bims-cebooc Biomed News
on Cell biology of oocytes
Issue of 2024–09–08
25 papers selected by
Gabriele Zaffagnini, Centre for Genomic Regulation
  1. Transcriptomic analysis of the spatiotemporal axis of oogenesis and fertilization in C. elegans.
  2. Nsun2 is critical for mouse oocyte meiosis and early embryonic development.
  3. The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.
  4. Distinct checkpoint and homolog biorientation pathways regulate meiosis I in Drosophila oocytes.
  5. Calcium signaling in oocyte quality and functionality and its application.
  6. Spindle morphology changes between meiosis and mitosis driven by CK2 regulation of the Ran pathway.
  7. Single-nucleus atlas of the Artemia female reproductive system suggests germline repression of the Z chromosome.
  8. Transcriptomic analysis of meiotic genes during the mitosis-to-meiosis transition in Drosophila females.
  9. Integrin signaling in pluripotent cells acts as a gatekeeper of mouse germline entry.
  10. Self-organizing ovarian somatic organoids preserve cellular heterogeneity and reveal cellular contributions to ovarian aging.
  11. A novel heterozygous missense variant of PANX1 causes human oocyte death and female infertility.
  12. Biallelic variants in α-tubulin isotypes cause female infertility characterised as recurrent preimplantation embryo arrest.
  13. Identification of the Polo-like kinase substrate required for homologous synapsis in C. elegans.
  14. TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis.
  15. In vitro production of viable eggs from undeveloped oocytes in mouse preantral follicles by reconstructing granulosa cell-oocyte complexes†.
  16. Unveiling inter-embryo variability in spindle length over time: Towards quantitative phenotype analysis.
  17. Generation of rat offspring from ovarian oocytes by xenotransplantation.
  18. A chromosome-coupled ubiquitin-proteasome pathway is required for meiotic surveillance.
  19. A sperm-enriched 5'fragment of tRNA-Valine regulates preimplantation embryonic transcriptome and development.
  20. pSTAT3 activation of Foxl2 initiates the female pathway underlying temperature-dependent sex determination.
  21. Quantifying DNA damage following light sheet and confocal imaging of the mammalian embryo.
  22. Genetic regulation of ovulation rate and multiple births.
  23. Traffic Jam activates the Flamenco piRNA cluster locus and the Piwi pathway to ensure transposon silencing and Drosophila fertility.
  24. 5-Formylcytosine is an activating epigenetic mark for RNA Pol III during zygotic reprogramming.
  25. Automatic ploidy prediction and quality assessment of human blastocysts using time-lapse imaging.
  1. Front Cell Dev Biol. 2024 ;12 1436975
    Transcriptomic analysis of the spatiotemporal axis of oogenesis and fertilization in C. elegans.
    Yangqi Su, Jonathan Shea, Darla Destephanis, Zhengchang Su.
      Caenorhabditis elegans hermaphrodite presents a unique model to study the formation of oocytes. However, the size of the model animal and difficulties in retrieval of specific stages of the germline have obviated closer systematic studies of this process throughout the years. Here, we present a transcriptomic level analysis into the oogenesis of C. elegans hermaphrodites. We dissected a hermaphrodite gonad into seven sections corresponding to the mitotic distal region, the pachytene region, the diplotene region, the early diakinesis region and the 3 most proximal oocytes, and deeply sequenced the transcriptome of each of them along with that of the fertilized egg using a single-cell RNA-seq (scRNA-seq) protocol. We identified specific gene expression events as well as gene splicing events in finer detail along the gonad and provided novel insights into underlying mechanisms of the oogenesis process. Furthermore, through careful review of relevant research literature coupled with patterns observed in our analysis, we delineate transcripts that may serve functions in the interactions between the germline and cells of the somatic gonad. These results expand our knowledge of the transcriptomic space of the C. elegans germline and lay a foundation on which future studies of the germline can be based upon.
    Keywords:  C. elegans; ScRNA-seq; oocyte; oogenesis; single cell; transcriptome
    DOI:  https://doi.org/10.3389/fcell.2024.1436975
  2. Reproduction. 2024 Sep 01. pii: REP-24-0235. [Epub ahead of print]
    Nsun2 is critical for mouse oocyte meiosis and early embryonic development.
    Xu Tengteng, Min Gao, Ling Zhang, Tianqi Cao, Yanling Qiu, Simiao Liu, Wenlian Wu, Yitong Zhou, Haiying Liu, Rui Zhang, Xiaohong Ruan, Junjiu Huang.
      Biallelic variants in the NSUN2 gene cause a rare intellectual disability and female infertility in humans. However, the function and mechanism of NSUN2 during mouse oocyte meiotic maturation and early embryonic development are unknown. Here, we show that NSUN2 is important for mouse oocyte meiotic maturation and early embryonic development. Specifically, NSUN2 is required for ovarian development and oocyte meiosis, and deletion of Nsun2 reduces oocyte maturation and increases the rates of misaligned chromosomes and aberrant spindles. In addition, Nsun2 deficiency results in a low blastocyst rate and impaired blastocyst quality. Strikingly, loss of Nsun2 leads to approximately 35% of embryos being blocked at the 2-cell stage, and Nsun2 knockdown impairs zygotic genome activation at the 2-cell stage. Taken together, these findings suggest that NSUN2 plays a critical role in mouse oocyte meiotic maturation and early embryonic development, and provide key resources for elucidating female infertility with NSUN2 mutations.
    DOI:  https://doi.org/10.1530/REP-24-0235
  3. PLoS Genet. 2024 Sep 03. 20(9): e1011373
    The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.
    Emily R Czajkowski, Yuntong Zou, Nikita S Divekar, Sarah M Wignall.
      Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.
    DOI:  https://doi.org/10.1371/journal.pgen.1011373
  4. bioRxiv. 2024 Aug 21. pii: 2024.08.21.608908. [Epub ahead of print]
    Distinct checkpoint and homolog biorientation pathways regulate meiosis I in Drosophila oocytes.
    Joanatta G Shapiro, Neha Changela, Janet K Jang, Jay N Joshi, Kim S McKim.
      Mitosis and meiosis have two mechanisms for regulating the accuracy of chromosome segregation: error correction and the spindle assembly checkpoint (SAC). We have investigated the function of several checkpoint proteins in meiosis I of Drosophila oocytes. Evidence of a SAC response by several of these proteins is found upon depolymerization of microtubules by colchicine. However, unattached kinetochores or errors in biorientation of homologous chromosomes does not induce a SAC response. Furthermore, the metaphase I arrest does not depend on SAC genes, suggesting the APC is inhibited even if the SAC is silenced. Two SAC proteins, ROD of the ROD-ZW10-Zwilch (RZZ) complex and MPS1, are also required for the biorientation of homologous chromosomes during meiosis I, suggesting an error correction function. Both proteins aid in preventing or correcting erroneous attachments and depend on SPC105R for localization to the kinetochore. We have defined a region of SPC105R, amino acids 123-473, that is required for ROD localization and biorientation of homologous chromosomes at meiosis I. Surprisingly, ROD removal, or "streaming", is independent of the dynein adaptor Spindly and is not linked to the stabilization of end-on attachments. Instead, meiotic RZZ streaming appears to depend on cell cycle stage and may be regulated independently of kinetochore attachment or biorientation status. We also show that dynein adaptor Spindly is also required for biorientation at meiosis I, and surprisingly, the direction of RZZ streaming.
    Author Summary: The Spindle Assembly Checkpoint (SAC) is known to delay cell cycle progression until chromosomes are properly attached to microtubules. Meiotic cells often have modified cell cycle phases, and natural arrest points such as metaphase I in Drosophila . We show that in Drosophila oocytes, the SAC is sensitive to loss of microtubules, but not sensitive to a variety of kinetochore attachment errors. Thus, the function of the SAC appears to be limited to monitoring oocyte spindle assembly, and not required for accurate chromosome segregation. However, two of the SAC genes, rod and Mps1 , are required for the biorientation of homologous chromosomes during meiosis I, suggesting an error correction function. Rod is part of the RZZ complex and is notable for its property of streaming off the kinetochores. However, our results show that streaming off the kinetochore may not contribute to RZZ regulation of microtubule attachments, and only be associated with SAC function. Instead, the establishment of stable end-on attachments may occur while RZZ is still present at kinetochore. We suggest that RZZ interacts with multiple motors to promote bidirectional movement of kinetochores along microtubules, which allows chromosomes to find and attach to the correct pole.
    DOI:  https://doi.org/10.1101/2024.08.21.608908
  5. Front Endocrinol (Lausanne). 2024 ;15 1411000
    Calcium signaling in oocyte quality and functionality and its application.
    Chen Chen, Zefan Huang, Shijue Dong, Mengqian Ding, Jinran Li, Miaomiao Wang, Xuhui Zeng, Xiaoning Zhang, Xiaoli Sun.
      Calcium (Ca2+) is a second messenger for many signal pathways, and changes in intracellular Ca2+ concentration ([Ca2+]i) are an important signaling mechanism in the oocyte maturation, activation, fertilization, function regulation of granulosa and cumulus cells and offspring development. Ca2+ oscillations occur during oocyte maturation and fertilization, which are maintained by Ca2+ stores and extracellular Ca2+ ([Ca2+]e). Abnormalities in Ca2+ signaling can affect the release of the first polar body, the first meiotic division, and chromosome and spindle morphology. Well-studied aspects of Ca2+ signaling in the oocyte are oocyte activation and fertilization. Oocyte activation, driven by sperm-specific phospholipase PLCζ, is initiated by concerted intracellular patterns of Ca2+ release, termed Ca2+ oscillations. Ca2+ oscillations persist for a long time during fertilization and are coordinately engaged by a variety of Ca2+ channels, pumps, regulatory proteins and their partners. Calcium signaling also regulates granulosa and cumulus cells' function, which further affects oocyte maturation and fertilization outcome. Clinically, there are several physical and chemical options for treating fertilization failure through oocyte activation. Additionally, various exogenous compounds or drugs can cause ovarian dysfunction and female infertility by inducing abnormal Ca2+ signaling or Ca2+ dyshomeostasis in oocytes and granulosa cells. Therefore, the reproductive health risks caused by adverse stresses should arouse our attention. This review will systematically summarize the latest research progress on the aforementioned aspects and propose further research directions on calcium signaling in female reproduction.
    Keywords:  Ca2+ oscillations; calcium; female fertility; fertilization; oocyte activation; oocyte maturation
    DOI:  https://doi.org/10.3389/fendo.2024.1411000
  6. bioRxiv. 2024 Jul 25. pii: 2024.07.25.605073. [Epub ahead of print]
    Spindle morphology changes between meiosis and mitosis driven by CK2 regulation of the Ran pathway.
    Helena Cantwell, Hieu Nguyen, Arminja Kettenbach, Rebecca Heald.
      The transition from meiotic divisions in the oocyte to embryonic mitoses is a critical step in animal development. Despite negligible changes to cell size and shape, following fertilization the small, barrel-shaped meiotic spindle is replaced by a large zygotic spindle that nucleates abundant astral microtubules at spindle poles. To probe underlying mechanisms, we applied a drug screening approach using Ciona eggs and found that inhibition of Casein Kinase 2 (CK2) caused a shift from meiotic to mitotic-like spindle morphology with nucleation of robust astral microtubules, an effect reproduced in cytoplasmic extracts prepared from Xenopus eggs. In both species, CK2 activity decreased at fertilization. Phosphoproteomic differences between Xenopus meiotic and mitotic extracts that also accompanied CK2 inhibition pointed to RanGTP-regulated factors as potential targets. Interfering with RanGTP-driven microtubule formation suppressed astral microtubule growth caused by CK2 inhibition. These data support a model in which CK2 activity attenuation at fertilization leads to activation of RanGTP-regulated microtubule effectors that induce mitotic spindle morphology.
    DOI:  https://doi.org/10.1101/2024.07.25.605073
  7. PLoS Genet. 2024 Aug 30. 20(8): e1011376
    Single-nucleus atlas of the Artemia female reproductive system suggests germline repression of the Z chromosome.
    Marwan Elkrewi, Beatriz Vicoso.
      Our understanding of the molecular pathways that regulate oogenesis and define cellular identity in the Arthropod female reproductive system and the extent of their conservation is currently very limited. This is due to the focus on model systems, including Drosophila and Daphnia, which do not reflect the observed diversity of morphologies, reproductive modes, and sex chromosome systems. We use single-nucleus RNA and ATAC sequencing to produce a comprehensive single nucleus atlas of the adult Artemia franciscana female reproductive system. We map our data to the Fly Cell Atlas single-nucleus dataset of the Drosophila melanogaster ovary, shedding light on the conserved regulatory programs between the two distantly related Arthropod species. We identify the major cell types known to be present in the Artemia ovary, including germ cells, follicle cells, and ovarian muscle cells. Additionally, we use the germ cells to explore gene regulation and expression of the Z chromosome during meiosis, highlighting its unique regulatory dynamics and allowing us to explore the presence of meiotic sex chromosome silencing in this group.
    DOI:  https://doi.org/10.1371/journal.pgen.1011376
  8. Genetics. 2024 Sep 03. pii: iyae130. [Epub ahead of print]
    Transcriptomic analysis of meiotic genes during the mitosis-to-meiosis transition in Drosophila females.
    Ana Maria Vallés, Thomas Rubin, Nicolas Macaisne, Laurine Dal Toe, Anahi Molla-Herman, Christophe Antoniewski, Jean-René Huynh.
      Germline cells produce gametes, which are specialized cells essential for sexual reproduction. Germline cells first amplify through several rounds of mitosis before switching to the meiotic program, which requires specific sets of proteins for DNA recombination, chromosome pairing, and segregation. Surprisingly, we previously found that some proteins of the synaptonemal complex, a prophase I meiotic structure, are already expressed and required in the mitotic region of Drosophila females. Here, to assess if additional meiotic genes were expressed earlier than expected, we isolated mitotic and meiotic cell populations to compare their RNA content. Our transcriptomic analysis reveals that all known meiosis I genes are already expressed in the mitotic region; however, only some of them are translated. As a case study, we focused on mei-W68, the Drosophila homolog of Spo11, to assess its expression at both the mRNA and protein levels and used different mutant alleles to assay for a premeiotic function. We could not detect any functional role for Mei-W68 during homologous chromosome pairing in dividing germ cells. Our study paves the way for further functional analysis of meiotic genes expressed in the mitotic region.
    Keywords:  Spo11; cell cycle; development; germline; oocyte; recombination
    DOI:  https://doi.org/10.1093/genetics/iyae130
  9. Sci Adv. 2024 Sep 06. 10(36): eadk2252
    Integrin signaling in pluripotent cells acts as a gatekeeper of mouse germline entry.
    Aly Makhlouf, Anfu Wang, Nanami Sato, Viviane S Rosa, Marta N Shahbazi.
      Primordial germ cells (PGCs) are the precursors of gametes and the sole mechanism by which animals transmit genetic information across generations. In the mouse embryo, the transcriptional and epigenetic regulation of PGC specification has been extensively characterized. However, the initial event that triggers the soma-germline segregation remains poorly understood. Here, we uncover a critical role for the basement membrane in regulating germline entry. We show that PGCs arise in a region of the mouse embryo that lacks contact with the basement membrane, and the addition of exogenous extracellular matrix (ECM) inhibits both PGC and PGC-like cell (PGCLC) specification in mouse embryos and stem cell models, respectively. Mechanistically, we demonstrate that the engagement of β1 integrin with laminin blocks PGCLC specification by preventing the Wnt signaling-dependent down-regulation of the PGC transcriptional repressor, Otx2. In this way, the physical segregation of cells away from the basement membrane acts as a morphogenetic fate switch that controls the soma-germline bifurcation.
    DOI:  https://doi.org/10.1126/sciadv.adk2252
  10. bioRxiv. 2024 Aug 10. pii: 2024.08.10.607456. [Epub ahead of print]
    Self-organizing ovarian somatic organoids preserve cellular heterogeneity and reveal cellular contributions to ovarian aging.
    Shweta S Dipali, Madison Q Gowett, Pratik Kamat, Aubrey Converse, Emily J Zaniker, Abigail Fennell, Teresa Chou, Michele T Pritchard, Mary Zelinski, Jude M Phillip, Francesca E Duncan.
      Ovarian somatic cells are essential for reproductive function, but no existing ex vivo models recapitulate the cellular heterogeneity or interactions within this compartment. We engineered a novel ovarian somatic organoid model by culturing a stroma-enriched fraction of mouse ovaries in scaffold-free agarose micromolds. Ovarian somatic organoids self-organized, maintained diverse cell populations, produced extracellular matrix, and secreted hormones. Organoids generated from reproductively old mice exhibited reduced aggregation and growth compared to young counterparts, as well as differences in cellular composition. Interestingly, matrix fibroblasts from old mice demonstrated upregulation of pathways associated with the actin cytoskeleton and downregulation of cell adhesion pathways, indicative of increased cellular stiffness which may impair organoid aggregation. Cellular morphology, which is regulated by the cytoskeleton, significantly changed with age and in response to actin depolymerization. Moreover, actin depolymerization rescued age-associated organoid aggregation deficiency. Overall, ovarian somatic organoids have advanced fundamental knowledge of cellular contributions to ovarian aging.
    DOI:  https://doi.org/10.1101/2024.08.10.607456
  11. J Ovarian Res. 2024 Sep 04. 17(1): 180
    A novel heterozygous missense variant of PANX1 causes human oocyte death and female infertility.
    Juepu Zhou, Ruolin Mao, Meng Wang, Rui Long, Limin Gao, Xiangfei Wang, Lei Jin, Lixia Zhu.
      Pannexin1 (PANX1) is a highly glycosylated membrane channel-forming protein, which has been found to implicate in multiple physiological and pathophysiological functions. Variants in the PANX1 gene have been reported to be associated with oocyte death and recurrent in vitro fertilization failure. In this study, we identified a novel heterozygous PANX1 variant (NM_015368.4 c.410 C > T (p.Ser137Leu)) associated with the phenotype of oocyte death in a non-consanguineous family, followed by an autosomal dominant (AD) mode. We explored the molecular mechanism of the novel variant and the variant c.976_978del (p.Asn326del) that we reported previously. Both of the variants altered the PANX1 glycosylation pattern in cultured cells, led to aberrant PANX1 channel activation, affected ATP release and membrane electrophysiological properties, which resulted in mouse and human oocyte death in vitro. For the first time, we presented the direct evidence of the effect of the PANX1 variants on human oocyte development. Our findings expand the variant spectrum of PANX1 genes associated with oocyte death and provide new support for the genetic diagnosis of female infertility.
    Keywords:   PANX1 ; Female infertility; Oocyte death; Variant
    DOI:  https://doi.org/10.1186/s13048-024-01462-9
  12. J Med Genet. 2024 Aug 29. pii: jmg-2024-110163. [Epub ahead of print]
    Biallelic variants in α-tubulin isotypes cause female infertility characterised as recurrent preimplantation embryo arrest.
    Huiling Hu, Xian Wan, Honghui Zhang, Jiaqi Sun, Fei Meng, Shuoping Zhang, Yifan Gu, Fei Gong, Han Zhao, Ge Lin, Wei Zheng.
       BACKGROUND: Recurrent preimplantation embryo developmental arrest (RPEA) is the most common phenotype in assisted reproductive technology treatment failure associated with identified genetic abnormalities. Currently known maternal genetic variants explain only a limited number of cases. Variants of the β-tubulin subunit gene, TUBB8, cause oocyte meiotic arrest and RPEA through a broad spectrum of spindle defects. In contrast, α-tubulin subunit genes are poorly studied in the context of preimplantation embryonic development.
    METHODS: Whole exome sequencing was performed on the PREA cohort. Functional characterisations of the identified candidate disease-causing variants were validated using Sanger sequencing, bioinformatics, in vitro functional analyses and single-cell RNA-sequencing of arrested embryos.
    RESULTS: Four homozygous variants were identified in the PREA cohort: two of TUBA1C (p.Gln358Ter and p.Asp444Metfs*42) and two of TUBA4A (p.Arg339Cys and p.Tyr440Ter). These variants cause varying degrees of spindle assembly defects. Additionally, we characterised changes in the human arrested embryo transcriptome carrying TUBA4A variants, with a particular focus on spindle organisation, chromosome segregation and mRNA decay.
    CONCLUSION: Our findings identified TUBA1C as a novel genetic marker and expanded the genetic and phenotypic spectrum of TUBA4A in female infertility and RPEA, which altogether highlighted the importance of α-tubulin isotypes in preimplantation embryonic development.
    Keywords:  Genetic Variation; Women's Health
    DOI:  https://doi.org/10.1136/jmg-2024-110163
  13. bioRxiv. 2024 Aug 13. pii: 2024.08.13.607834. [Epub ahead of print]
    Identification of the Polo-like kinase substrate required for homologous synapsis in C. elegans.
    Ariel L Gold, Matthew E Hurlock, Alicia M Guevara, Lilah Y Z Isenberg, Yumi Kim.
      The synaptonemal complex (SC) is a zipper-like protein structure that aligns homologous chromosome pairs and regulates recombination during meiosis. Despite its conserved appearance and function, how synapsis occurs between chromosome axes remains elusive. Here, we demonstrate that Polo-like kinases (PLKs) phosphorylate a single conserved residue in the disordered C-terminal tails of two paralogous SC subunits, SYP-5 and SYP-6, to establish an electrostatic interface between the SC central region and chromosome axes in C. elegans . While SYP-5/6 phosphorylation is dispensable for the ability of SC proteins to self-assemble, local phosphorylation by PLKs at the pairing center is crucial for SC elongation between homologous chromosome axes. Additionally, SYP-5/6 phosphorylation is essential for asymmetric SC disassembly and proper PLK-2 localization after crossover designation, which drives chromosome remodeling required for homolog separation during meiosis I. This work identifies a key regulatory mechanism by which localized PLK activity mediates the SC-axis interaction through phosphorylation of SYP-5/6, coupling synapsis initiation to homolog pairing.
    DOI:  https://doi.org/10.1101/2024.08.13.607834
  14. Elife. 2024 Aug 29. pii: RP92195. [Epub ahead of print]12
    TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis.
    Jessica Y Chotiner, N Adrian Leu, Fang Yang, Isabella G Cossu, Yongjuan Guan, Huijuan Lin, P Jeremy Wang.
      Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. Mouse TRIP13 and its ortholog Pch2 are instrumental in remodeling HORMA domain proteins. HORMAD proteins are associated with unsynapsed chromosome axes but depleted from the synaptonemal complex (SC) of synapsed homologs. Here we report that TRIP13 localizes to the synapsed SC in early pachytene spermatocytes and to telomeres throughout meiotic prophase I. Loss of TRIP13 leads to meiotic arrest and thus sterility in both sexes. Trip13-null meiocytes exhibit abnormal persistence of HORMAD1 and HOMRAD2 on synapsed SC and chromosome asynapsis that preferentially affects XY and centromeric ends. These major phenotypes are consistent with reported phenotypes of Trip13 hypomorph alleles. Trip13 heterozygous mice exhibit meiotic defects that are less severe than the Trip13-null mice, showing that TRIP13 is a dosage-sensitive regulator of meiosis. Localization of TRIP13 to the synapsed SC is independent of SC axial element proteins such as REC8 and SYCP2/SYCP3. Terminal FLAG-tagged TRIP13 proteins are functional and recapitulate the localization of native TRIP13 to SC and telomeres. Therefore, the evolutionarily conserved localization of TRIP13/Pch2 to the synapsed chromosomes provides an explanation for dissociation of HORMA domain proteins upon synapsis in diverse organisms.
    Keywords:  TRIP13; cell biology; fertility; meiosis; mouse; spermatogenesis
    DOI:  https://doi.org/10.7554/eLife.92195
  15. Biol Reprod. 2024 Aug 21. pii: ioae125. [Epub ahead of print]
    In vitro production of viable eggs from undeveloped oocytes in mouse preantral follicles by reconstructing granulosa cell-oocyte complexes†.
    Tomohiro Kohama, Ikuo Tomioka, Kanako Morohaku.
      In vitro culture of ungrown oocytes in preantral follicles is one of the intriguing subjects being pursued to produce viable eggs in assisted reproductive technology. Previous studies have succeeded in obtaining mature eggs after in vitro culture of preantral follicles, while denuded undeveloped oocytes, which are obtained occasionally when collecting preantral follicles, seem to be almost useless. Moreover, methods to culture them efficiently to produce viable eggs have not been established yet. The present study was conducted to demonstrate in vitro culture of mouse denuded undeveloped oocytes by reconstructing granulosa cell-oocyte complexes, and to analyze cellular communication in reconstructed granulosa cell-oocyte complexes. Single denuded undeveloped oocytes were aggregated with 1 × 104 granulosa cells in wells with U-shaped bottoms in a low-binding cell culture plate for 8 days under either 20% or 5% O2, and then the reconstructed granulosa cell-oocyte complexes formed were cultured on a collagen-coated culture membrane insert for 4 days under 5% O2. At day 8 of culture, the rates of reconstructed granulosa cell-oocyte complexes formation were significantly higher in the culture group under 5% O2 (64.9%) than that under 20% O2 (42.3%; P < 0.001); furthermore, the formation of transzonal projections was observed. After maturation and fertilization, we produced matured eggs and blastocysts at higher rates (>90% and 61.9%, respectively) in the group cultured under 5% O2. After transferring 126 two- to four-cell stage embryos, six live pups were obtained. This is the first report that demonstrates production of viable eggs after in vitro culture of denuded undeveloped oocytes from preantral follicles by reconstruction of granulosa cell-oocyte complexes.
    Keywords:  granulosa cell–oocyte complexes; in vitro culture; reconstructed GOCs; viable eggs
    DOI:  https://doi.org/10.1093/biolre/ioae125
  16. PLoS Comput Biol. 2024 Sep;20(9): e1012330
    Unveiling inter-embryo variability in spindle length over time: Towards quantitative phenotype analysis.
    Yann Le Cunff, Laurent Chesneau, Sylvain Pastezeur, Xavier Pinson, Nina Soler, Danielle Fairbrass, Benjamin Mercat, Ruddi Rodriguez-Garcia, Zahraa Alayan, Ahmed Abdouni, Gary de Neidhardt, Valentin Costes, Mélodie Anjubault, Hélène Bouvrais, Christophe Héligon, Jacques Pécréaux.
      How can inter-individual variability be quantified? Measuring many features per experiment raises the question of choosing them to recapitulate high-dimensional data. Tackling this challenge on spindle elongation phenotypes, we showed that only three typical elongation patterns describe spindle elongation in C. elegans one-cell embryo. These archetypes, automatically extracted from the experimental data using principal component analysis (PCA), accounted for more than 95% of inter-individual variability of more than 1600 experiments across more than 100 different conditions. The two first archetypes were related to spindle average length and anaphasic elongation rate. The third archetype, accounting for 6% of the variability, was novel and corresponded to a transient spindle shortening in late metaphase, reminiscent of kinetochore function-defect phenotypes. Importantly, these three archetypes were robust to the choice of the dataset and were found even considering only non-treated conditions. Thus, the inter-individual differences between genetically perturbed embryos have the same underlying nature as natural inter-individual differences between wild-type embryos, independently of the temperatures. We thus propose that beyond the apparent complexity of the spindle, only three independent mechanisms account for spindle elongation, weighted differently in the various conditions. Interestingly, the spindle-length archetypes covered both metaphase and anaphase, suggesting that spindle elongation in late metaphase is sufficient to predict the late anaphase length. We validated this idea using a machine-learning approach. Finally, given amounts of these three archetypes could represent a quantitative phenotype. To take advantage of this, we set out to predict interacting genes from a seed based on the PCA coefficients. We exemplified this firstly on the role of tpxl-1 whose homolog tpx2 is involved in spindle microtubule branching, secondly the mechanism regulating metaphase length, and thirdly the central spindle players which set the length at anaphase. We found novel interactors not in public databases but supported by recent experimental publications.
    DOI:  https://doi.org/10.1371/journal.pcbi.1012330
  17. Sci Rep. 2024 08 29. 14(1): 20109
    Generation of rat offspring from ovarian oocytes by xenotransplantation.
    Hiroaki Taketsuru, Runa Hirayama, Ena Nakatsukasa, Rie Natsume, Keizo Takao, Manabu Abe, Kenji Sakimura.
      The idea of utilizing unused oocytes present in the ovaries has been tested in various ways to produce offspring. However, only a limited number of studies succeeded in offspring generation. They include transplantation of ovaries into autologous or allogeneic animals, and acquisition of pups from oocytes obtained by transplanting mouse ovaries into immunodeficient rats. Here we report successful production of rat oocytes by transplanting rat ovaries under the kidney capsule of immunodeficient mice with addition of hormone administration to the mice. In addition, these oocytes were developed by in vitro fertilization, and transplanted into the oviducts of pseudopregnant rats, resulting in successful delivery of pups. The modified gene of the donor rat was confirmed to be correctly inherited to the pups. These results show that xenotransplantation of ovarian tissue makes it possible to leave offspring, beginning a new phase in developmental engineering.
    Keywords:  Embryo; Immunodeficient mouse; Offspring production; Ovary; Rat; Xenotransplantation
    DOI:  https://doi.org/10.1038/s41598-024-71030-0
  18. Cell Death Differ. 2024 Sep 05.
    A chromosome-coupled ubiquitin-proteasome pathway is required for meiotic surveillance.
    Ruirui Zhang, Bohan Liu, Yuqi Tian, Mingyu Xin, Qian Li, Xiuhua Huang, Yuanyuan Liu, Li Zhao, Feifei Qi, Ruoxi Wang, Xiaoqian Meng, Jianguo Chen, Jun Zhou, Jinmin Gao.
      Defects in meiotic prophase can cause meiotic chromosome missegregation and aneuploid gamete formation. Meiotic checkpoints are activated in germ cells with meiotic defects, and cells with unfixed errors are eliminated by apoptosis. How such a surveillance process is regulated remains elusive. Here, we report that a chromosome-coupled ubiquitin-proteasome pathway (UPP) regulates meiotic checkpoint activation and promotes germ cell apoptosis in C. elegans meiosis-defective mutants. We identified an F-box protein, FBXL-2, that functions as a core component within the pathway. This chromosome-coupled UPP regulates meiotic DSB repair kinetics and chromosome dynamic behaviors in synapsis defective mutants. Disrupted UPP impairs the axial recruitment of the HORMA domain protein HIM-3, which is required for efficient germ cell apoptosis in synapsis defective mutants. Our data suggest that an efficient chromosome-coupled UPP functions as a part of the meiotic surveillance system by enhancing the integrity of the meiotic chromosome axis.
    DOI:  https://doi.org/10.1038/s41418-024-01375-6
  19. bioRxiv. 2024 Aug 27. pii: 2024.08.08.607197. [Epub ahead of print]
    A sperm-enriched 5'fragment of tRNA-Valine regulates preimplantation embryonic transcriptome and development.
    Simeiyun Liu, Andrew D Holmes, Sol Katzman, Upasna Sharma.
      Sperm small RNAs have been implicated in intergenerational epigenetic inheritance of paternal environmental effects; however, their biogenesis and functions remain poorly understood. We previously identified a 5' fragment of tRNA-Valine-CAC-2 (tRFValCAC) as one of the most abundant small RNA in mature sperm. tRFValCAC is specifically enriched in sperm during post-testicular maturation in the epididymis, and we found that it is delivered to sperm from epididymis epithelial cells via extracellular vesicles. Here, we investigated the mechanistic basis of tRFValCAC delivery to sperm and its functions in the early embryo. We show that tRFValCAC interacts with an RNA binding protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB), in the epididymis, and this interaction regulates the sorting and packing of tRFValCAC into extracellular vesicles. In the embryo, we found that tRFValCAC regulates early embryonic mRNA processing and splicing. Inhibition of tRFValCAC in preimplantation embryos altered the transcript abundance of genes involved in RNA splicing and mRNA processing. Importantly, tRFValCAC-inhibited embryos showed altered mRNA splicing, including alternative splicing of various splicing factors and genes important for proper preimplantation embryonic development. Finally, we find that inhibition of tRFValCAC in zygotes delayed preimplantation embryonic development. Together, our results reveal a novel function of a sperm-enriched tRF in regulating alternating splicing and preimplantation embryonic development and shed light on the mechanism of sperm small RNA-mediated epigenetic inheritance.
    DOI:  https://doi.org/10.1101/2024.08.08.607197
  20. Proc Natl Acad Sci U S A. 2024 Sep 10. 121(37): e2401752121
    pSTAT3 activation of Foxl2 initiates the female pathway underlying temperature-dependent sex determination.
    Pengfei Wu, Xifeng Wang, Chutian Ge, Lin Jin, Zihan Ding, Fang Liu, Ju Zhang, Fei Gao, Weiguo Du.
      Ovarian development was traditionally recognized as a "default" sexual outcome and therefore received much less scientific attention than testis development. In turtles with temperature-dependent sex determination (TSD), how the female pathway is initiated to induce ovary development remains unknown. In this study, we have found that phosphorylation of the signal transducer and activator of transcription 3 (pSTAT3) and Foxl2 exhibit temperature-dependent sexually dimorphic patterns and tempo-spatial coexpression in early embryos of the red-eared slider turtle (Trachemys scripta elegans). Inhibition of pSTAT3 at a female-producing temperature of 31 °C induces 64.7% female-to-male sex reversal, whereas activation of pSTAT3 at a male-producing temperature of 26 °C triggers 75.6% male-to-female sex reversal. In addition, pSTAT3 directly binds to the locus of the female sex-determining gene Foxl2 and promotes Foxl2 transcription. Overexpression or knockdown of Foxl2 can rescue the sex reversal induced by inhibition or activation of pSTAT3. This study has established a direct genetic link between warm temperature-induced STAT3 phosphorylation and female pathway initiation in a TSD system, highlighting the critical role of pSTAT3 in the cross talk between female and male pathways.
    Keywords:  Foxl2; STAT3; Trachemys scripta; female pathway; temperature-dependent sex determination
    DOI:  https://doi.org/10.1073/pnas.2401752121
  21. Sci Rep. 2024 Sep 05. 14(1): 20760
    Quantifying DNA damage following light sheet and confocal imaging of the mammalian embryo.
    Darren J X Chow, Erik P Schartner, Stella Corsetti, Avinash Upadhya, Josephine Morizet, Frank J Gunn-Moore, Kylie R Dunning, Kishan Dholakia.
      Embryo quality assessment by optical imaging is increasing in popularity. Among available optical techniques, light sheet microscopy has emerged as a superior alternative to confocal microscopy due to its geometry, enabling faster image acquisition with reduced photodamage to the sample. However, previous assessments of photodamage induced by imaging may have failed to measure more subtle impacts. In this study, we employed DNA damage as a sensitive indicator of photodamage. We use light sheet microscopy with excitation at a wavelength of 405 nm for imaging embryo autofluorescence and compare its performance to laser scanning confocal microscopy. At an equivalent signal-to-noise ratio for images acquired with both modalities, light sheet microscopy reduced image acquisition time by ten-fold, and did not induce DNA damage when compared to non-imaged embryos. In contrast, imaging with confocal microscopy led to significantly higher levels of DNA damage within embryos and had a higher photobleaching rate. Light sheet imaging is also capable of inducing DNA damage within the embryo but requires multiple cycles of volumetric imaging. Collectively, this study confirms that light sheet microscopy is faster and safer than confocal microscopy for imaging live embryos, indicating its potential as a label-free diagnostic for embryo quality.
    DOI:  https://doi.org/10.1038/s41598-024-71443-x
  22. Reprod Fertil Dev. 2024 Sep;pii: RD24083. [Epub ahead of print]36
    Genetic regulation of ovulation rate and multiple births.
    G W Montgomery.
      Ovulation rate in many mammalian species is controlled to regulate the numbers of offspring and maximise reproductive success. Pathways that regulate ovulation rate still respond to genetic and environmental factors and show considerable variation within and between species. Genetic segregation, positional cloning, and association studies have discovered numerous mutations and genetic risk factors that contribute to this variation. Notable among the discoveries has been the role of mutations in bone morphogenetic protein 15 (BMP15 ), growth differentiation factor 9 (GDF9 ) and bone morphogenetic protein receptor type 1B (BMPR1B ) from the intra-ovarian signalling pathway contributing to the evidence that signalling from the oocyte is the key driver in follicle regulation rather than circulating gonadotrophin concentrations. Multiple variants in different domains of BMP15 and GDF9 result in partial or complete loss of function of the proteins providing insights into their functional roles and differential regulation contributing to species differences in ovulation rate. Early success encouraged many more studies in prolific strains of sheep, cattle and goats providing a valuable catalogue of genetic variants of large effect increasing ovulation rate and litter size. More recently, genetic association studies are beginning to identify genetic risk factors with smaller effects. Most genes implicated are from pathways with defined roles in regulation of the ovarian function. However, some genomic regions suggest regulation by novel genes. Continuing genetic and related functional studies will add further to our understanding of the detailed regulation of ovulation rate and litter size with implications for health and animal production systems.
    DOI:  https://doi.org/10.1071/RD24083
  23. bioRxiv. 2024 Aug 16. pii: 2024.08.15.608167. [Epub ahead of print]
    Traffic Jam activates the Flamenco piRNA cluster locus and the Piwi pathway to ensure transposon silencing and Drosophila fertility.
    Austin Rivera, Jou-Hsuan Roxie Lee, Shruti Gupta, Linda Yang, Raghuveera Kumar Goel, Joseph Zaia, Nelson C Lau.
      Flamenco (Flam) is the most prominent piRNA cluster locus expressed in Drosophila ovarian follicle cells, and it is required for female fertility to silence gypsy/mdg4 transposons. To determine how Flam is regulated, we used promoter-bashing reporter assays in OSS cells to uncover novel enhancer sequences within the first exons of Flam . We confirmed the enhancer sequence relevance in vivo with new Drosophila Flam deletion mutants of these regions that compromised Flam piRNA expression and lowered female fertility from activated transposons. Our proteomic analysis of proteins associated with these enhancer sequences discovered the transcription factor Traffic Jam (TJ). Tj knockdowns in OSS cells caused a decrease in Flam transcripts, Flam piRNAs, and multiple Piwi pathway genes. A TJ ChIP-seq analysis from whole flies and OSS cells confirmed TJ binding exactly at the enhancer that was deleted in the new Flam mutant as well as at multiple Piwi pathway gene enhancers. Interestingly, TJ also bound the Long Terminal Repeats of transposons that had decreased expression after Tj knockdowns in OSS cells. Our study reveals the integral role TJ plays in the on-going arms race between selfish transposons and their suppression by the host Piwi pathway and the Flam piRNA cluster locus.
    DOI:  https://doi.org/10.1101/2024.08.15.608167
  24. Cell. 2024 Aug 28. pii: S0092-8674(24)00902-4. [Epub ahead of print]
    5-Formylcytosine is an activating epigenetic mark for RNA Pol III during zygotic reprogramming.
    Eleftheria Parasyraki, Medhavi Mallick, Victoria Hatch, Viviana Vastolo, Michael U Musheev, Emil Karaulanov, Alexandr Gopanenko, Simon Moxon, Maria Méndez-Lago, Dandan Han, Lars Schomacher, Debasish Mukherjee, Christof Niehrs.
      5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression.
    Keywords:  5-formylcytosine; B-box; RNA Pol III; TDG; TET; Xenopus; ZGA; perinucleolar compartment; tRNA; tRNA-iMet
    DOI:  https://doi.org/10.1016/j.cell.2024.08.011
  25. Nat Commun. 2024 Sep 05. 15(1): 7756
    Automatic ploidy prediction and quality assessment of human blastocysts using time-lapse imaging.
    Suraj Rajendran, Matthew Brendel, Josue Barnes, Qiansheng Zhan, Jonas E Malmsten, Pantelis Zisimopoulos, Alexandros Sigaras, Kwabena Ofori-Atta, Marcos Meseguer, Kathleen A Miller, David Hoffman, Zev Rosenwaks, Olivier Elemento, Nikica Zaninovic, Iman Hajirasouliha.
      Assessing fertilized human embryos is crucial for in vitro fertilization, a task being revolutionized by artificial intelligence. Existing models used for embryo quality assessment and ploidy detection could be significantly improved by effectively utilizing time-lapse imaging to identify critical developmental time points for maximizing prediction accuracy. Addressing this, we develop and compare various embryo ploidy status prediction models across distinct embryo development stages. We present BELA, a state-of-the-art ploidy prediction model that surpasses previous image- and video-based models without necessitating input from embryologists. BELA uses multitask learning to predict quality scores that are thereafter used to predict ploidy status. By achieving an area under the receiver operating characteristic curve of 0.76 for discriminating between euploidy and aneuploidy embryos on the Weill Cornell dataset, BELA matches the performance of models trained on embryologists' manual scores. While not a replacement for preimplantation genetic testing for aneuploidy, BELA exemplifies how such models can streamline the embryo evaluation process.
    DOI:  https://doi.org/10.1038/s41467-024-51823-7
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