bims-cebooc 2025-08-03 papers

bims-cebooc

Biomed News

on Cell biology of oocytes

Issue of 2025–08–03
thirty papers selected by
Gabriele Zaffagnini, Universität zu Köln

Assessment of Chromosome Congression Defects in Mammalian Oocytes.
Highly Multiplexed Immunofluorescence Imaging of Mouse Oocytes.
Nuclear Tracking During Early Embryonic Divisions.
RNA-Fluorescence In Situ Hybridization for the Detection of RNA in Mammalian Oocytes and Embryos.
Imaging and Quantifying F-Actin Bundles in the Cortex of Mammalian Oocytes.
Machine Learning Methods to Characterize Mouse and Human Oocytes.
Ca2+-driven cytoplasmic backflow ensures spindle anchoring in fertilized mouse eggs.
Optical Coherence Microscopy for Oocyte Imaging.
PAL-AI reveals genetic determinants that control poly(A)-tail length during oocyte maturation, with relevance to human fertility.
Electron Microscopy of Human Oocytes.
Whole Mount Oligopaint Fluorescence In Situ Hybridization of Mouse Oocytes, Eggs, or Early-Stage Embryos.
The E3 ubiquitin ligase adaptor KLHL8 targets ZAR1 to regulate maternal mRNA degradation in oocytes.
Multi-omics analysis reveals single-cell meiotic hotspot dynamics and epigenomic regulations in female mammals.
USP17L promotes the 2-cell-like program through deubiquitination of H2AK119ub1 and ZSCAN4.
Discordant effects of maternal age on the human MII oocyte transcriptome.
Live imaging endogenous transcription factor dynamics reveals mechanisms of epiblast and primitive endoderm fate segregation.
Collection of Growing Bovine Oocytes and Library Preparation for Single-Cell RNA Sequencing.
"JessTM Simple WesternTM": A New Technology to Assess Protein Abundance in Mouse Oocytes.
N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation.
Trade-off between antibacterial immune defense and oogenesis progression in female Drosophila melanogaster.
Using Photoactivatable GFP to Study Cytokinesis Dynamics in Early Development.
Biallelic BRCA2 variants induce premature ovarian insufficiency by impaired meiotic homologous recombination.
Micro magnetic resonance spectroscopy for noninvasive metabolic screening of mammalian embryos and oocytes.
In Vitro Maturation and Time-Lapse Imaging of Immature Human Oocytes: A Direction Toward Clinical Applications.
Design and Use of a Microfluidic Device for Oocyte Quality Screening.
Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst.
On-Valve μSFE-SFC-MS Enables Fast Analysis of Single Oocyte Lipidome.
Frequency and Distribution of Crossovers in Caenorhabditis elegans Meiosis by SNP Genotyping using Real-time PCR.
Stress granule clearance mediated by V-ATPase-interacting protein NCOA7 mitigates ovarian aging.
Isolation of Sheep Ovarian Cortex and In Vitro Culture to Study Folliculogenesis.

Methods Mol Biol. 2025 ;2946 125-135

Assessment of Chromosome Congression Defects in Mammalian Oocytes.

Adela Horakova, Marketa Konecna, Martin Anger.

  Congression of all chromosomes on the equatorial plane is in mitosis required for metaphase-anaphase transition, and for successful segregation of sister chromatids during anaphase. In contrast to somatic cells, mouse oocytes, which are undergoing the first meiotic division, are capable to undergo metaphase-anaphase transition without congression of all chromosomes. Consequently, oocytes exhibit an increased frequency of chromosome segregation errors, which might lead to aneuploidy. In various experimental conditions, for example, during gene function studies, or during evaluation of the effects of pharmaceutical inhibitors, it might be beneficial to assess chromosome congression in live oocytes. In this chapter, we describe all steps necessary for performing such experiments, including isolation of mouse oocytes, microinjection-free labeling of chromosomes and spindle, the time-lapse recording of chromosome division during meiosis I, using confocal live cell microscopy, and also steps necessary for data analysis.

Keywords:  Chromosome congression; Chromosomes; Live imaging; Metaphase; Oocyte; Spindle

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_10
Methods Mol Biol. 2025 ;2946 163-173

Highly Multiplexed Immunofluorescence Imaging of Mouse Oocytes.

Cyprien Noble, Typhaine Esteves, Adel Al Jord.

  Highly multiplexed immunofluorescence imaging methods advanced our understanding of biology across scales, from tissues down to molecules. By enabling the visualization of tens of proteins in a single sample, highly multiplexed imaging is especially relevant for scarce and challenging biospecimens like mammalian oocytes. However, most methods remain relatively costly and complex to implement. Here, we provide a cost-effective simple protocol, based on iterative indirect immunofluorescence imaging (4i), allowing to capture the distribution and abundance of tens of proteins in a single mouse oocyte. Our approach is adaptable to other mammalian oocytes or analogously large non-adherent cells like the early embryo.

Keywords:  4i; Germ cells; Iterative indirect immunofluorescence imaging; Mammalian oocyte; Meiosis I; Multiplex immunofluorescence; Multiplexed imaging; Non-adherent cells; Oocyte

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_13
Methods Mol Biol. 2025 ;2946 151-161

Nuclear Tracking During Early Embryonic Divisions.

Yunan Ye, Hayden A Homer.

  Positioning of the largest organelle, the nucleus, provides important cues for the geometry of cell divisions and cell fate determination. A primary function of nuclear positioning is to determine the spindle position and cleavage plane localization during cell division. Thus, centrally located nuclei lead to symmetrical divisions typical of mitosis, and off-center spindles culminate in symmetry-breaking as in mammalian oocytes. More recently, nuclear positioning has also been implicated in regulating mechanotransduction and gene expression in mouse oocytes. In human embryos, equal blastomere size arising from symmetrical cleavage-stage divisions is associated with favorable developmental outcomes, whereas off-center nuclear positioning affects division symmetry that can derail development. Therefore, investigating the mechanisms involved in nuclear positioning is critical for understanding embryo biology and the basis for clinical infertility. Here, we describe a method involving confocal imaging for performing nuclear tracking during early embryonic divisions in mouse embryos.

Keywords:  Embryo; Nuclear positioning; Nuclear tracking; Oocyte; Time-lapse imaging; TrackMate

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_12
Methods Mol Biol. 2025 ;2946 57-68

RNA-Fluorescence In Situ Hybridization for the Detection of RNA in Mammalian Oocytes and Embryos.

Denisa Jansova, Fatima J Berro, Andrej Susor.

  RNA metabolism plays an essential role in the development of both oocytes and embryos. Their dependence on stored maternal transcripts is due to a long period of silenced transcription in which the oocyte undergoes meiotic maturation and fertilization. Although maternal RNAs are unusually stable, they should be replaced by "zygotic" transcripts during the transition from oocyte to zygote. Analysis of ncRNA and mRNA distribution in the oocyte can provide clues to the fate of RNA in the single-cell environment.Our work focuses on the visualization of the subcellular distribution of specific RNAs in mammalian oocytes and early embryos. The localization of many RNAs in the oocyte and embryo is still not fully understood. In this chapter, we describe an optimized protocol for RNAscope, a type of RNA fluorescence in situ hybridization (RNA FISH), which is a valuable technique for exploring mammalian oocytes and embryos. The method is based on a specific probe design strategy that enables signal amplification together with simultaneous background suppression. Additionally, it is suitable for quantitative spatio-temporal analysis of specific RNA transcripts. RNAscope, a simple and a reliable protocol, contributes to advancing our understanding of RNA biology in the context of germ cell development.

Keywords:  Embryo; Oocyte; RNA FISH; Single-molecule imaging; mRNA

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_5
Methods Mol Biol. 2025 ;2946 115-124

Imaging and Quantifying F-Actin Bundles in the Cortex of Mammalian Oocytes.

Ghislain de Labbey, Elvira Nikalayevich.

  We describe the method of collection and microinjection of oocytes, acquisition of images of the oocyte cortex, as well as our image analysis pipeline to quantify the F-actin bundles in the oocyte cortex from these images. The metric is based on spatial correlations of the image intensity gradient.

Keywords:  Bundles; Cortex; F-actin; Nematic; Spatial correlations

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_9
Methods Mol Biol. 2025 ;2946 193-201

Machine Learning Methods to Characterize Mouse and Human Oocytes.

Gaëlle Letort.

  We describe the steps of our machine learning pipeline to phenotype oocyte based on images/movies acquired noninvasively in transmitted light. The pipeline allows to extract hundreds of morphological/dynamical features describing individual oocytes by segmenting the oocyte contours and measuring the corresponding features. Oocytes are then characterized by running feature-based machine learning algorithm on the features.

Keywords:  Features; Machine learning; Morphology; Oocyte; Segmentation

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_15
Curr Biol. 2025 Jul 23. pii: S0960-9822(25)00859-0. [Epub ahead of print]

Ca2+-driven cytoplasmic backflow ensures spindle anchoring in fertilized mouse eggs.

Takaya Totsuka, Miho Ohsugi, Takashi Akera.

  Female meiosis is highly asymmetric, producing a large egg and a small polar body to preserve maternal storage essential for embryogenesis. To achieve asymmetric division, the egg spindle must maintain its cortical position until fertilization completes meiosis. In mice, fertilization triggers chromosome segregation, followed by spindle rotation to achieve the perpendicular orientation relative to the cortex, leading to the extrusion of one set of chromosomes. However, it was unknown how the spindle maintains its cortical position while rotating. Here, we developed a high-resolution live-imaging method to investigate spindle dynamics during fertilization. Our results indicate that Ca2+ oscillations put the brakes on spindle rotation by transiently reversing cytoplasmic streaming and that this cytoplasmic backflow secures the spindle localization at the cortex. Mechanistically, Ca2+ oscillations drive cortical actomyosin contraction to induce the cytoplasmic backflow. Altogether, this work revealed a previously unknown role of Ca2+ oscillations in maintaining spindle position, ensuring the highly asymmetric divisions inherent to female meiosis.

Keywords:  Ca(2+) oscillations; actomyosin contraction; cytoplasmic streaming; fertilization; live imaging; meiosis II; mouse egg; second polar body extrusion; spindle dynamics

DOI:  https://doi.org/10.1016/j.cub.2025.06.073
Methods Mol Biol. 2025 ;2946 87-102

Optical Coherence Microscopy for Oocyte Imaging.

Anna Ajduk, Szymon Tamborski, Maciej Szkulmowski.

  Optical coherence microscopy (OCM) is a novel approach to fluorophore-free 3D live imaging of cells, particularly mammalian oocytes and embryos. It allows for 3D high-resolution visualization of the intracellular architecture: nuclei with nucleoli, metaphase spindles, and networks of membranous structures. Moreover, as it is compatible with time-lapse imaging, it enables monitoring and quantitative analysis of the dynamic behavior of these organelles over time. Importantly, OCM, when imaging settings are properly optimized, is safe for oocytes and embryos and does not negatively affect their developmental capabilities. Therefore, OCM is an interesting alternative to currently used imaging techniques, not only in basic research but also in clinical applications. In the present chapter, we describe the main principles of spectral OCM and show how this technique can be applied to visualize mouse oocytes.

Keywords:  Embryo; Image processing; Imaging; Mouse; Nucleolus; Nucleus; Oocyte; Optical coherence microscopy; Spindle

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_7
Nat Commun. 2025 Aug 01. 16(1): 7079

PAL-AI reveals genetic determinants that control poly(A)-tail length during oocyte maturation, with relevance to human fertility.

Kehui Xiang, David P Bartel.

In oocytes of mammals and other animals, gene regulation is mediated primarily through changes in poly(A)-tail length. Here, we introduce PAL-AI, an integrated neural network machine-learning model that accurately predicts tail-length changes in maturing oocytes of frogs and mammals. We show that PAL-AI learned known and previously unknown sequence elements and their contextual features that control poly(A)-tail length, enabling it to predict tail-length changes resulting from 3'-untranslated region single-nucleotide substitutions. It also predicted tail-length-mediated translational changes, allowing us to nominate genes important for oocyte maturation. When comparing predicted tail-length changes in human oocytes with genomic datasets of the All of Us Research Program and gnomAD, we found that genetic variants predicted to disrupt tail lengthening have been under negative selection in the human population, thereby linking mRNA tail lengthening to human female fertility.

DOI: https://doi.org/10.1038/s41467-025-62171-5
Methods Mol Biol. 2025 ;2946 103-114

Electron Microscopy of Human Oocytes.

Zuzana Holubcová, Barbora Baďurová, Zuzana Trebichalská.

  Electron microscopy represents a powerful visualizing technique, capable of a million times magnification. Prior to imaging, biological samples must undergo complex preparation to withstand the exposition to electrons in the vacuum inside the electron microscope. Here, we describe a preparation technique allowing preservation of scarce and delicate human oocytes for ultrastructural investigation.

Keywords:  Electron microscopy; Focused ion beam scanning electron microscopy; Human oocytes; Transmission electron microscopy; Ultrastructure

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_8
Methods Mol Biol. 2025 ;2946 69-86

Whole Mount Oligopaint Fluorescence In Situ Hybridization of Mouse Oocytes, Eggs, or Early-Stage Embryos.

Frances E Clark, Duilio M Z A Silva, Morgan Skinner, Leah F Rosin, Naomi L Greenberg, Takashi Akera.

  Fluorescence in situ hybridization (FISH) to visualize DNA or chromosomes in cells is well recognized as a valuable and versatile technique. The recent development of Oligopaint FISH is particularly useful for studies of chromosome structure, function, and evolution. Here, we describe a series of protocols for applying the Oligopaint FISH technique to mouse oocytes, eggs, and early-stage embryos. We describe techniques to adhere these cell types to microscopy slides, improving the ease of applying the FISH technique. We also provide an option to combine this with immunofluorescence (IF).

Keywords:  FISH; Mouse embryo; Mouse oocyte; Oligopaint; Zona pellucida

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_6
EMBO Rep. 2025 Jul 28.

The E3 ubiquitin ligase adaptor KLHL8 targets ZAR1 to regulate maternal mRNA degradation in oocytes.

Huizhen Fan, Ruyi Liu, Ran Yu, Biaobang Chen, Qiaoli Li, Jian Mu, Weijie Wang, Tianyu Wu, Lin He, Lei Wang, Qing Sang, Zhihua Zhang.

  Maternal protein homeostasis and timely degradation of maternal mRNAs are essential for meiotic cell-cycle progression and subsequent embryonic development, but the mechanisms of maternal protein degradation are poorly understood. Here, we show that KLHL8, a substrate adaptor of Cullin-RING E3 ubiquitin ligases, is highly expressed in mouse oocytes and co-localizes with mitochondria. Oocyte-specific deletion of Klhl8 causes oocyte maturation defects and female infertility. ZAR1, an RNA binding protein that is required for mitochondria-associated ribonucleoprotein domain (MARDO) dissolution, is specifically recognized and degraded by KLHL8-mediated ubiquitination. In Klhl8-deficient oocytes, ZAR1 accumulation causes abnormal MARDO and mitochondria clustering, correlating with impaired maternal mRNA decay. Supplementation with exogenous Klhl8 mRNA rescues the degradation of ZAR1 and the dissolution of the MARDO in Klhl8oo-/- oocytes. Taken together, our study shows that KLHL8 mediates the ubiquitination and degradation of ZAR1, thus regulating maternal mRNA clearance during oocyte maturation. These findings provide new insights into the roles of the ubiquitin proteasome system during oocyte maturation and establish an interaction network between ubiquitination modification, RNA binding proteins, and maternal mRNA.

Keywords:  KLHL8; Maternal mRNA Decay; Oocyte Maturation; Ubiquitination; ZAR1

DOI:  https://doi.org/10.1038/s44319-025-00537-y
Cell Rep. 2025 Jul 29. pii: S2211-1247(25)00853-8. [Epub ahead of print]44(8): 116082

Multi-omics analysis reveals single-cell meiotic hotspot dynamics and epigenomic regulations in female mammals.

Jingyi Li, Jiansen Lu, Jiayu Chen, Qianzheng Fu, Zhenhuan Jiang, Shaorong Gao, Fuchou Tang.

  Meiotic recombination initiates via DNA double-strand breaks (DSBs) at specialized hotspots, while the regulation of meiotic recombination hotspots in females remain elusive due to the scarcity of embryonic stage germ cells (EGCs). Here, we mapped genome-wide active recombination hotspots and estimated their activities in female EGCs at single-cell resolution, revealing the high variability in hotspot usage frequency among individual germ cells. Further investigation of nucleosome positioning and histone modifications at recombination hotspots revealed that PRDM9-mediated open chromatin and flanking H3K4me3 established earlier at high-frequency hotspots compared with less frequently used ones. Unexpectedly, although recombination hotspots usually distributed outside of heterochromatin, H3K9me3 was clearly enriched around hotspots in females, forming a unique H3K4me3/H3K9me3 bivalent state. And we showed that an appropriate H3K9me3 level may be required for downstream DSB repairs. Together, our results provided understanding about the landscape and epigenomic regulation of recombination hotspots in females.

Keywords:  CP: Developmental biology; H3K4me3-H3K9me3 bivalency; ULI-NChIP-seq; embryonic stage germ cells; low-input MNase-seq; meiotic recombination hotspots; single-cell ATAC-seq

DOI:  https://doi.org/10.1016/j.celrep.2025.116082
Nat Commun. 2025 Aug 01. 16(1): 7071

USP17L promotes the 2-cell-like program through deubiquitination of H2AK119ub1 and ZSCAN4.

Panpan Shi, Xukun Lu, Kairang Jin, Linlin Liu, Guoxing Yin, Wenying Wang, Jiao Yang, Lijuan Wang, Lijun Dong, Wei Xie, Lin Liu.

In mouse, minor zygotic genome activation (ZGA) precedes and is essential for major ZGA in two-cell (2C) embryos. A subset of ZGA genes (known as "2C" genes) are also activated in a rare population of embryonic stem cells (ESCs) (2C-like cells). However, the functions of the 2C genes are not fully understood. Here, we find that one family of the 2C genes, Usp17l, plays critical roles in transcriptional and post-translational regulation of the 2C-like state in mESCs. Specifically, USP17LE, a member of the USP17L family, deubiquitinates H2AK119ub1 and promotes the expression of Dux and the downstream 2C genes and retrotransposons. Moreover, USP17LE deubiquitinates and stabilizes ZSCAN4. In mouse pre-implantation embryos, Dux is marked by strong H2AK119ub1 except for the 1-cell and early 2-cell stages. Usp17le overexpression reduces H2AK119ub1 and promotes Dux and 2C gene activation. Thus, our findings identify USP17L as a potential regulator of the 2C program.

DOI: https://doi.org/10.1038/s41467-025-62303-x
Mol Hum Reprod. 2025 Jul 29. pii: gaaf038. [Epub ahead of print]

Discordant effects of maternal age on the human MII oocyte transcriptome.

Xiaorui Zhang, Jiao Yang, Wenting Yang, Nan Cui, Tingting Duan, Shan Li, Jing Cao, Stephen J Bush, Guoqing Tong.

  While advanced maternal age is associated with significant changes in oocyte gene expression, these are not global changes but limited to a fraction of the transcriptome. However, there is little consensus on the specific genes affected, and on the transcriptomic signatures of age-related declines in oocyte quality. To characterise the effects of age on the human MII oocyte transcriptome, here we take a two-part approach. We first generated single-oocyte Smart-seq2 datasets from ten younger (21-29 years) and ten older (37-43 years) donors, identifying genes differentially expressed between the two groups, then cross-referenced our results with those of 12 studies (9 human, 3 mouse) performing equivalent analyses using a variety of single-cell transcriptomic or microarray platforms. Technical differences notwithstanding, we found considerable discordance between the datasets, suggesting that age-related signatures of differential gene expression are not easily reproducible. Independent corroboration of age-associated changes in expression was limited to few genes, with the vast majority only supported by one of the 13 datasets, including our own. Nevertheless, we identified 40 genes whose expression significantly altered with age in multiple studies, highlighting common processes underlying ageing, including dysregulated proteostasis. As human Smart-seq2 oocyte libraries are challenging to procure and rare in public archives, we next implemented a meta-analytic method for their re-use, combining our 20 oocytes with 130 pre-existing libraries sourced from 12 different studies and representing a continuous age range of 18-43 years. We identified 25 genes whose expression level significantly correlated with age and corroborated 14 of these genes with RT-PCR, including the proteasomal subunits PSMA1 and PSMA2, both of which were downregulated in older oocytes. Overall, our findings are consistent with both pronounced inter-oocyte heterogeneity in transcription and with oocyte ageing being a multifactorial process to which bona fide transcriptomic changes may only play a restricted role, while proteomic changes play more pronounced roles.

Keywords:  differential gene expression; meta-analysis; oocyte ageing; oocyte quality; proteasome

DOI:  https://doi.org/10.1093/molehr/gaaf038
Curr Biol. 2025 Jul 26. pii: S0960-9822(25)00897-8. [Epub ahead of print]

Live imaging endogenous transcription factor dynamics reveals mechanisms of epiblast and primitive endoderm fate segregation.

Rebecca P Kim-Yip, David Denberg, Denis F Faerberg, Hayden Nunley, Isabella Leite, Madeleine Chalifoux, Bradley Joyce, Jared Toettcher, Bin Gu, Eszter Posfai.

  The segregation of the epiblast (EPI) and primitive endoderm (PE) cell types in the preimplantation mouse embryo is not only a crucial decision that sets aside the precursors of the embryo proper from extraembryonic cells, respectively, but also has served as a central model to study a key concept in mammalian development: how much of developmental patterning is predetermined vs. stochastically emergent. Here, we address this question by quantitative live imaging of multiple endogenously tagged transcription factors key to this fate decision and trace their dynamics at a single-cell resolution through the formation of EPI and PE cell fates. Strikingly, we reveal an initial symmetry breaking event, the formation of a primary EPI cell lineage, and show that this is linked to the dynamics of the prior inner cell mass/trophectoderm fate decision through the expression of SOX2. This primary EPI lineage, through fibroblast growth factor (FGF) signaling, induces an increase in the transcription factor GATA6 in other inner cell mass cells, setting them on the course toward PE differentiation. Interestingly, this trajectory can switch during a defined developmental window, leading to the emergence of secondary EPI cells. Finally, we show that early expression levels of NANOG, which are seemingly stochastic, can bias whether a cell's trajectory switches to secondary EPI or continues as PE. Our data give unique insight into how fate patterning is initiated and propagated during unperturbed embryonic development through the interplay of lineage-history-biased and stochastic cell-intrinsic molecular features, unifying previous models of EPI/PE segregation.

Keywords:  GATA6; ICM; NANOG; SOX2; blastocyst; epiblast; live imaging; mouse; preimplantation embryo; primitive endoderm

DOI:  https://doi.org/10.1016/j.cub.2025.07.031
Methods Mol Biol. 2025 ;2946 43-55

Collection of Growing Bovine Oocytes and Library Preparation for Single-Cell RNA Sequencing.

Lais B Latorraca, Antônio Galvão, Gavin Kelsey, Trudee Fair.

  The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized the study of gene expression in individual cells, providing unprecedented insights into cellular heterogeneity and developmental processes. The application of scRNA-seq to oocyte biology has facilitated the identification of species-specific transcriptional signatures and developmental trajectories, enhancing our understanding of oogenesis. This chapter presents a detailed protocol for scRNA-seq analysis of growing bovine oocytes.

Keywords:  Bovine oocytes; Cellular heterogeneity; Gene expression; scRNA-seq

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_4
Methods Mol Biol. 2025 ;2946 175-192

"JessTM Simple WesternTM": A New Technology to Assess Protein Abundance in Mouse Oocytes.

Damien Cladière, Katja Wassmann, Sandra A Touati.

  Traditional Western blot technology coupled with two-dimensional SDS-PAGE has been used for decades to detect specific proteins in extracts. Here we describe an automated Western technology based on protein separation by capillary assay. Only 15 mouse oocytes are sufficient to obtain a quantitative protein signal in the assay we used here. This automated process is a good alternative to classical western blotting, as it is less time consuming, highly reproducible and requires less material.

Keywords:  JessTM Simple WesternTM; Mouse oocytes; Protein quantification

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_14
Elife. 2025 Jul 29. pii: RP92068. [Epub ahead of print]13

N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation.

Alaknanda Emery, Orest W Blaschuk, Doan T Dinh, Tim McPhee, Rouven Becker, Andrew D Abell, Krzysztof M Mrozik, Andrew C W Zannettino, Rebecca L Robker, Darryl L Russell.

  The cell adhesion molecule N-cadherin (CDH2) is a membrane component of adherens junctions which regulates tissue morphogenesis and architecture. In the follicles of mammalian ovaries, N-cadherin adherens junctions are present between granulosa cells, cumulus cells, and at the interface of cumulus cell transzonal projections and the oocyte. We demonstrate a mechanosensory role of N-cadherin integrating tissue structure and hormonal regulation of follicular morphogenic events including expansion of the cumulus-oocyte complex (COC) matrix, oocyte maturation, and ovulation. Two small molecule N-cadherin antagonists inhibited COC maturation in vitro. Transcriptome profiling revealed that targets of β-catenin and YAP1 pathways were dysregulated by N-cadherin antagonists. In vivo, N-cadherin antagonist significantly reduced ovulation in mice compared to controls (11 vs 26 oocytes/ovary; p = 5.8 × 10-6). Ovarian follicles exhibited structural dysgenesis with granulosa and cumulus cell layers becoming disorganised and the connection between cumulus cells and the oocyte disrupted and the transcriptome again indicated altered mechanical sensing causing dysregulation of the Hippo/YAP and β-catenin pathways and extracellular matrix reorganisation. Granulosa-specific N-cadherin depletion in Cdh2Fl/FL;Amhr2Cre/+ also showed significantly altered mechanosensitive gene expression and reduced ovulation. Our findings demonstrate a critical role for N-cadherin in ovarian follicular development and ovulation, and the potential to inhibit ovulation through targeting this signalling mechanism.

Keywords:  cell biology; cell signalling; contraception; mechanotransduction; mouse; oocyte; ovary; ovulation

DOI:  https://doi.org/10.7554/eLife.92068
Genetics. 2025 Aug 01. pii: iyaf151. [Epub ahead of print]

Trade-off between antibacterial immune defense and oogenesis progression in female Drosophila melanogaster.

Kathleen E Gordon, Shravasti Ray, Jeremy M Marcin, Patrick M Gonzales, Mona L Li, Crystal Liang, Mariana F Wolfner, Brian P Lazzaro.

  Trade-offs between reproduction and immunity are common in animals, potentially due to preferential allocation of limiting resources. In Drosophila melanogaster, mating stimulates egg production but also triggers a rapid and persistent decrease in female immune defense. Proteins essential for both processes are produced in fat body tissue, which may result in competition for cellular resources that could drive a functional trade-off between reproduction and immune defense. We predicted that arrest of oogenesis prior to egg provisioning would alleviate post-mating immune suppression because cellular stress would be relieved, but that post-mating immune suppression would be observed in genotypes that fully provision eggs even if fertility is compromised. In the present work, we test these predictions by evaluating post-mating immune competence in mated D. melanogaster mutants that arrest oogenesis either prior to, or subsequent to, vitellogenesis. Consistent with our prediction, we find that mated female immune defense is maintained when egg development is arrested prior to vitellogenesis. We find that progression through the vitellogenic stages of oogenesis results in post-mating immune suppression, except in the case of a mutant with an egg-retention phenotype, where we infer that the failure to lay eggs results in feedback that inhibits subsequent egg development. We additionally show that elimination of yolk protein synthesis in the fat body and follicle cells of the ovary partially restores female immune capacity. Nevertheless, females that lack yolk protein genes still experience partially reduced immune capacity after mating, suggesting that other reproductive demands also suppress immune defense.

Keywords:   Drosophila ; Antimicrobial Peptides; Immune Defense; Life History; Mating; Reproduction; Trade-Off; Yolk Protein

DOI:  https://doi.org/10.1093/genetics/iyaf151
Methods Mol Biol. 2025 ;2946 137-149

Using Photoactivatable GFP to Study Cytokinesis Dynamics in Early Development.

Filip Vasilev, Greg FitzHarris.

  Fluorescent protein photoconversion is a powerful approach that can be adapted to investigate various aspects of cellular behavior in diverse contexts. In somatic cells, fluorescent photoactivation of photoactivatable-GFP (PAGFP) has been used to accurately determine the timing of plasma membrane abscission, the final step of cell division that establishes two cytoplasmically separate cells. Diffusion of photoactivated PAGFP from one cell into its sister cell shortly after mitosis indicates that abscission has not occurred, whereas photoactivated PAGFP that will not pass between cells suggests successful abscission. Here we have adapted this approach to enable investigation of abscission timing in the context of early mammalian embryos.

Keywords:  Cell division; Cytokinesis; Live cell imaging; Mouse embryo; Preimplantation development

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_11
Commun Biol. 2025 Jul 25. 8(1): 1104

Biallelic BRCA2 variants induce premature ovarian insufficiency by impaired meiotic homologous recombination.

Xinyi Wu, Qian Zhang, Chang Li, Shuning Zhuang, Hongyuan Liu, Xue Jiao, Shidou Zhao, Yingying Qin, Ting Guo.

The DNA damage response plays a pivotal role in ovarian aging. Breast cancer susceptibility gene 2 (BRCA2), which participates in homologous recombination (HR), is a key regulator of natural menopause. Rare BRCA2 variants have been identified in patients with premature ovarian insufficiency (POI). However, the underlying molecular mechanisms are not well understood. Using a viable mouse model, Brca2c.68-1G>C/c.4384-4394del, carrying compound heterozygous variants mirroring the ones identified in a POI pedigree, we illustrated the essential role of BRCA2 in primordial follicle pool establishment. Germline deficiency of BRCA2 did not affect primordial germ cell (PGC) proliferation but impaired the recruitment of RAD51 and DMC1 to programmed DNA double-strand breaks (DSBs) during meiotic HR, causing postnatal oocyte depletion. Moreover, Brca2c.68-1G>C/c.4384-4394del mice presented increased tumor susceptibility. These findings confirmed the pathogenicity of BRCA2 biallelic variants in POI, revealing the dual impact on germ cell development and somatic cancer risk, underscoring the necessity of tumor surveillance in POI patients with BRCA2 mutations.

DOI: https://doi.org/10.1038/s42003-025-08426-9
Proc Natl Acad Sci U S A. 2025 Aug 05. 122(31): e2424459122

Micro magnetic resonance spectroscopy for noninvasive metabolic screening of mammalian embryos and oocytes.

Giulia Sivelli, Arthur Barakat, Kathryn B Marable, Guillaume Gruet, Serena L Bitetti, Barry Behr, Valentina Lodde, Alberto Maria Luciano, Carolina Herrera, Michelle Blom, Marco Grisi.

  Analyzing cellular health and metabolism without compromising cell integrity is a major challenge. We present a noninvasive technique using micro magnetic resonance spectroscopy (micro MRS) for nondestructive metabolic fingerprinting at the single-cell scale. This is an application of micro MRS to bovine preimplantation embryos (~8 cells) and oocytes (single cell), with measurements performed on a total of over 150 samples. Among various applications, this method holds significant potential for assisted reproductive technologies (ART), where metabolic assessments of preimplantation embryos could improve treatment outcomes. Early results indicate that classification models using micro MRS data effectively distinguish embryos with high developmental potential and show correlation with oocytes maturity. Furthermore, a multigenerational safety study in a mouse model revealed no adverse effects from embryo exposure to static magnetic field. These findings indicate that micro MRS is a promising, safe tool for assessing embryo metabolism, potentially improving the efficiency and outcomes of ART.

Keywords:  Magnetic Resonance Spectroscopy; metabolic fingerprinting; micro MRS; non-invasive embryo screening; single-cell MRS

DOI:  https://doi.org/10.1073/pnas.2424459122
Methods Mol Biol. 2025 ;2946 21-41

In Vitro Maturation and Time-Lapse Imaging of Immature Human Oocytes: A Direction Toward Clinical Applications.

Xavier Pollet-Villard, Mohamed Yahiatene, Alpesh Doshi.

  Recently, multiple teams reported the successful utilization of denuded oocytes matured in vitro after controlled ovarian stimulation, as well as their small but significant contribution to cumulative live-birth rates after intracytoplasmic sperm injection (ICSI). Published data are in favor of rescue ICSI when mature oocytes are obtained one day after oocyte pickup, the clinical contribution being higher for patient with advanced maternal age, low ovarian response to COS, low maturation rate, or low fertilization rate. From a strictly resource-oriented perspective, rescue ICSI of immature oocytes may not be cost-effective as it would require additional expenses related to consumables, media, and embryologist time and as the developmental potential of such oocytes is consistently decreased across reported studies compared to their sibling mature oocytes. Being able to select the right clinical context as well as the right oocytes to fertilize to optimize outcome and resources spent is thus of significant clinical value. In that regard, even if more costly than conventional culture, time-lapse imaging may play an important role as morphokinetic markers of oocyte maturation and oocyte developmental potential have been described. However, the limited number of reported cases warrants for the restricted use of rescue ICSI only in registered clinical trials with long-term follow-up of children obtained from this still experimental procedure.

Keywords:  Controlled ovarian stimulation; Germinal vesicle oocytes; ICSI; IVF; In vitro maturation; Metaphase 1 oocytes; Metaphase 2 oocytes; Rescue-ICSI; Time-lapse imaging

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_3
Methods Mol Biol. 2025 ;2946 203-211

Design and Use of a Microfluidic Device for Oocyte Quality Screening.

Rose Bulteau, Lucie Barbier.

  Oocyte quality is a major factor limiting female fertility. In the context of medically assisted reproduction (MAR), the assessment of oocyte quality is not yet well established, and researchers are screening for characteristics likely to influence pregnancy rate using a wide variety of approaches based on chemistry, -omics, optics, and physical parameters. In this context, microfluidics appears to be a promising technology as it allows the incorporation of multiple sensors on an easy-to-use platform, thus offering practical solutions for oocyte assessment in MAR routines. However, while a variety of microfluidic systems have been established for the manipulation of thousands or millions of cells, few are applicable to single cells of typical oocyte size, limiting the use of microfluidic devices in assisted reproductive technologies. We propose the design of a microfluidic device and a protocol for operating a single oocyte to any sensor integrated into the device and then retrieving it for fertilization. Our approach does not induce morphological alterations in oocytes and is compatible with standard MAR protocols.

Keywords:  Microfabrication; Oocyte evaluation; Single-cell handling in microfluidic device

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_16
Nat Commun. 2025 Jul 28. 16(1): 6922

Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst.

Claire S Simon, Afshan McCarthy, Laura Woods, Desislava Staneva, Martin Proks, Nazmus Salehin, Georgia Lea, Qiulin Huang, Madeleine Linneberg-Agerholm, Alex Faulkner, Athanasios Papathanasiou, Kay Elder, Phil Snell, Leila Christie, Patricia Garcia, Valerie Shaikly, Mohamed Taranissi, Meenakshi Choudhary, Mary Herbert, Courtney W Hanna, Joshua M Brickman, Kathy K Niakan.

Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naïve human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and maintain molecular markers of the epiblast. Our functional study demonstrates the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner.

DOI: https://doi.org/10.1038/s41467-025-61830-x
Anal Chem. 2025 Jul 30.

On-Valve μSFE-SFC-MS Enables Fast Analysis of Single Oocyte Lipidome.

Jinlei Yang, Tianyi Liao, Pingping Zhang, Runsong Cheng, Jing Wang, Siyuan Pan, Murong Du, Boya Zhao, Weiliang Liu, Liying Yan, Sichun Zhang, Xinrong Zhang.

Lipid regulation and remodeling are pivotal in follicle development and oocyte maturation. Performing lipidome analysis on single oocytes is essential for optimizing assisted reproductive techniques and improving pregnancy outcomes. Conventional organic solvent extraction faces challenges in single-cell analysis, including excessive sample dilution and time-consuming processing. In this study, we developed an on-valve microflow supercritical fluid extraction and chromatography-mass spectrometry (μSFE-SFC-MS) method. Lipids in intact single oocytes are extracted online by supercritical carbon dioxide fluid, separated by a C18 capillary column, ionized, and annotated. Notably, this method significantly reduces sample pretreatment and chromatographic separation time to just 15 min per sample, compared to about 2 h of the conventional method. We analyzed of 276 lipid species from a single oocyte. Lipidomic differences in oocyte maturation stages clarify distinct metabolic remodeling of phospholipids into sphingomyelins and glycerides. The differential analysis suggests that several lipid species can be used as criteria for determining oocyte maturation at the single-cell level. The present work offers a fast and high-coverage lipidomic analysis for single oocytes, also providing a workflow for other single-cell and trace samples.

DOI: https://doi.org/10.1021/acs.analchem.5c03365
J Vis Exp. 2025 Jul 11.

Frequency and Distribution of Crossovers in Caenorhabditis elegans Meiosis by SNP Genotyping using Real-time PCR.

Miku Koishihara, Akihide Onishi, Akari Hirose, Takamune T Saito.

Caenorhabditis elegans is an excellent model organism for studying meiosis. In addition to general advantages, such as a short reproductive cycle, many progenies, and a transparent body for imaging, there are six pairs of homologous holocentric chromosomes. There is strong crossover interference and regulation of crossover distribution. To measure the crossover frequency, visible marker mapping, snip-SNP mapping, and quantification of cytological markers as precursors of crossover formation have been developed. Here, we introduce a modified SNP genotyping method to measure the crossover frequency and distribution in C. elegans oogenesis. This method can omit the laborious steps of restriction digestion and gel electrophoresis and avoid the ambiguous judgment of uncut DNA bands. Single-nucleotide polymorphism (SNP) genotyping is performed using the Bristol/Hawaiian hybrid strain. Crossing hermaphrodites with myo-3p::GFP-expressing males enables us to focus on oogenesis. A single worm lysate produces sufficient DNA template for all six chromosomes. The 5' exonuclease-based TaqMan chemistry and SNP-specific minor groove binder (MGB) probes allow the precise detection of SNP at a genotyping rate of approximately 97.7%. We utilize the accurate detection of SNPs to measure crossover frequency in C. elegans. This method can also be applied to determine the crossover frequency in male or crossover-defective mutants, as well as for more specific chromosomal intervals.

DOI: https://doi.org/10.3791/68183
Nat Aging. 2025 Jul 31.

Stress granule clearance mediated by V-ATPase-interacting protein NCOA7 mitigates ovarian aging.

Ting Dong, Nianyu Li, Huirui Wang, Hanbing Zhu, Yinghui Gao, Yue Liu, Fang Fang, Xiaojie Fu, Pinxin Si, Cheng Li, Mei Li, Fei Wang, Shidou Zhao, Ting Guo, Linlin Cui, Xinyi Jiang, Xiaohui Liu, Han Zhao, Yingying Qin, Zi-Jiang Chen, Hongxiang Lou, Xue Jiao.

Reproductive longevity is essential for female fertility and healthy aging; however, the role of stress response, especially stress granule accumulation, in ovarian aging remains elusive and interventions are lacking. Here, we identified deleterious mutations and decreased expression of NCOA7, a stress-response protein related to granulosa cell senescence in women with physiological and pathological ovarian aging. NCOA7 deletion accelerates oxidative stress-related cellular senescence, ovarian aging and fecundity decline in mice. Mechanistically, NCOA7 partitions into the stress granule containing G3BP1-V-ATPase and facilitates autophagic degradation of stress granules to relieve stress. Boosting granulophagy with rapamycin or lipid nanoparticle-based mRNA delivery of NCOA7 accelerates stress granule clearance, alleviating cellular senescence in human granulosa cells and delaying ovarian aging in mice. This study depicts a mechanism for ovarian resilience to stress and provides potential targets for therapeutic strategies to alleviate ovarian aging.

DOI: https://doi.org/10.1038/s43587-025-00927-w
Methods Mol Biol. 2025 ;2946 1-7

Isolation of Sheep Ovarian Cortex and In Vitro Culture to Study Folliculogenesis.

Elsa Labrune, Odile Lepinasse, Julie Fiscus.

  Folliculogenesis is a complex and precisely regulated physiological process. Performing a complete in vitro folliculogenesis remains challenging and has not been yet achieved in the sheep. Successfully accomplishing folliculogenesis in a large animal such as the sheep is an essential step in validating in vitro folliculogenesis technique and its further application to fertility preservation in human. The main knowledge gap hindering this achievement is a better comprehension of mechanical and biochemical signals involved in the initiation of follicular growth from the reserve pool of primordial follicles. Here, we describe the dissection and stretching methods as well as the in vitro culture of sheep ovarian cortex tissue in view to initiate folliculogenesis.

Keywords:  Follicle culture; In vitro folliculogenesis; Ovary; Sheep; Stretching

DOI:  https://doi.org/10.1007/978-1-0716-4658-8_1