like, GV, and MII oocytes (n = 5 per group) was performed. DEseq2 was used to identify differentially expressed genes. Further, a Gene set enrichment analysis and a transcriptomic meta-analysis between bovine and human oocytes were performed. RESULTS: In control cows, we found 2223 differentially expressed genes between the GV and MII stages. Specifically, the affected genes were related to RNA processing and transport, protein synthesis, organelle remodeling and reorganization, and metabolism. The meta-analysis with a set of young human oocytes at different maturation stages revealed 315 conserved genes through the GV-MII transition in cows and humans, mostly related to meiotic progression and cell cycle. Gene expression analysis between GV and MII of POI-like oocytes showed no differences in terms of differentially expressed genes, pointing towards a substantial failure to properly remodel the transcriptome in the POI model, and with the clustering analysis indicating that the cow's genetic background had a higher impact than the oocyte's maturation stage. CONCLUSION: Overall, we have identified and characterized a valuable animal model of POI, paving the way to identifying new molecular mechanisms involved in POI.

Novel PATL2 variants cause female infertility with oocyte maturation defect.

PURPOSE: Oocyte maturation defect (OOMD) is a rare cause of in vitro fertilization failure characterized by the production of immature oocytes. Compound heterozygous or homozygous PATL2 mutations have been associated with oocyte arrest at the germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as morphological changes. METHODS: In this study, we recruited three OOMD cases and conducted a comprehensive multiplatform laboratory investigation. RESULTS: Whole exome sequence (WES) revealed four diagnostic variants in PATL2, nonsense mutation c.709C > T (p.R237*) and frameshift mutation c.1486_1487delinsT (p.A496Sfs*4) were novel mutations that have not been reported previously. Furthermore, the pathogenicity of these variants was predicted using in silico analysis, which indicated detrimental effects. Molecular dynamic analysis suggested that the A496S variant disrupted the hydrophobic segment, leading to structural changes that affected the overall protein folding and stability. Additionally, biochemical and molecular experiments were conducted on cells transfected with wild-type (WT) or mutant PATL2 (p.R237* and p.A496Sfs*4) plasmid vectors. CONCLUSIONS: The results demonstrated that PATL2A496Sfs*4 and PATL2R237* had impacts on protein size and expression level. Interestingly, expression levels of specific genes involved in oocyte maturation and early embryonic development were found to be simultaneously deregulated. The findings in our study expand the variation spectrum of the PATL2 gene, provide solid evidence for counseling on future pregnancies in affected families, strongly support the application of in the diagnosis of OOMD, and contribute to the understanding of PATL2 function.

Maternal regulation of the vertebrate oocyte-to-embryo transition.

Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the acquisition of oocyte and egg developmental competence to ensure the production of viable embryos. However, their molecular nature and functional importance remain poorly understood. Here, we present a collection of 9 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oocyte-to-embryo transition. Four genes, over easy, p33bjta, poached and black caviar, were found to control initial steps in yolk globule sizing and protein cleavage during oocyte maturation that act independently of nuclear maturation. The krang, kazukuram, p28tabj, and spotty genes play distinct roles in egg activation, including cortical granule biology, cytoplasmic segregation, the regulation of microtubule organizing center assembly and microtubule nucleation, and establishing the basic body plan. Furthermore, we cloned two of the mutant genes, identifying the over easy gene as a subunit of the Adaptor Protein complex 5, Ap5m1, which implicates it in regulating intracellular trafficking and yolk vesicle formation. The novel maternal protein Krang/Kiaa0513, highly conserved in metazoans, was discovered and linked to the function of cortical granules during egg activation. These mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-embryo transition, fertility, and human disease. Additionally, our genetic adult screen not only contributes to the existing knowledge in the field but also sets the basis for future investigations. Thus, the identified maternal genes represent key players in the coordination and execution of events prior to fertilization.