bims-cebooc 2025-11-30 papers

bims-cebooc

Biomed News

on Cell biology of oocytes

Issue of 2025–11–30
twenty-two papers selected by
Gabriele Zaffagnini, Universität zu Köln

An mTOR-Stat3-Stathmin pathway controls centrosome and microtubule dynamics for oocyte polarization.
Spatiotemporal Crosstalk Between Oocyte and the Microenvironment Governs Preovulatory Follicle Aging.
MEIOC prevents continued mitotic cycling and promotes meiotic entry during mouse oogenesis.
Acetyl-CoA carboxylase maintains energetic balance for functional oogenesis.
Lysosomal control of proteostasis and reproductive capacity by conserved LMD-3 protein in C. elegans.
Analysis of Bush Cricket Oogenesis Provides an Insight into the Function and Anagenesis of an Enigmatic Organelle Assemblage: The Balbiani Body.
Apoptosis promotes fertility in C. elegans by maintaining functional germline morphology.
A proximity labeling approach to identify proteins that associate with synaptonemal complex components in Drosophila melanogaster females.
BAF-1-VRK-1 mediated release of meiotic chromosomes from the nuclear periphery is important for genome integrity.
The SPN-4 Rbfox RNA-binding protein selects maternal mRNAs for CCR4-NOT-dependent clearance in early Caenorhabditis elegans embryos.
Dynamic Remodeling of the Zona Pellucida: Implications for Oocyte Competence and Assisted Reproduction.
Newly Evolved Endogenous Retroviruses Prime the Ovarian Reserve for Activation.
TRIP13 fosters both transcriptional silencing and DSB repair during meiosis.
Leveraging CRISPR-Cas13d in an inducible knockdown system to interrogate Drosophila germ granule mRNAs.
Pause and play: insights into mammalian embryonic diapause.
Cell size reduction scales spindle elongation but not chromosome segregation in C. elegans.
The Everlasting Ovary: Decoding the Mechanisms of Lifelong Oogenesis in the Naked Mole-Rat.
Multi-step implementation of meiotic crossover patterning.
Spatial Partitioning of Core Glycolysis Enables Tissue-Specific Metabolic Programs In Vivo.
Lipid Nanoparticle-mediated mRNA delivery system into preimplantation embryos.
Transcription start sites experience a high influx of heritable variants fueled by early development.
In Vitro Fertilization Accelerates Female Reproductive Aging Through Early Ovarian Failure.

Curr Biol. 2025 Nov 26. pii: S0960-9822(25)01466-6. [Epub ahead of print]

An mTOR-Stat3-Stathmin pathway controls centrosome and microtubule dynamics for oocyte polarization.

Amal Shawahny, Yoel Bogoch, Neta Hart, Yaniv M Elkouby.

  In animals and plants, egg production is essential for fertility, reproduction, and embryonic development. However, our understanding of the regulatory mechanisms underlying the cellular and developmental programs of oogenesis is lacking. Here, we aimed to identify overlooked regulators of early oogenesis in zebrafish. First, we established a long-term ovary culture system that enables physiological oocyte development from oogonia to primordial follicles, providing an effective ex vivo platform for rapid investigation. Next, we utilized this system for functional screening of candidates from stage-specific oocyte transcriptomic data. We identified mammalian target of rapamycin (mTOR), signal transducer and activator of transcription 3 (Stat3), and the microtubule-destabilizing protein Stathmin as novel regulators of oocyte polarity. In zebrafish and most species, oocyte polarity is essential for oogenesis and embryonic development. In polarization, microtubules control the localization of the polarity regulator Buc to the centrosome, as well as its condensation into the Balbiani body, an oocyte membraneless compartment. Through a combination of genetics and pharmacological manipulations in vivo and ex vivo, we show that loss of mTOR or Stat3 functions, as well as overactivation of Stathmin, disrupted centrosome regulation and destabilized microtubules, leading to dispersed Buc condensates and loss of polarity. We demonstrate that mTOR acts upstream of Stat3 in oocytes, and inhibition of Stathmin in either stat3-/- or mTOR-deficient ovaries rescued both cytoskeletal and polarity defects. Thus, we established an unpredicted but essential mTOR-Stat3-Stathmin pathway that controls centrosome and microtubule dynamics to drive oocyte polarity. We propose that through cytoskeletal regulation, this pathway provides oocytes with polarization competence, a likely common step in cell polarity.

Keywords:  Balbiani body; Buc molecular condensation; Bucky ball molecular condensation; Stat3; Stathmin; aebrafish oogenesis and ovary development; cell polarity; centrosome MTOC; mTOR; microtubules

DOI:  https://doi.org/10.1016/j.cub.2025.10.085
Aging Cell. 2025 Nov 23. e70302

Spatiotemporal Crosstalk Between Oocyte and the Microenvironment Governs Preovulatory Follicle Aging.

Xin Yi Koh, Kah Junn Tan, Zeng Hao Lim, Shi Chee Ong, Sophie Emma Tan, Jing Xuan Lu, Zhongwei Huang, Jun Wei Pek.

  Preovulatory follicle aging is the period between formation and ovulation of a mature follicle. Previous studies had shown that mammalian preovulatory follicle aging is associated with chromosomal abnormalities and developmental defects such as decreased implantation, increased malformation and mortality and lower embryonic weight. Our understanding of the molecular events governing this process has been hampered by the difficulty in accessing them in vivo under natural conditions. We hypothesize that the quality of the mature oocyte is regulated by crosstalk between the oocyte and the somatic microenvironment during extended storage prior to ovulation. By combining temporal profiling and tissue-specific functional analyzes in Drosophila, we characterize a spatiotemporal crosstalk between the oocyte and the granulosa cells that governs preovulatory follicle aging in vivo. Preovulatory follicle aging is characterized by two distinct phases-early oocyte protective and late degenerative phases. The degenerative phase involves a positive feedback loop between oocyte mitochondrial dysfunction mediated by a mitochondrial-localized microprotein PIGBOS, and granulosa cell functional decline through a circular RNA circdlg1. Activation of the feedback loop is suppressed by germline Sestrin during the early phase. Our findings highlight that natural preovulatory follicle aging in vivo is governed by a mechanism that represses an oocyte-degenerative positive feedback loop between oocyte and granulosa cells.

Keywords:   drosophila ; sestrin ; PIGBOS; Preovulatory oocyte; aging; circRNA; granulosa cell; mitochondria

DOI:  https://doi.org/10.1111/acel.70302
Development. 2025 Nov 25. pii: dev.205037. [Epub ahead of print]

MEIOC prevents continued mitotic cycling and promotes meiotic entry during mouse oogenesis.

Esther G Ushuhuda, Jenniluyn T Nguyen, Natalie G Pfaltzgraff, Shelbie M Wenner, Matthew Kofron, Maria M Mikedis.

  To generate haploid gametes, germ cells must transition from mitosis to meiosis. In mammals, transcriptional activator STRA8-MEIOSIN mediates the decision to enter the meiotic cell cycle, but how germ cells prevent continued mitotic cycling before meiotic entry remains unclear. MEIOC was previously shown to repress the mitotic program after meiotic entry. Here, we investigate MEIOC's role in the mitosis-to-meiosis transition during mouse oogenesis. Using cell proliferation analysis and cell cycle transcriptomics, we demonstrate that MEIOC prevents continued mitotic cycling prior to meiotic entry in oogenic cells. We find that G1/S cyclin CCNA2 is downregulated during the mitosis-to-meiosis transition, and MEIOC contributes to this downregulation. MEIOC also promotes entry into meiosis by increasing Meiosin transcript abundance and consequently activating STRA8-MEIOSIN. We also demonstrate that BMP signaling halts mitotic cycling and promotes meiotic entry by upregulating MEIOC. Thus, in mouse oogenic cells, the transition from mitosis to meiosis occurs as two molecularly regulated steps- (i) halt of mitotic cycling and (ii) entry into the meiotic cell cycle - and that MEIOC modifies the cell cycle program to facilitate both steps in this transition.

Keywords:  Fetal oocytes; MEIOC; MEIOSIN; Meiosis; Ovarian germ cell; STRA8

DOI:  https://doi.org/10.1242/dev.205037
Nat Commun. 2025 Nov 27. 16(1): 10677

Acetyl-CoA carboxylase maintains energetic balance for functional oogenesis.

Oyundari Amartuvshin, Chi-Hung Lin, Yi-Ting Ke, Han-Jung Lee, Kreeti Kajal, Tsai-Ling Huang, Wen-Der Wang, Tsai-Ming Lu, Ling-Huei Yih, Chen-Yuan Tseng, Hwei-Jan Hsu.

Reproduction is tightly linked to nutrient availability and metabolic homeostasis, yet how specific metabolic pathways coordinate with cellular signaling to control oogenesis remains unclear. Through a targeted RNAi screen in the Drosophila germline, we identify Acetyl-CoA Carboxylase (Acc), the rate-limiting enzyme in fatty acid synthesis (FAS), as an essential regulator of germline stem cell (GSC) maintenance and oocyte development. Acc loss shifts cellular metabolism toward fatty acid oxidation (FAO), fueling the TCA cycle and electron transport chain, which elevates ATP levels and hyperactivates TOR signaling. This metabolic reprogramming induces excessive protein synthesis, disrupting endosomal trafficking and fusome branching, a germline-specific organelle essential for synchronized cell divisions and oocyte selection. These defects are rescued by inhibiting FAO, suppressing TOR activity, reducing protein synthesis, or restricting dietary protein intake. Our study establishes a direct metabolic-signaling-structural axis in the female germline and highlights Acc as a key metabolic checkpoint that safeguards energy balance, intracellular trafficking, and oocyte fate.

DOI: https://doi.org/10.1038/s41467-025-65708-w
Sci Adv. 2025 Nov 28. 11(48): eadz3192

Lysosomal control of proteostasis and reproductive capacity by conserved LMD-3 protein in C. elegans.

Yile Zhai, Tiantian Wang, Jiangxue Han, Mengxiao Wu, Meixi Gong, Wenfei Li, Zhe Zhang.

Human female fertility declines markedly with age, a pattern mirrored in C. elegans fecundity. This shared vulnerability stems from evolutionarily conserved molecular pathways. A growing body of evidence links impaired proteostasis-the cell's ability to manage its proteins-to this age-related fertility drop in both species, although the underlying mechanisms are not fully understood. Here, we identify that LMD-3, a LysM domain protein, is a critical regulator of proteostasis and reproductive capacity in C. elegans. Deficiency of lmd-3 leads to notable defects in oxidation resistance and constitutively high cellular stress responses. We demonstrate that LMD-3, localized to the lysosome, interacts with vitellogenin and a proton-pumping V-type ATPase via its TLDc domain to regulate lysosomal function and maintain yolk protein homeostasis. We also found that supplementing with vitamin B12 can restore fertility in lmd-3 mutants by reducing oxidative stress and improving lysosomal function. These findings establish a model for studying reproductive health and finding potential therapeutic strategies.

DOI: https://doi.org/10.1126/sciadv.adz3192
Results Probl Cell Differ. 2026 ;76 167-182

Analysis of Bush Cricket Oogenesis Provides an Insight into the Function and Anagenesis of an Enigmatic Organelle Assemblage: The Balbiani Body.

Szczepan M Bilinski, Malgorzata Sekula, Magda Sochaczewska, Waclaw Tworzydlo.

Balbiani body (Bb) is an intricate, oocyte-specific organelle complex described in the ooplasm of nearly all examined vertebrates and invertebrates. The Bb is devoid of a limiting membrane and consists of such organelles as elements of endoplasmic reticulum (ER), Golgi complexes (GCs), mitochondria, and characteristic accumulations of fibrillo-granular material, termed the "nuage." Despite numerous studies, the functioning of the Bb remains not completely understood. Until now at least four disparate functions have been attributed to the Bb: (1) delivery of germinal granules and localized mRNAs to the oocyte vegetal cortex; (2) transfer of mitochondria to the polar (germ) plasm; (3) selective elimination of dysfunctional (damaged, containing mutated mitochondrial DNA (mtDNA)) mitochondrial units in female germline cells; and (4) formation of oocyte reserve materials, e.g., lipid droplets. Such functional variability obviously raises several intriguing questions for students of developmental and cellular biology. Here we present the results of our studies on oogenesis of bush crickets that advance our understanding of the Bb function and anagenesis of this organelle complex during the evolution of bilateral animals.

DOI: https://doi.org/10.1007/978-3-032-06766-1_9
bioRxiv. 2025 Oct 23. pii: 2025.10.22.683972. [Epub ahead of print]

Apoptosis promotes fertility in C. elegans by maintaining functional germline morphology.

U N Saydee-Onwubiko, M E Werner, G C Trapp, A S Maddox.

Programmed cell death (apoptosis) during oogenesis is conserved across metazoans and linked to regulation of oocyte number and quality. In oogenic germlines, the removal of developing oocytes by apoptosis ensures that oocytes do not contain DNA damage or multiple nuclei. Beyond this chromatin-quality control assurance role, it was unknown how apoptosis contributes to oocyte quality. We used the nematode Caenorhabditis elegans ( C. elegans ) to study the consequences of loss of apoptosis on oogenesis. Blocking apoptosis reduced fecundity in hermaphrodites at peak fertility and caused germline architectural defects such as abnormal rachis morphology and perturbed the arrangement and distribution of oogenic germline compartments. We posit that the defects associated with the loss of germline apoptosis arise due to lack of sufficient space for normal oogonia growth. In support of this idea, oocytes and embryos are abnormally small and exhibit low viability in animals unable to execute apoptosis. These findings suggest that in addition to preventing ploidy defects during oogenesis, apoptosis contributes to fertility by preserving homeostatic germline structure required for the fidelity of oogenesis.
Summary Statement: Blocking apoptosis during oogenesis disrupts germline structure over time, causing morphological defects and infertility, highlighting the role of apoptosis in maintaining syncytial architecture and reproductive function.

DOI: https://doi.org/10.1101/2025.10.22.683972
bioRxiv. 2025 Oct 15. pii: 2025.10.14.682398. [Epub ahead of print]

A proximity labeling approach to identify proteins that associate with synaptonemal complex components in Drosophila melanogaster females.

Stacie E Hughes, Christopher Viermann, Morgan James, Charles Banks, R Scott Hawley.

  Organisms use a specialized cell division called meiosis for the creation of haploid gametes. Multiple carefully orchestrated steps must occur at specific times and places for meiosis to be successful, including chromosome pairing, meiotic entry, recombination, synapsis, and two rounds of chromosome segregation. The regulation and molecular mechanisms for many of the steps of meiosis have not been fully elucidated. During synapsis, the synaptonemal complex (SC) builds along the entire lengths of the homologs to maintain the pairing of the homologs and promote the formation of the crossovers that help ensure proper segregation of homologs at the meiosis I division in many organisms. The SC is a large tripartite structure that is believed to function as a biomolecular condensate. To attempt to identify proteins that interact with SC components during female meiosis in Drosophila melanogaster, a protein of the lateral element, C(2)M, and a protein of the central element, Cona, were tagged with the APEX2 enzyme, which can biotinylate nearby proteins under the appropriate conditions. Under biotinylating promoting conditions, biotin labeled proteins were observed to be associated with the SC by immunofluorescence. Biotinylated proteins were isolated for mass spectrometry analysis, and multiple proteins were found to be enriched compared to control samples. RNAi knockdown lines targeting a subset of enriched proteins were examined for phenotypes in early Drosophila female meiosis. RNAi knockdown of Cpsf5, an mRNA cleavage factor, caused delayed or defective SC formation, as well as additional meiotic defects, indicating a role for maturation of mRNA in regulating processes of female meiosis. These results support proximity labeling as a strategy for identifying additional meiotic proteins.

Keywords:  meiosis; recombination; synaptonemal complex

DOI:  https://doi.org/10.1101/2025.10.14.682398
Nat Commun. 2025 Nov 25. 16(1): 10446

BAF-1-VRK-1 mediated release of meiotic chromosomes from the nuclear periphery is important for genome integrity.

Dimitra Paouneskou, Antoine Baudrimont, Réka Kelemen, Marwan Elkrewi, Angela Graf, Shehab Moukbel Ali Aldawla, Claudia Kölbl, Irene Tiemann-Boege, Beatriz Vicoso, Verena Jantsch.

Rapid prophase chromosome movements ensure faithful alignment of the parental homologous chromosomes and successful synapsis formation during meiosis. These movements are driven by cytoplasmic forces transmitted to the nuclear periphery, where chromosome ends are attached through transmembrane proteins. During many developmental stages a specific genome architecture with chromatin nuclear periphery contacts mediates specific gene expression. Whether chromatin is removed from the nuclear periphery as a consequence of chromosome motions or by a specific mechanism is not fully understood. Here, we identify a mechanism to remove chromatin from the nuclear periphery through vaccinia related kinase (VRK-1)-dependent phosphorylation of Barrier to Autointegration Factor 1 (BAF-1) in Caenorhabditis elegans early prophase of meiosis. Interfering with chromatin removal delays chromosome pairing, impairs synapsis, produces oocytes with abnormal chromosomes and elevated apoptosis. Long read sequencing reveals deletions and duplications in offspring lacking VRK-1 underscoring the importance of the BAF-1-VRK-1 module in preserving genome stability in gametes during rapid chromosome movements.

DOI: https://doi.org/10.1038/s41467-025-65420-9
bioRxiv. 2025 Oct 15. pii: 2025.10.15.682621. [Epub ahead of print]

The SPN-4 Rbfox RNA-binding protein selects maternal mRNAs for CCR4-NOT-dependent clearance in early Caenorhabditis elegans embryos.

Caroline A Spike, Dylan M Parker, Tatsuya Tsukamoto, Naly Torres-Mangual, Erika C Tsukamoto, Karissa Coleman, Micah D Gearhart, David Greenstein, Erin Osborne Nishimura.

The elimination of maternal mRNAs is an essential feature of the maternal-to-zygotic transition. We report an essential pathway that clears many maternal transcripts from early C. elegans embryos using the Rbfox-related SPN-4 RNA-binding protein as a specificity factor and the CCR4-NOT deadenylase complex as an effector. We biochemically identified SPN-4-associated mRNAs from late-stage oocytes and found that the set of SPN-4-associated transcripts is enriched for maternal mRNAs that undergo early decay. Single-molecule fluorescence in situ hybridization experiments established that many SPN-4-associated mRNAs fail to be eliminated in the absence of SPN-4. In the 3'UTRs of two target mRNAs, we identified Rbfox motifs that are required for SPN-4-dependent clearance in vivo and bind SPN-4 in vitro . In a genetic screen to identify factors that work with SPN-4, we isolated mutant alleles of CCR4-NOT components. Auxin-induced degradation of the LET-711/NOT1 scaffold and the CCF-1 deadenylase disrupted clearance of two SPN-4-associated transcripts. Our results support a model in which SPN-4 initiates expression in late-stage oocytes, associates with maternal mRNA targets through RNA sequences in their 3'UTRs and promotes CCR4-NOT-mediated decay during early embryogenesis.

DOI: https://doi.org/10.1101/2025.10.15.682621
Int J Mol Sci. 2025 Nov 17. pii: 11108. [Epub ahead of print]26(22):

Dynamic Remodeling of the Zona Pellucida: Implications for Oocyte Competence and Assisted Reproduction.

Daniel de la Fuente, Michela Prestianni, Paula Navarrete-López, Cristina García-Merino, Miriam Balastegui-Alarcón, Pilar Soria, Manuel Avilés, Dimitrios Rizos, Alfonso Gutiérrez-Adan.

  The zona pellucida (ZP) is a glycoprotein-rich extracellular matrix essential for fertilization, early embryonic development, and implantation. Beyond its core functions, the ZP undergoes dynamic remodeling during oocyte maturation, involving regulated synthesis, assembly, and conformational changes. This complex and tightly controlled process ensures the biomechanical integrity of the oocyte, providing both protection and selective permeability essential for reproductive success. Oviductal glycoprotein 1 (OVGP1; oviductin) integrates into the ZP, modulating pore size, glycan composition, and structural homogeneity. This glycoprotein establishes a species-specific barrier that prevents polyspermy and fine-tunes sperm-oocyte interactions. Recent evidence suggests that OVGP1 not only contributes to ZP stabilization but also influences sperm capacitation, acrosome reaction, and early zygotic signaling, thereby linking oviductal physiology to gamete compatibility. Exploiting ZP remodeling in assisted reproductive technologies (ART) offers new opportunities to enhance fertilization efficiency, embryo quality, and implantation success, including through assisted hatching or modulating ZP properties to overcome fertility limitations. Moreover, advances in proteomic and glycomic profiling of the ZP are enabling the identification of novel biomarkers of oocyte competence and reproductive potential. These advances provide critical insights into species-specific reproductive mechanisms and pave the way for improved ART protocols and fertility preservation strategies.

Keywords:  assisted reproductive techniques; fertilization; hatching; maturation; ovastacin; oviductin; sperm binding; zona pellucida glycoproteins

DOI:  https://doi.org/10.3390/ijms262211108
bioRxiv. 2025 Nov 12. pii: 2025.11.10.687753. [Epub ahead of print]

Newly Evolved Endogenous Retroviruses Prime the Ovarian Reserve for Activation.

Yasuhisa Munakata, Mengwen Hu, Raissa G Dani, Naokazu Inoue, Richard M Schultz, Satoshi H Namekawa.

Female mammals are born with a finite pool of non-growing oocytes (NGOs) housed in primordial follicles, which form the ovarian reserve that determines reproductive lifespan. Mechanisms underlying the reserve's long-term maintenance and subsequent follicular activation remain elusive. Using total RNA sequencing and de novo transcriptome assembly, we first captured the comprehensive oocyte transcriptome across perinatal oogenesis in mice. We show that NGOs establish accessible chromatin at gene regulatory elements-including promoters and enhancers-partly driven by newly evolved endogenous retroviruses (ERVs). In NGOs, epigenetic priming for follicular activation involves prior loading of transcription factors TCF3 and TCF12 and non-phosphorylated form of RNA polymerase II at these sites. This primed state is counteracted by repression via Polycomb Repressive Complex 1-mediated H2AK119 ubiquitylation. We propose that ERV-mediated epigenetic priming underlies the ovarian reserve's long-term maintenance and establishes a transcriptionally competent yet repressive configuration that enables rapid gene activation upon oocyte growth.

DOI: https://doi.org/10.1101/2025.11.10.687753
bioRxiv. 2025 Oct 20. pii: 2025.10.20.683374. [Epub ahead of print]

TRIP13 fosters both transcriptional silencing and DSB repair during meiosis.

Marina Marcet-Ortega, Ana Martínez-Marchal, Andros Maldonado-Linares, Mercè Encinas, Anna Esteve Codina, Maria Jasin, Scott Keeney, Ignasi Roig.

During meiosis, the coordinated suppression of global gene transcription and induction of double-strand breaks (DSBs) genome wide is crucial for successful gamete formation. The AAA+ ATPase TRIP13 is a master meiotic regulator involved in numerous cellular processes, including DSB repair, organization of the meiotic chromosome axis, and meiotic silencing of unsynapsed axes. Here, we examine the involvement of TRIP13 in transcription suppression and DSB repair during meiotic prophase I in mice. Because TRIP13 is required for silencing of sex chromosomes at pachynema, we hypothesized that it may also be involved in the global transcription suppression that begins at the onset of meiotic prophase. Indeed, as predicted, we observed increased incorporation of 5-ethynyl uridine in early stages of meiotic prophase I and a higher presence of the active form of RNA polymerase II in Trip13 mutant spermatocytes. RNA sequencing data revealed that more than two-thirds of upregulated transcripts came from the sex chromosomes. Given this more general role in transcription silencing, we also tested whether TRIP13 promotes the transcription block that causes meiotic arrest in synapsis-deficient oocytes. However, Trip13 ablation did not rescue Spo11 -/- oocytes, which experience asynapsis without DSB repair defects. Instead, Trip13 mutation did increase the number of oocytes in Dmc1 -/- Chk2 -/- mice, in which DSBs persist unrepaired. This rescue seems attributable to a previously unknown function of TRIP13 in preventing DSB repair by non-canonical pathways, as we observed fewer homologous recombination markers along with an increased presence of non-homologous end joining markers in the triple mutant cells. These findings underscore TRIP13's multifaceted role in transcription and DSB repair regulation during meiosis, highlighting its importance and potential for further meiotic regulation and fertility research.

DOI: https://doi.org/10.1101/2025.10.20.683374
bioRxiv. 2025 Oct 30. pii: 2024.09.09.611872. [Epub ahead of print]

Leveraging CRISPR-Cas13d in an inducible knockdown system to interrogate Drosophila germ granule mRNAs.

Zoya A Gauhar, Aaron J Duthoy, Seema Chatterjee, Rodrigo Berber-Pulido, Cameron Myhrvold, Elizabeth R Gavis.

Ribonucleoprotein (RNP) germ granules are hallmarks of germ cells across the animal kingdom and are thought to be hubs for post-transcriptional regulation that promote formation of the germ cell precursors. While numerous RNAs are associated with germ granules in Drosophila , the functions of many in germline development are poorly understood. Current methods for RNA knockdown, such as RNAi, do not allow local depletion of transcripts such as those found in the germ granules. We leveraged CRISPR-Cas13 to create a subcellular RNA knockdown system and tested it on two mRNAs, nanos (nanos) and sarah (sra) , whose abundance in germ granules differs. Because Cas13 has both cis and trans cleavage activities, we evaluated the effect of target abundance on off-target RNA depletion. We show on and off-target RNA depletion is coupled when targeting the more abundant nanos germ granule transcripts. Off-target RNA knockdown is less potent when the system is used for less abundant sra transcripts. When sra is knocked down in germ granules, we observed an increase in the calcium indicator GCaMP at the posterior and defective primordial germ cell migration, consistent with sra encoding a negative regulator of calcium signaling. In sum, we report an in vivo Cas13-based system for subcellular knockdown, evaluate its feasibility, and uncover a novel function for sra germ granule transcripts in promoting germline development.

DOI: https://doi.org/10.1101/2024.09.09.611872
Development. 2025 Nov 15. pii: dev205121. [Epub ahead of print]152(22):

Pause and play: insights into mammalian embryonic diapause.

Rui Chen, Ivan Bedzhov.

  Typically, embryonic development is a continuous process. However, in some species, embryos can halt their development and enter a dormant state known as diapause. During this period, the embryo retains its viability and developmental capacity to resume transient embryogenesis. While the diapausing embryo appears to be in a state of suspended animation, recent studies have revealed a more dynamic picture of modulated signalling responses, metabolic rewiring and slow-paced but active tissue morphogenesis. In this Spotlight, we discuss the emerging concepts of the molecular and cellular mechanisms that govern mammalian embryonic diapause, focusing on the mouse as a model system.

Keywords:  Blastocyst; Diapause; Dormancy; Embryo; Quiescence

DOI:  https://doi.org/10.1242/dev.205121
bioRxiv. 2025 Oct 14. pii: 2025.10.13.681585. [Epub ahead of print]

Cell size reduction scales spindle elongation but not chromosome segregation in C. elegans.

Chukwuebuka William Okafornta, Reza Farhadifar, Gunar Fabig, Hai-Yin Wu, Maria Köckert, Martin Vogel, Daniel Baum, Robert Haase, Michael J Shelley, Daniel J Needleman, Thomas Müller-Reichert.

How embryos adapt their internal cellular machinery to reductions in cell size during development remains a fundamental question in cell biology 1-11 . Here, we use high-resolution lattice light-sheet fluorescence microscopy and automated image analysis to quantify lineage-resolved mitotic spindle and chromosome segregation dynamics from the 2-to 64-cell stages in Caenorhabditis elegans embryos. While spindle length scales with cell size across both wild-type and size-perturbed embryos, chromosome segregation dynamics remain largely invariant, suggesting that distinct mechanisms govern these mitotic processes. Combining femtosecond laser ablation 12,13 with large-scale electron tomography 14 , we find that central spindle microtubules mediate chromosome segregation dynamics and remain uncoupled from cell size across all stages of early development. In contrast, spindle elongation is driven by cortically anchored motor proteins and astral microtubules, rendering it sensitive to cell size 12,13,15-17 . Incorporating these experimental results into an extended stoichiometric model for both the spindle and chromosomes, we find that allowing only cell size and microtubule catastrophe rates to vary reproduces elongation dynamics across development. The same model also accounts for centrosome separation and pronuclear positioning in the one-cell C. elegans embryo 18 , spindle-length scaling across nematode species spanning ~100 million years of divergence 17 , and spindle rotation in human cells 19 . Thus, a unified stoichiometric framework provides a predictive, mechanistic account of spindle and nuclear dynamics across scales and species.

DOI: https://doi.org/10.1101/2025.10.13.681585
Results Probl Cell Differ. 2026 ;76 47-63

The Everlasting Ovary: Decoding the Mechanisms of Lifelong Oogenesis in the Naked Mole-Rat.

Odei Barreñada, Andrew C Pearson, Miguel A Brieño-Enríquez.

The naked mole-rat (Heterocephalus glaber) is a fascinating model organism which challenges conventional paradigms in evolutionary developmental biology. As one of the two known eusocial mammals with a reproductive hierarchy akin to social insects, the naked mole-rat presents an exceptional system for studying the interplay between social structure, environmental adaptation, and developmental plasticity. This chapter explores how the species' unique reproductive strategies-including lifelong fertility, postnatal oogenesis, and social suppression of reproduction-reshape our understanding of mammalian reproductive aging. The queen, the sole breeding female within a colony, maintains an exceptionally large ovarian reserve throughout life, defying the prevailing dogma of a fixed oocyte pool and progressive depletion. Unlike other mammals, germ cells in the naked mole-rat continue to proliferate postnatally, offering unprecedented insights into the regulation of ovarian function and reproductive longevity. Additionally, the integration of genomic, epigenetic, and neuroendocrine mechanisms underlying eusociality provides a rare perspective on how developmental processes can be shaped by cooperative behaviors and environmental constraints. By situating these traits within an evo-devo framework, this chapter underscores the naked mole-rat's potential to advance research in several fields such as aging, reproductive biology, and the evolution of complex social systems.

DOI: https://doi.org/10.1007/978-3-032-06766-1_4
bioRxiv. 2025 Nov 13. pii: 2025.11.12.687980. [Epub ahead of print]

Multi-step implementation of meiotic crossover patterning.

Ivana Čavka, Alexander Woglar, Yu-Le Wu, Emine Berna Durmus, Lauren Sloat, Aafke Gros, Cristina Piñeiro López, Felix Hecht, Anne M Villeneuve, Jonas Ries, Simone Köhler.

Crossover formation during meiosis is a tightly controlled process in which genetic information is exchanged between homologous chromosomes to increase the diversity of the progeny. In this process, an excess of double-strand breaks is introduced, but only a limited subset is ultimately processed into crossovers. Imbalances in the distribution of crossovers can lead to errors in chromosome segregation, with devastating consequences on the health of the progeny. However, the selection of which breaks are designated to become crossovers is still poorly understood, as both its timing and the ultimate molecular mechanisms are under debate. Here, we used 3D dual-color single-molecule localization microscopy and real-time confocal imaging, combined with advanced image analysis, to investigate the timing and mechanism of crossover designation in C. elegans . We show that meiotic crossover patterning is not established by a single decision point but depends on a dynamic, multi-layered regulation process. An initial, early selection process restricts potential crossovers to a small subset of double-strand break sites that already exhibit basic patterning features, including assurance and interference. A second, later step fine-tunes this pattern to ultimately ensure genome integrity and promote accurate chromosome segregation. Real-time imaging reveals that although the full process takes more than seven hours, key molecular events occur within minutes, high-lighting how rapid local dynamic changes can give rise to an overall slow but extremely robust crossover regulation program.

DOI: https://doi.org/10.1101/2025.11.12.687980
bioRxiv. 2025 Nov 10. pii: 2025.11.09.687253. [Epub ahead of print]

Spatial Partitioning of Core Glycolysis Enables Tissue-Specific Metabolic Programs In Vivo.

Ian J Gonzalez, Aaron D Wolfe, Benjamin Clark, Michael Hanna, Quishi Sun, Snusha Ravikumar, Anastasia Tsives, Sarah E Emerson, Stephan Siebel, Richard Kibbey, Daniel Colón-Ramos.

Tissues exhibit metabolic heterogeneity that tailors metabolism to their physiological demands. How the conserved pathways of metabolism achieve metabolic heterogeneity is not well understood, particularly in vivo. We established a system in Caenorhabditis elegans to investigate tissue-specific requirements for glucose 6-phosphate isomerase (GPI-1), a conserved glycolytic enzyme that also regulates the pentose phosphate pathway (PPP). Using CRISPR-Cas9 genome editing, we found that gpi-1 knockout animals display germline defects consistent with impaired PPP, and somatic defects consistent with impaired glycolysis. We discovered that two GPI-1 isoforms are differentially expressed and localized: GPI-1A is expressed in most tissues, where it displays cytosolic localization, whereas GPI-1B is primarily expressed in the germline, where it localizes to subcellular foci near the endoplasmic reticulum. GPI-1B expression alone is sufficient to maintain wild type levels of reproductive fitness, but insufficient to reconstitute wild-type glycolytic dynamics. Our findings uncover isoform-specific, spatially-compartmentalized functions of GPI-1 that underpin tissue-specific anabolic and catabolic metabolism in vivo , underscoring roles for subcellular localization in achieving tissue-specific metabolic flux.

DOI: https://doi.org/10.1101/2025.11.09.687253
Biol Reprod. 2025 Nov 27. pii: ioaf262. [Epub ahead of print]

Lipid Nanoparticle-mediated mRNA delivery system into preimplantation embryos.

Chihiro Emori, Hiroki Tanaka, Tatsuya Nakagawa, Hidetaka Akita, Masahito Ikawa.

  Lipid nanoparticles (LNPs) have emerged as a nonviral mRNA delivery vehicle for both basic and clinical applications. In the present study, we showed that the LNPs passed through the zona pellucida (ZP), the extracellular matrix surrounding the egg, and efficiently transduced preimplantation embryos. We first tested different types of surface polarity in LNPs carrying EGFP (LNP-EGFP) by changing cholesterol and found that the LNPs composed with neutral or anionic cholesterol are less toxic and more suitable for mRNA delivery into zygotes. Next, we transferred Cre mRNA using neutral LNP (LNP-Cre) and saw the floxed allele recombination in reporter mTmG transgenic embryos. After treatment with LNP-Cre at a 10 ng/μL concentration for 20 hrs at the zygote stage, about 76% mTmG embryos expressed the reporter EGFP at the blastocyst stage, and about 82 % of embryos expressed EGFP in both the placenta and fetus at E12.5. Finally, we treated mTmG blastocyst stage embryos with LNP-Cre at a 50 ng/μL concentration for 20 hrs, and found that about 86% of them expressed the reporter EGFP. Intriguingly, the EGFP fluorescence was only observed in trophectoderm (TE) cells, but not in the inner cell mass (ICM). Subsequently, we observed placenta-specific reporter EGFP expression in about 65% of the later-stage embryos (E12.5). Collectively, our study shows that LNPs provide a novel mRNA delivery system into preimplantation embryos that can be used for studying gene functions in pre-, peri-, and post-implantation development.

Keywords:  Lipid nanoparticle; gene delivery; preimplantation embryo; zygote

DOI:  https://doi.org/10.1093/biolre/ioaf262
Nat Commun. 2025 Nov 26. 16(1): 10120

Transcription start sites experience a high influx of heritable variants fueled by early development.

Miguel Cortés Guzmán, David Castellano, Clàudia Serrano Colomé, Vladimir Seplyarskiy, Donate Weghorn.

Mutations drive evolution and genetic diversity, with the most consequential mutations occurring in coding exons and regulatory regions. However, the impact of transcription on germline mutagenesis remains poorly understood. Here, we identify a mutational hotspot at transcription start sites (TSSs) in the human germline, spanning several hundred base pairs in both directions. Notably, the hotspot is absent in de novo mutation data. We reconcile this by showing that TSS mutations are significantly enriched with early mosaic variants, many of which are excluded from de novo mutation calls, indicating that the hotspot partly arises during early embryogenesis. We associate the TSS mutational hotspot with divergent transcription, RNA polymerase II stalling, R-loops, and mitotic-but not meiotic-double-strand breaks, suggesting a recombination-independent mechanism distinct from known processes. Our findings are reinforced by mutational signature analysis, which highlights alternative double-strand break repair and transcription-associated mutagenesis. These insights reveal a germline mutational phenomenon with evolutionary and biomedical implications, particularly affecting genes linked to cancer and developmental phenotypes.

DOI: https://doi.org/10.1038/s41467-025-66201-0
bioRxiv. 2025 Oct 14. pii: 2025.10.13.682153. [Epub ahead of print]

In Vitro Fertilization Accelerates Female Reproductive Aging Through Early Ovarian Failure.

Eric A Rhon-Calderon, Cassidy N Hemphill, Alexandra J Savage, Ana Domingo-Muelas, Zhengfeng Liu, Christopher J Krapp, Laren Riesche, Nicolas D Plachta, Richard M Schultz, Marisa S Bartolomei.

Reproductive aging is characterized by the progressive decline of reproductive function, with broad implications for overall health and longevity. Environmental factors, including assisted reproductive technologies (ART), can accelerate reproductive aging by promoting premature ovarian failure in females. In vitro fertilization (IVF), though widely used and generally considered safe, is associated with lasting effects on offspring health. Using a mouse model that closely approximates human IVF, we demonstrate that IVF accelerates reproductive aging in female offspring by inducing premature ovarian failure. IVF-conceived females exhibit altered ovarian function, disrupted endocrine profiles, and transcriptomic and epigenetic changes consistent with premature reproductive decline. These findings reveal long-term consequences of IVF on female reproductive health and highlight the need to understand how early-life interventions influence reproductive longevity.

DOI: https://doi.org/10.1101/2025.10.13.682153