bims-cebooc 2025-11-16 papers

bims-cebooc

Biomed News

on Cell biology of oocytes

Issue of 2025–11–16
nineteen papers selected by
Gabriele Zaffagnini, Universität zu Köln

The bottleneck for maternal transmission of mtDNA is linked to purifying selection by autophagy.
Trigonelline activates NRF2 and awakens dormant ovarian follicles to promote pregnancy in aging mice.
Reprogramming of H3K36me2 guides lineage-specific post-implantation de novo DNA methylation.
Meiotic cohesion requires Sirt1 and preserving its activity in aging oocytes reduces missegregation.
Early female germline development in Xenopus laevis: Stem cells, nurse cells, and germline cysts.
Targeted RNAi screen reveals novel regulators of RNA-binding protein phase transitions in Caenorhabditis elegans oocytes.
Regulation of Xvelo assembly by centrosomal proteins.
The emerging role of the oocyte cortical domain in maturation, fertilization, and development.
Local nuclear to cytoplasmic ratio regulates H3.3 incorporation via cell cycle state during zygotic genome activation.
Phosphorylation of YWHAZ at Serine 58 by PKB/Akt is associated with resumption of meiotic arrest in mouse oocytes.
Collection of Human Follicular Fluid, Follicle Somatic Cells, and Immature Oocytes from Individuals Undergoing In Vitro Fertilization.
The ORB2 RNA-binding protein negatively regulates its target transcripts during the Drosophila maternal-to-zygotic transition via its functionally conserved Zinc-binding 'ZZ' domain.
Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture.
Mitochondrial metabolism influences meiotic maturation in human oocytes of young and advanced maternal age women.
A mammalian-like piRNA pathway in Axolotl reveals the origins of piRNA-directed DNA methylation.
A primordial germ cell-like-cell platform enables CRISPRi screen for epigenetic fertility modifiers.
The synaptonemal complex component corolla regulates meiotic crossover formation in Drosophila melanogaster.
Developmental spatiotemporal switching of the germline-specific subunits of heterodimeric NAC protein in Drosophila melanogaster.
Investigating metabolic activity during oocyte and early embryo development through label-free metabolic imaging: a systematic approach for timelapse applications.

Sci Adv. 2025 Nov 14. 11(46): eaea4660

The bottleneck for maternal transmission of mtDNA is linked to purifying selection by autophagy.

Laura S Kremer, Zoe Golder, Tom Barton-Owen, Polyxeni Papadea, Camilla Koolmeister, Patrick F Chinnery, Nils-Göran Larsson.

Mammalian mitochondrial DNA (mtDNA) inheritance differs fundamentally from nuclear inheritance owing to exclusive maternal transmission, high mutation rate, and lack of recombination. Two key mechanisms shape this inheritance: the bottleneck, which drives stochastic transmission of maternal mtDNA variants, and purifying selection, which actively removes mutant mtDNA. Whether these mechanisms interact has been unresolved. To address this question, we generated a series of mouse models with random mtDNA mutations alongside alleles altering mtDNA copy number or decreasing autophagy. We demonstrate that tightening the mtDNA bottleneck increases heteroplasmic variance between individuals, causing lower mutational burden and nonsynonymous-to-synonymous ratios. In contrast, reduced autophagy weakens purifying selection, leading to decreased interoffspring heteroplasmic variance and increased mutational burden with higher nonsynonymous-to-synonymous ratios. These findings provide experimental evidence that the mtDNA bottleneck size modulates the efficacy of purifying selection. Our findings yield fundamental insights into the processes governing mammalian mtDNA transmission with direct implications for the origin and propagation of mtDNA mutations causing human disease.

DOI: https://doi.org/10.1126/sciadv.aea4660
iScience. 2025 Nov 21. 28(11): 113717

Trigonelline activates NRF2 and awakens dormant ovarian follicles to promote pregnancy in aging mice.

Mahboobeh Amoushahi, Emil Hagen Ernst, Anders Heuck, Margit Dueholm, Erik Ernst, Karin Lykke-Hartmann.

  Aging ovaries exhibit increased oxidative stress, contributing to infertility through cellular and hormonal changes. Nuclear factor E2-related factor 2 (NRF2), a key transcription factor, regulates antioxidant responses. This study investigates NRF2 in dormant (primordial) ovarian follicles to determine if NRF2 activation accounts for primordial follicle activation. We show that trigonelline (TRG) transiently activates NRF2, promoting primordial follicle activation in the non-hormonal phase of follicle development. Indeed, TRG enhances egg quality in aged mice. In human ovarian tissues, TRG increased activation of primordial follicles, resulting in more primary and secondary follicles. Mechanistically, TRG induces NRF2 nuclear translocation and upregulates NRF2-responsive genes, including Egf. Elevated epidermal growth factor (EGF) levels activate EGF receptor (EGFR), increasing protein kinase B (AKT) phosphorylation, which leads to FOXO3A nuclear extrusion and primordial follicle activation. These findings demonstrate that transient NRF2 activation is sufficient to initiate primordial follicle activation and follicle growth and development.

Keywords:  Cell biology; Reproductive medicine

DOI:  https://doi.org/10.1016/j.isci.2025.113717
Nat Cell Biol. 2025 Nov 11.

Reprogramming of H3K36me2 guides lineage-specific post-implantation de novo DNA methylation.

Xukun Lu, Lijuan Wang, Bofeng Liu, Xiaoyu Hu, Zhengmao Wang, Ling Liu, Guang Yu, Lijun Dong, Feng Kong, Qiang Fan, Yu Zhang, Wei Xie.

In mammals, DNA methylation is re-established after implantation following post-fertilization global erasure. Yet, the underlying mechanism remains elusive. Here we investigate H3K36me2 reprogramming in mouse early development and its role in post-implantation DNA methylation re-establishment. In oocytes, H3K36me2 accumulates in gene bodies upon transcription silencing and partially persists to the eight-cell stage. De novo H3K36me2 occurs at enhancers after zygotic genome activation, before spreading genome-wide after implantation, except on the inactive X chromosome. Mutation of the H3K36me2 methyltransferase NSD1 compromises global DNA methylation after implantation preferentially in extra-embryonic lineages and that at methylation-prone promoters, including those of germline-specific genes. However, DNA methylation establishment partially bypasses H3K36me2 through upregulated DNMT3B, a 'leaky' H3K36me2/3 reader. This contrasts with DNMT3A, which strictly requires H3K36me2/3 for DNA methylation through its PWWP domain. Finally, DNA methylation valleys escape de novo DNA methylation via PRC1/H2AK119ub1-mediated H3K36me2 exclusion. Thus, H3K36me2 reprogramming regulates lineage- and locus-specific post-implantation DNA methylation establishment.

DOI: https://doi.org/10.1038/s41556-025-01805-8
EMBO Rep. 2025 Nov 10.

Meiotic cohesion requires Sirt1 and preserving its activity in aging oocytes reduces missegregation.

Zihan Meng, Nicholas G Norwitz, Sharon E Bickel.

  Chromosome segregation errors in human oocytes increase dramatically as women age and premature loss of meiotic cohesion is one factor that contributes to a higher incidence of segregation errors in older oocytes. Here we show that knockdown of the NAD+-dependent deacetylase Sirt1 during meiotic prophase in Drosophila oocytes causes premature loss of arm cohesion and chromosome segregation errors. We demonstrate that acetylation of the Sirt1 substrate H4K16 increases significantly in sirt1 null and Sirt1 knockdown oocytes and use this as a marker for Sirt1 activity in vivo. When oocytes undergo aging, the H4K16ac signal increases significantly, consistent with an aging-dependent decline in Sirt1 deacetylase activity. However, if females are fed the Sirt1 activator SRT1720 as their oocytes age, the H4K16ac signal on oocyte DNA remains low in aged oocytes, consistent with preservation of Sirt1 activity during aging. Strikingly, age-dependent segregation errors are significantly reduced if mothers are fed SRT1720 while their oocytes age. Our data suggest that maintaining Sirt1 activity in aging oocytes may provide a viable therapeutic strategy to decrease age-dependent segregation errors.

Keywords:   Drosophila ; Maternal Age Effect; Meiosis; SRT1720; Sister Chromatid Cohesion

DOI:  https://doi.org/10.1038/s44319-025-00634-y
Proc Natl Acad Sci U S A. 2025 Nov 18. 122(46): e2522343122

Early female germline development in Xenopus laevis: Stem cells, nurse cells, and germline cysts.

Asya Davidian, Allan C Spradling.

  In Drosophila, germline cysts arise through synchronous mitotic divisions and acquire a polarized architecture organized by the fusome, which guides oocyte specification and supports meiotic progression. Similar cyst structures exist in nonmammalian vertebrate ovaries, but their polarity and function have remained uncertain. Using single-cell RNA sequencing and high-resolution imaging, we reconstructed the germ cell differentiation trajectory in Xenopus laevis and uncovered striking parallels with invertebrate and mouse cyst development. We identified a distinct germline stem cell (GSC) population marked by piwil4, low translational activity, and expression of neuronal-specific and transposon-silencing genes. Downstream from GSCs, during cyst development, an asymmetric fusome-like structure composed of stable microtubules forms a rosette-like connection between cystocytes and contains Golgi vesicles and endoplasmic reticulum, suggesting polarized trafficking. In contrast to previous claims, ~80% of cyst cells turned over rather than forming oocytes, consistent with a nurse cell fate. The striking parallels described here between cyst and fusome formation, polarization, cyst breakdown, and nurse-like cell turnover to produce relatively few oocytes argue that amphibian cysts have important functions in female gametogenesis.

Keywords:  Xenopus; germ cell; germline cyst; germline stem cell; ovary

DOI:  https://doi.org/10.1073/pnas.2522343122
G3 (Bethesda). 2025 Nov 10. pii: jkaf266. [Epub ahead of print]

Targeted RNAi screen reveals novel regulators of RNA-binding protein phase transitions in Caenorhabditis elegans oocytes.

Mohamed T Elaswad, Grace M Thomas, Corrin Hays, Nicholas J Trombley, Jennifer A Schisa.

  The ability of oocytes to maintain their quality is essential for successful reproduction. One critical aspect of oocyte quality and successful embryogenesis after fertilization is the proper regulation of the stores of maternal mRNA by RNA-binding proteins. Many RNA-binding proteins undergo regulated phase transitions during oogenesis, and alterations of the protein phase can disrupt its ability to regulate mRNA stability and translation. In C. elegans, regulators of RNA-binding protein phase transitions in maturing oocytes of young adult hermaphrodites remain poorly characterized. However, a few recently identified genes are also required for the clearance of damaged proteins during maturation, suggesting coordination between these processes. To explore this relationship and gain insight into the regulation of phase transitions, we conducted a targeted RNAi screen of genes required for removal of protein aggregates in maturing oocytes. Here, we identify six novel regulators of phase transitions of the KH-domain protein MEX-3. We present strong evidence that the regulation of MEX-3 phase transitions in the oocyte overlaps with, but is distinct from, the regulatory network of protein aggregate clearance.

Keywords:   Caenorhabditis elegans ; Animalia; MEX-3; RNA-binding proteins; WormBase; condensates; oocyte quality; phase transitions

DOI:  https://doi.org/10.1093/g3journal/jkaf266
iScience. 2025 Nov 21. 28(11): 113805

Regulation of Xvelo assembly by centrosomal proteins.

Divyanshi, Meining Li, Elvan Böke, Jing Yang.

  The oocyte Balbiani body (Bb) is a conserved membraneless organelle required for germline specification in many species. Here we report that when overexpressed in mammalian somatic cells, Xenopus Velo (Xvelo), the Bb matrix protein could reconstitute a "Bb-like" structure, which shares key features of the oocyte Bb. Using this system, we investigated the potential mechanism of Bb assembly. We focused on the involvement of centrosomal proteins in this process, as both endogenous oocyte Bb and the "Bb-like" structure induced by Xvelo overexpression form around the centrosome. Our results reveal that multiple components of the centrosome can interact with Xvelo and promote Xvelo assembly to varying degrees. Moreover, knockdown of these proteins reduces Xvelo assembly. Our findings thus suggest that the interaction between centrosomal proteins and Xvelo may facilitate the initial aggregation of Xvelo, leading to the assembly of Bb around the centrosome in the oocyte.

Keywords:  biochemistry; cell biology; molecular biology

DOI:  https://doi.org/10.1016/j.isci.2025.113805
Hum Reprod. 2025 Nov 06. pii: deaf208. [Epub ahead of print]

The emerging role of the oocyte cortical domain in maturation, fertilization, and development.

Giovanni Coticchio, Valentina Casciani, Laura Rienzi, Danilo Cimadomo, Chiara Cosseddu, Marilena Taggi, Sergio Ledda, Daniela Bebbere.

  The cell cortex, a cytoskeletal network with regulatory signalling pathways, is localized beneath the cell membrane: it is especially prominent in mammalian oocytes. As in other cells, the cortex ensures appropriate shape and robustness of oocytes. It is also involved in other key and more specific functions. The cortex is part of the interface between the germinal and somatic cell compartments; as such, it participates in the delicate bi-directional interaction by which oocytes regulate cumulus cell function and in return, oocytes receive nutrients and regulative factors from cumulus cells. During oocyte maturation, fertilization, and early development, the cortex undergoes major structural and functional modifications. Such changes are needed to support crucial processes, including meiotic spindle localization, polar body extrusion, chromosome segregation, and pronuclear formation. Cortex dysregulation may be also implicated in blastomere fragmentation during early embryo development. Mechanical properties of the cortex are associated with oocyte quality and developmental competence; with appropriate technology, such properties could be harnessed to develop new approaches to non-invasive oocyte assessment in human IVF.

Keywords:  cortex; cytoskeleton; embryo; fertilization; meiosis; oocyte

DOI:  https://doi.org/10.1093/humrep/deaf208
EMBO Rep. 2025 Nov 11.

Local nuclear to cytoplasmic ratio regulates H3.3 incorporation via cell cycle state during zygotic genome activation.

Anusha D Bhatt, Madeleine G Brown, Aurora B Wackford, Yuki Shindo, Amanda A Amodeo.

  Early embryos often have unique chromatin states prior to zygotic genome activation (ZGA). In Drosophila, ZGA occurs after 13 reductive nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio grows exponentially. Previous work found that histone H3 chromatin incorporation decreases while its variant H3.3 increases leading up to ZGA. In other cell types, H3.3 is associated with sites of active transcription and heterochromatin, suggesting a link between H3.3 and ZGA. Here, we test what factors regulate H3.3 incorporation at ZGA. We find that H3 nuclear availability falls more rapidly than H3.3 leading up to ZGA. We generate H3/H3.3 chimeric proteins at the endogenous H3.3 A locus and observe that chaperone binding, but not gene structure, regulates H3.3 behavior. We identify the N/C ratio as a major determinant of H3.3 incorporation. To isolate how the N/C ratio regulates H3.3 incorporation we test the roles of genomic content, zygotic transcription, and cell cycle state. We determine that cell cycle regulation, but not H3 availability or transcription, controls H3.3 incorporation. Overall, we propose that local N/C ratios control histone variant usage via cell cycle state during ZGA.

Keywords:  Cell Cycle; Chromatin; Histones; Nuclear to Cytoplasmic Ratio; Transcription

DOI:  https://doi.org/10.1038/s44319-025-00596-1
Mol Hum Reprod. 2025 Nov 14. pii: gaaf056. [Epub ahead of print]

Phosphorylation of YWHAZ at Serine 58 by PKB/Akt is associated with resumption of meiotic arrest in mouse oocytes.

Jianchao Huo, Yifan Zhao, Zengyou Ma, Haoyi Zheng, Hui Peng.

  Mammalian oocyte maturation is a tightly regulated process essential for successful fertilisation and embryonic development. Meiotic resumption in mammalian oocytes is controlled by various regulatory factors, including the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHA/14-3-3). However, the specific functions of individual YWHA isoforms in oocyte meiosis remain poorly understood. In this study, we revealed that knockdown of Ywhaz, one of the isoforms of YWHA, using short interfering RNA (siRNA) or morpholino oligomers (MOs), accelerates meiotic resumption in mouse oocytes. To elucidate the mechanism underlying YWHAZ-mediated meiotic resumption, we thus explored its interactions with potential target proteins. Co-immunoprecipitation and immunofluorescence analyses demonstrated a physical interaction between YWHAZ and phosphorylated CDC25B. Additionally, we identified the protein kinases responsible for YWHAZ phosphorylation at distinct residues. Specifically, JNK1, CSNK1A1/CKIα and protein kinase B (PKB/Akt) were found to phosphorylate YWHAZ at Serine 184/186, Threonine 232 and Serine 58, respectively. Notably, phosphorylation of YWHAZ at serine 58 by PKB/Akt promoted meiotic resumption in mouse oocytes. Furthermore, we found the formation of a heterodimer between YWHAZ and YWHAQ. Our results provide insights into the PKB/Akt-YWHAZ-CDC25B signalling pathway and illuminate the functional influence of YWHAZ phosphorylation in meiotic regulation.

Keywords:  CDC25B; PKB/Akt; YWHAZ; meiosis; mouse; oocyte

DOI:  https://doi.org/10.1093/molehr/gaaf056
J Vis Exp. 2025 Oct 24.

Collection of Human Follicular Fluid, Follicle Somatic Cells, and Immature Oocytes from Individuals Undergoing In Vitro Fertilization.

Dilan Gokyer, Natasha Salpeter, Lydia Hughes, Hoi Chang Lee, Meredith J Lohman, Tomiris Atazhanova, Anna Kleinhans, Joan K Riley, MaryEllen Pavone, Francesca E Duncan, Emily Zaniker-Gomez, Elnur Babayev.

Human ovary follicular contents, including follicular fluid (FF), oocytes, cumulus cells, and somatic cells, offer a powerful yet underutilized opportunity to study ovarian physiology. Assisted reproductive technologies (ART) routinely generate these biological byproducts, which are often discarded after mature oocytes are used for clinical care. However, they provide valuable insights into fertility, reproductive aging, and ovarian dysfunction. Here, we present a standardized protocol for the systematic collection and downstream processing of follicular components from women undergoing oocyte retrieval. Fully expanded cumulus-oocyte complexes (COCs) are microdissected to isolate cumulus cells without enzymatic denudation, preserving transcriptomic integrity. FF is centrifuged, with the supernatant analyzed for cytokines, hormones, metabolites, and environmental toxicants, while the pellet is cryopreserved and processed via density gradient centrifugation to isolate viable granulosa cells. A stepwise protocol enables the preparation of single-cell suspensions from fresh FF for single-cell RNA sequencing (scRNA-Seq), including enzymatic digestion, serial filtration, and red blood cell lysis, and yields >65% viable cells representing a diverse composition, including granulosa cells, macrophages, T cells, smooth muscle-like cells, and antigen-presenting cells. In addition, immature germinal vesicle (GV) oocytes can be matured in vitro, with 56% reaching the MII stage within 24 h. Together, these protocols yield high-quality RNA, enriched granulosa cell populations, and complex single-cell datasets that capture the heterogeneity of the ovarian microenvironment. They provide a framework to leverage discarded reproductive tissues for translational research in human fertility and reproductive biology.

DOI: https://doi.org/10.3791/69122
Genetics. 2025 Nov 06. pii: iyaf241. [Epub ahead of print]

The ORB2 RNA-binding protein negatively regulates its target transcripts during the Drosophila maternal-to-zygotic transition via its functionally conserved Zinc-binding 'ZZ' domain.

Timothy C H Low, Hua Luo, Chalini Weerasooriya, Zhuyi Wang, Sichun Lin, Angelo Karaiskakis, Stephane Angers, Craig A Smibert, Howard D Lipshitz.

  Post-transcriptional regulation is particularly prominent during the maternal-to-zygotic transition (MZT), a developmental phase during which a large proportion of maternally provided mRNAs is repressed and cleared from metazoan embryos. RNA-binding proteins (RBPs) are key components of the post-transcriptional regulatory machinery. We show that the ORB2 RBP, the Drosophila ortholog of human Cytoplasmic Polyadenylation Element Binding Protein (hCPEB) 2-4 protein subfamily, binds to hundreds of maternally provided, rare-codon-enriched mRNAs in early embryos; that ORB2 targets are translationally repressed and unstable during the MZT; identify a U-rich motif enriched in ORB2 targets' 3'UTRs; and show that this motif confers ORB2 binding and repression to a luciferase reporter mRNA in S2 tissue culture cells. When tethered to a luciferase reporter, ORB2 and hCPEB2 (but not ORB and hCPEB1) repress translation; the C-terminal Zinc-binding ('ZZ') domain of ORB2 is necessary and sufficient for repression. ORB2 interacts with a suite of post-transcriptional regulators in early embryos; a subset of these interactions is lost upon deletion of the ZZ domain, notably with the Cup repressive complex. ORB2-targets significantly overlap with those previously identified for the repressive RBP, Smaug (SMG). Analysis of the early embryo's translatome in the presence or absence of the endogenous ZZ domain shows that mRNAs bound by ORB2 but not by SMG move onto polysomes upon ZZ domain deletion whereas co-bound transcripts do not, consistent with co-regulation of the latter set of transcripts by both RBPs. Our results assign a function to the ZZ domain and position ORB2 in the post-transcriptional network that regulates maternal transcripts during the Drosophila MZT.

Keywords:  Cup; Cytoplasmic Polyadenylation Element Binding Protein (CPEB); ORB2; RNA-binding protein (RBP); S2 cell; embryo; maternal-to-zygotic transition (MZT); post-transcriptional regulation; pre-initiation complex (PIC); translational regulation

DOI:  https://doi.org/10.1093/genetics/iyaf241
J Vis Exp. 2025 Oct 24.

Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture.

Hannes Campo, Prianka H Hashim, Emily J Zaniker-Gomez, Samuel Gauthier, Zihang Yan, Hao F Zhang, James A Ankrum, Francesca E Duncan.

The ovarian follicle is the functional unit of the ovary that produces hormones and gametes needed to sustain female reproductive function and health. The ability to recapitulate folliculogenesis, ovulation, and luteinization in vitro has broad basic, translational, and clinical utility. The most advanced in vitro follicle growth systems maintain the follicle's three-dimensional (3D) architecture, which is crucial for the development of meiotically competent metaphase II oocytes in humans. Recently, a scaffold-free method for in vitro follicle growth of mouse multilayer secondary follicles was developed and validated. For this, custom 3D printed molds were used to micropattern agarose with microwells that accommodate the volumetric expansion of follicles. Follicles grown in this scaffold-free environment showed comparable hormone production and viability relative to well-established alginate-based encapsulated in vitro follicle growth (eIVFG) systems. Importantly, agarose microwells are a scalable method, less technically demanding, and show improved follicle growth and ovulation rates relative to eIVFG. This methodology produces customizable molds that are biocompatible with the oocyte, a cell highly sensitive to material-specific leachates and other environmental contaminants. Further, follicles in this system are cultured in the same focal plane, enabling real-time timelapse imaging and analysis. To increase the accessibility of this new approach, this article details the methods needed to design and 3D-print master molds, create silicone molds for 24- or 96-well plates, and culture isolated multilayer secondary ovarian follicles in the agarose molds. This setup can also be integrated with a cost-effective time-lapse imaging system, enabling morphokinetic analysis. In addition, molds can be paraffin-embedded for downstream histological analyses. Overall, this user-friendly method is a versatile tool for follicle culture and can be customized further to promote the differentiation and maturation of germ cells within the context of the follicle to sustain complete in vitro gametogenesis.

DOI: https://doi.org/10.3791/68871
Hum Reprod. 2025 Nov 06. pii: deaf207. [Epub ahead of print]

Mitochondrial metabolism influences meiotic maturation in human oocytes of young and advanced maternal age women.

Sara Pietroforte, Marion Martins, Montserrat Barragan, Elena Ibañez, Rita Vassena, Mina Popovic, Tim Sanchez, Denny Sakkas, Filippo Zambelli.

   STUDY QUESTION: Is there a relationship between the mitochondrial activity and the meiotic progression of oocytes from germinal vesicle (GV) to metaphase II (MII) stages in young and advanced maternal age (AMA) women?
SUMMARY ANSWER: Poor mitochondrial metabolism impairs the meiotic progression of human GV oocytes, contributing to a lower oocyte maturation capacity of AMA oocytes.
WHAT IS KNOWN ALREADY: AMA oocytes are characterized by diminished quality, mostly due to the higher rates of chromosomal segregation errors occurring during meiosis I. Another hallmark of AMA oocytes is impaired mitochondrial metabolism. Studies in mice have suggested a link between metabolic dysfunction and meiotic failure, but this relationship has not been fully elucidated in humans. Metabolic dynamics can be visualized by indirect measurements through mitochondrial staining and quantified more directly using fluorescence lifetime imaging microscopy (FLIM). This live-imaging approach can generate metabolic timelapse profiles of oocytes throughout meiosis. In the present study, we explored mitochondrial distribution and functionality in human oocytes at the GV and MII stages, obtained from young and AMA women, to establish the role of mitochondrial metabolism in meiosis progression.
STUDY DESIGN, SIZE, DURATION: A total of 340 GV oocytes from young (≤34 years) and AMA (>37 years) women were included in the study. Denuded GVs were matured in vitro in G2-plus medium for 30 h. Maturation was determined by the presence of the extruded first polar body (PB1). The collected oocytes were processed for mitochondrial protein imaging (n = 80), or for live imaging (n = 171). Moreover, 89 oocytes were used for loss-of-function analysis by treating young GVs with 1 μM trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP) for 30 min before in vitro maturation.
PARTICIPANTS/MATERIALS, SETTING, METHODS: The proteins dihydrolipoamide-S-acetyltransferase (D-LAT) and translocase-of-outer mitochondrial-membrane (TOMM20) were analyzed in young and AMA oocytes by immunofluorescence to assess mitochondrial activity and localization, respectively. Fluorescence mean intensities (arbitrary-unit, AU) were quantified with ImageJ and compared by t-test; maturation rates were compared by chi-squared test. FLIM comprehensive metabolism (NAD(P)H; FAD+) was taken at GV stage. Different FLIM parameters (fluorescence intensity, fraction bound, short/long lifetime) and the Redox ratio (NAD(P)H intensity/FAD+ intensity) were evaluated.
MAIN RESULTS AND THE ROLE OF CHANCE: The findings revealed that active mitochondria are specifically localized in the subcortical area, while mitochondria in general are distributed across the whole oocyte. This pattern was substantially maintained in AMA oocytes, which were in turn characterized by a lower mitochondrial activity (D-LAT intensity of 78 614 ± 58 534 AU in young, 12 517 ± 10 187 AU in AMA, P = 0.003), while a lower number of mitochondria was observed In AMA patients but the difference did not reach statistical significance (TOMM20 intensity of 61 674 ± 24 322 AU in young, 32 186 ± 33 414 AU in AMA, P = 0.195). Using non-invasive FLIM, we assessed the metabolic dynamics of maturing oocytes (Redox ratio in young 2e + 00 ± 0.15, in AMA 1e + 00 ± 0.16, P = 2.969e-05), confirming a similar pattern observed by immunofluorescence. Specifically, FLIM microscopy revealed that GV oocytes from young women slightly increased their metabolism, by 4% on average, after the GV breakdown, and the increase was very consistent across different oocytes. On the contrary, in AMA maturing oocytes, little to no increase in metabolism was observed; they were characterized instead by higher variability, and more AMA oocytes failed to successfully reach the MII stage [AMA oocytes (62.3%; 38/61) compared with young oocytes (86.3%; 63/73; P = 0.002). These differential trends observed in AMA oocytes compared to the young oocytes suggest that impaired metabolic activity significantly compromises maturation capacity, revealing a functional link between adequate metabolic levels and successful meiosis progression.
LIMITATIONS, REASONS FOR CAUTION: Maturation rates were assessed by the presence of an extruded PB1 and variations in spindle assembly timings may have been overlooked. The quantification of mitochondrial activity in loss-of-function studies was assessed only by immunofluorescence staining. Additionally, the oocytes included in the present study were collected from women who underwent ovarian stimulation and may not faithfully recapitulate physiological maturation.
WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate the presence of a functional link between oocyte mitochondrial metabolism and meiosis progression, which may contribute to the decline of oocyte quality with aging. Overall, we provided evidence to understand the biological mechanisms in mitochondrial metabolism that might contribute to driving the decay in oocyte quality in AMA women.
STUDY FUNDING/COMPETING INTEREST(S): This project received intramural funding from the Eugin Group and funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 860960. T.S. is a former owner and former stock owner of Optiva Fertility Inc (company closed) and filed two patents for Optiva Fertility Inc (both abandoned). D.S.: Presenter EMD Senoro and Dep. Editor of Human Reproduction. All of the other authors (S.P., M.M., M.B., E.I., M.P., R.V., and F.Z.) have no conflicts of interest to declare. All of the authors contributed substantially to the manuscript and approve its submission.
TRIAL REGISTRATION NUMBER: N/A.

Keywords:  advanced maternal age; live imaging; metabolism; mitochondria; non-invasive technique; oocyte maturation

DOI:  https://doi.org/10.1093/humrep/deaf207
EMBO J. 2025 Nov 13.

A mammalian-like piRNA pathway in Axolotl reveals the origins of piRNA-directed DNA methylation.

Xinyu Xiang, Anni Gao, Dominik Handler, Francisco Falcon, Diego Rodriguez-Terrones, Sergej Nowoshilow, Wanlu Liu, Elly M Tanaka, Dónal O'Carroll.

  The piRNA pathway protects animal germlines from active transposons. Mammals employ a cytoplasmic pathway to destroy transposon transcripts during germline reprogramming. This post-transcriptional mechanism is ancient and found throughout the animal kingdom. A nuclear piRNA pathway mediates transposon DNA re-methylation, which is believed to be bespoke to mammals. However, when exactly piRNA-directed DNA methylation evolved remains unknown. We found that a mammalian-like piRNA pathway evolved early in tetrapod evolution and is found and expressed in its current configuration in the axolotl salamander. Analysis of axolotl testes and oocytes revealed diverse repertoires of piRNAs and pervasive post-transcriptional targeting of young transposons. We identified high levels of genome methylation in axolotl spermatozoa, with full-length transposons being heavily methylated. Our findings reveal that the mammalian nuclear piRNA pathway has ancient vertebrate origins, and it has likely been safeguarding the germline throughout most of tetrapod evolution. Thus, the emergence of piRNA-directed DNA methylation is a pivotal epigenetic evolutionary event that may have laid the foundation for germline reprogramming and genomic imprinting.

Keywords:  DNA Methylation; Genomic Imprinting; Germline; Germline Reprogramming; Transposon; piRNA

DOI:  https://doi.org/10.1038/s44318-025-00631-w
EMBO Rep. 2025 Nov 13.

A primordial germ cell-like-cell platform enables CRISPRi screen for epigenetic fertility modifiers.

Liangdao Li, Jingyi Gao, Dain Yi, Alex P Sheft, John C Schimenti, Xinbao Ding.

  Primordial germ cells (PGCs) are the precursors of gametes, and the ability to derive PGC-like cells (PGCLCs) from pluripotent stem cells has transformed germline research. A key limitation remains producing PGCLCs in sufficient numbers for large-scale applications. Here, we show that overexpression of Nanog plus three PGC master regulators - Prdm1, Prdm14, and Tfap2c - in mouse epiblast-like cells and formative embryonic stem cells yields abundant and highly enriched PGCLCs without costly recombinant cytokines. Nanog enhances the PGC regulatory network, suppresses somatic differentiation, and stabilizes PGCLC fate. Transcriptomically, these PGCLCs are developmentally more advanced than cytokine-induced counterparts and can be sustained long-term or differentiated into spermatogonia-like cells. Using this platform, we conduct a CRISPRi screen of 701 epigenetic genes to identify those needed for PGCLC formation. Downregulation of Ncor2, a histone deacetylase (HDAC) recruiter, has the greatest impact. Additionally, the HDAC inhibitors valproic acid and sodium butyrate suppress PGCLC formation and sperm counts of in utero-exposed animals. This work establishes a scalable system for functional screening of genes that influence germline development.

Keywords:  CRISPRi; Epigenome; Fertility; Histone Deacetylase Inhibition; PGCLCs

DOI:  https://doi.org/10.1038/s44319-025-00633-z
Chromosoma. 2025 Nov 10. 134(1): 10

The synaptonemal complex component corolla regulates meiotic crossover formation in Drosophila melanogaster.

Stefanie Williams, Grace McKown, Zulin Yu, Jennifer Gardner, Cynthia Staber, Matthew C Gibson, R Scott Hawley.

  In Drosophila melanogaster females, as in most organisms, the segregation of homologous chromosomes in meiosis depends on the formation of crossovers between them. In most cases, crossovers require the synaptonemal complex (SC), a conserved multi-protein structure that forms between homologous chromosomes in early meiosis. Recent studies leveraging partial-loss-of-function alleles suggest that the SC plays a more direct role in crossover formation. One SC protein that is involved in crossover formation is SYP-4 in nematodes, which we found is a likely ortholog of the D. melanogaster SC protein Corolla. To create a hypomorphic allele of corolla in D. melanogaster, we used CRISPR/Cas9 to replace it with its D. mauritiana ortholog, yielding corollamau. Since SC protein sequences are rapidly diverging while maintaining the SC's structure, we hypothesized that this replacement would enable SC assembly but show defects in crossover formation. Indeed, at 25 °C corollamau homozygous females exhibited defects in SC maintenance and crossover formation, resulting in moderate levels of chromosome missegregation. At 18 °C, SC maintenance was rescued, and recombination rates were improved, although they remained significantly lower than observed in wild type. Unexpectedly, in homozygotes we also observed unique polycomplexes composed of the SC proteins Corolla and Corona but lacking the transverse filament protein C(3)G. Overall, we report a novel hypomorphic allele of corolla that suggests Corolla regulates crossover formation. Further, the unique polycomplexes found in mutant flies may provide new insights into SC architecture and protein-protein interactions.

Keywords:  Crossover; Hypomorph; Meiosis; Recombination; Synaptonemal complex

DOI:  https://doi.org/10.1007/s00412-025-00839-z
Biochim Biophys Acta Mol Cell Res. 2025 Nov 09. pii: S0167-4889(25)00190-9. [Epub ahead of print] 120085

Developmental spatiotemporal switching of the germline-specific subunits of heterodimeric NAC protein in Drosophila melanogaster.

Elena A Mikhaleva, Natalia V Akulenko, Oxana M Olenkina, Yuri A Abramov, Sergey A Lavrov, Thoomas A Leinsoo, Sofia V Marfina, Georgij Arapidi, Victoria Shender, Sergei S Ryazansky, Galina L Kogan, Vladimir A Gvozdev.

  The ribosome-associated αβ heterodimeric ubiquitously expressed NAC protein is involved in protein homeostasis in eukaryotes. We previously reported that the germline-specific α and β subunits (gNACαβ) differ from ubiquitously expressed paralogs by the presence of extended intrinsically disordered regions. The embryonic precursors of the germline (pole cells) express both α and β gNAC subunits. CRISPR/Cas9-mediated knockout of the gNAC α- subunit gene resulted in the death of pole cells progenitors, implicating gNAC as an essential component of the germ plasm responsible for the germline development. Immunofluorescence detection was used to track changes in the expression of α- and β-subunits of gNAC during development. The bright fluorescence of the α-subunit in the germarium strongly decreases during oocyte specification and is replaced by the onset of increased β-subunit fluorescence, especially in the posterior part of mature oocytes, in the germ plasm region, where NACα-subunit fluorescent signal is absent. The visualized switches of gNAC subunits presence, as well as detection of separate cytoplasmic fluorescent puncta for α and β gNAC subunits in a germline cells (elongating spermatids in testis or embryonic pole cells), are explained by transient formation the hybrid NAC heterodimers composed of α or β germline subunit and a ubiquitous partner. The formation of hybrids, demonstrated by mass spectrometry analysis in testes, indicates a programed spatiotemporal functioning of both the ubiquitous and germline-specific NAC paralogs to support the germline-specific proteostasis.

Keywords:  Centrosome; Germline; IDP; Paralog switching; Proteostasis

DOI:  https://doi.org/10.1016/j.bbamcr.2025.120085
Hum Reprod. 2025 Nov 06. pii: deaf196. [Epub ahead of print]

Investigating metabolic activity during oocyte and early embryo development through label-free metabolic imaging: a systematic approach for timelapse applications.

F Horta, A Vuyyuru, H Newman, G Ballerin, S Mercer, E Rolfe, M Haft-Tananian, M Pangestu, P Temple-Smith, B Vollenhoven, R B Gilchrist, S Catt.

   STUDY QUESTION: Is it possible to assess label-free live cell metabolic imaging during early oocyte and embryo development?
SUMMARY ANSWER: Label-free metabolic imaging can be systematically used during early development, showing no differences between controls and illuminated oocytes and embryos in terms of early development, blastocyst formation, and embryo outgrowth.
WHAT IS KNOWN ALREADY: Non-invasive methods that are reliable to assess oocyte and embryo quality are a significant aim for ARTs. Changes in metabolic activity could lead to cell death or altered early development and low implantation potential. This could potentially be predicted by incorporating non-invasive measurements of metabolism. Metabolic imaging has been investigated through complex methodologies; however, scientific evidence for its utility during early oocyte and embryo development requires further investigation to assess potential translation in clinical settings. Measurements of metabolic activity could be a useful tool, as the autofluorescence of molecules such as nicotinamide adenine dinucleotide phosphate hydrogen (NAD(P)H) and flavin adenine dinucleotide (FAD) are a straightforward representation of mitochondrial function.
STUDY DESIGN, SIZE, DURATION: Female mice (n = 15) and super-ovulated female mice (n = 30) were used to produce oocytes and embryos, respectively. Oocytes and in-vivo produced embryos were divided into the control group, sham control group, and illuminated group. Illuminated samples were assessed for both NAD(P)H and FAD levels in oocytes and NAD(P)H levels during early embryo development every 3 h using arbitrary units of autofluorescence (AU). Produced blastocysts were assessed for total cell and inner-cell-mass (ICM) number (by immunostaining for Oct4) and embryo outgrowth assays. Furthermore, safety live birth studies were also conducted.
PARTICIPANTS/MATERIALS, SETTING, METHODS: F1 (C57BL6/CBA) mouse strain was used. NAD(P)H and FAD autofluorescence levels were measured during oocyte and embryo development using confocal microscopy (Olympus FV1200). A confocal Z-stacking function was used to record 15 focal planes, using a 20×/0.95 NA air objective of the entire oocytes and embryos and opening the confocal pinhole system completely. Images were then collected and analysed using FIJI software (version: 2.0.0-rc-69/1.52n; ImageJ). Developmental rates, blastocyst cell numbers, outgrowth rates (for 4 days post blastocyst formation), and live birth rates were assessed.
MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte IVM and embryo culture experiments showed no significant differences in developmental rates between study groups (P > 0.05). Similarly, the total number of cells from blastocysts (control: 82.9 ± 5.6; sham: 76.5 ± 3.3; Illuminated: 77.1 ± 4.2; ± SEM) and ICM cells (control: 10.8 ± 1.3; sham: 9.4 ± 0.7; Illuminated: 11.9 ± 0.8; ± SEM) did not differ between groups (P > 0.05). Outgrowth assays of the study groups presented similar outgrowth areas during Days 5-8 (post) blastocyst development (P > 0.05). Illumination of oocytes demonstrated a significant increase in metabolic activity during IVM, measured by the optical redox ratio (ORR: FAD/NAD(P)H + FAD; P < 0.001). Illumination of embryos demonstrated significantly different NAD(P)H activity levels during embryo development, particularly between the two-cell stage (987.1 ± 36.2 AU), morula stage (1226.0 ± 31.5 AU) and blastocyst stage (649 ± 42.9 AU; ± SEM; P < 0.05). Additionally, embryos that did not form blastocysts also presented significantly decreased NAD(P)H activity levels at the two-cell stage (normal development: 987.1 ± 36.2; no blastocyst: 726.9 ± 121.7 AU; P < 0.05) to the morula stage (normal development: 1226.0 ± 31.5; no blastocyst: 886.0 ± 150.4 AU; P < 0.05) when compared with normally developing embryos. Our study indicated that metabolic imaging during early oocyte and embryo development presents no negative effects on developmental rates, blastocyst quality, and embryo outgrowths. Subsequently, live birth rates and offspring health showed no differences between controls and illuminated embryos at the blastocyst stage. Current results provide significant useful information about metabolic activity during live cell imaging as a potential method for timelapse metabolic imaging.
LARGE SCALE DATA: N/A.
LIMITATIONS, REASONS FOR CAUTION: The study was conducted using a mouse model and focused on early oocyte and embryo development, embryo outgrowths, live birth, and early offspring health. Thus, further studies of long-term offspring health are required to fully assess safety and to further validate potential wider applications. Validation in ageing models is also required to assess potential applications for embryo selection.
WIDER IMPLICATIONS OF THE FINDINGS: Measurements of metabolic activity could be applied to determine oocyte and embryo metabolic activity using a variety of microscopy technology with low energy doses as described in this study. Further applications could link the use of metabolic imaging with timelapse technology and artificial intelligence applications to monitor culture conditions.
STUDY FUNDING/COMPETING INTEREST(S): This study was funded in part by a research/educational grant from Ferring Pharmaceuticals, awarded from the Fertility Society of Australia and New Zealand (FSANZ). Funding was also provided in part by the Education Program in Reproduction and Development (EPRD), Department of Obstetrics and Gynaecology, Monash University. F.H. and M.H.-T. have applied for a patent in the topic of metabolic imaging. R.B.G. declares speakers' fees from Gedeon Richter and Ferring. The other authors have nothing to declare.

Keywords:  ART; early development; embryology; metabolic imaging; oocytes; timelapse-imaging

DOI:  https://doi.org/10.1093/humrep/deaf196