Embryologie I OOGENESIS autumn 2024 Oocyte cytoskeleton Zuzana Holubcová Department of Histology and Embryology zholub@med.muni.cz Cytoskeleton Centrosome Centrosome Microtubules Chromosomes Centrosome = major Microtubule Organising Center (MTOC) in animal cells pericentriolar material 9 microtubule triplets ➢ Centrioles Interconnecting fibers Centrosome Centrosomes duplicate in coordination with DNA synthesis ➢ Centrrosome duplication cycle Centrosome Centrosome In animal somatic cells, centrosomes ➢ drive microtubule (MT) nucleation ➢ focus microtubule (-)ends at spindle poles and stabilize spindle poles ➢ assemble central bipolar spindle that evenly segregate sister chromatids during mitosis Centrosome overamplification Centrosome clustering - occurs in cancer cells - promotes genetic instability - acentriolar centrosomes (only PCM) capable to nucleate and capture microtubules Centrosomes define spindle geometry - overamplification of centrosomes generates multipolar spindle which produces gross aneuploidy - clustering of centrosomes enables bipolarization but persisting merotelic attachments favour chromoosome lagging during anaphase and create risk of chromosome missegregation Microtubule nucleation pathways Centrosomes present present absent ▪ plant cells ▪ animal mitotic cells Microtubule nucleation pathways ❖ Chromatin-driven microtubule (MT) nucleation Heald and Khodjakov, J Cell Biol, 2015 - RanGTP gradient promotes both de novo MT nucleation near kinetochores and amplification of MT growth toward chromosomes Female meiotic spindles lack centrosomes Mitosis Female meiosis Centrosomal spindle Acentrosomal spindle Symmetric division Asymmetric division Centrioles are eliminated during oogenesis Metazoan oocytes eliminate centrosomes during oogenesis in order to (1) ensure highly asymmetric cell division (2) avoid a superior number after fertilisation Gruss 2018 - PCM synthesized during oocyte maturation - sperm-derived centrioles recruit maternal PCM after fertilization to assemble first mitotic spindle - sperm-derived centrioles are destroyed - first mitosis with acentrosomal spindle - de novo centriole assembly during embryo cleavage stage - Centrioles paternally inherited Centrosome inheritance? Andrea Procházková (bakalářská práce 2020) , Katedra vizuální informatiky, Fakulta informatiky MU Centrioles are delivered by sperm during fertilization Centrosome inheritance?Human oocyte spindle lacks centrosome Holubcova et al, 2015 How are meiotic spindle poles assembled in the absence of centrosomes? GERMINAL VESICLE (GV) METAPHASE I (MI) METAPHASE II (MII) ANAPHASE I ANAPHASE II egg aneuploidyerrors in 1st meiotic division Functional spindle is required for chromosome segregation fidelity errors in 2nd meiotic division embryo aneuploidy Acentrosomal spindle drives chromosomal segregation during female meiosis Acentrosomal spindle assembly in mouse oocytes Melina Schuh Schuh and Ellenberg, 2007 - high-resolution confocal live cell imaging of mouse oocytes maturing in vitro showed that mouse oocyte spindle is assembled by multiple small acentriolar MTOCs that functionally replace canonical centrosomes DNA microtubules Acentrosomal spindle assembly in mouse oocytes Schuh and Ellenberg, 2007 Prophase microtubule network with low dynamics DNA microtubules - MTOC consists of PCM proteins (pericentrin, g-tubulin, Cep192, Cep120, Cep 125, NEDD1,..) - MTOCS cluster around nucleus before NEBD - MTOC nucleate MT „ball“ which carries chromosomes on its surface - MT mass elongates and chromosome congress - chromosome alignment after spindle bipolarization - spindle migration to the cortex - Ran activity overdriven by coordinated action of MTOCS Clift and Schuh, 2015. Multiple acentriolar MTOCs converge at spindle poles and stabilize them DNA MTOCs Acentrosomal spindle assembly in mouse oocytes Human oocyte Mouse oocyte Chromosomes Microtubules From mice to human 25 μm DIC (transmitted light) Chromosomes (H2B-mRFP) Microtubules (MAP4-EGFP)Holubcova et al. Science 2015. Acentrosomal spindle assembly in human oocytes Acentrosomal spindle assebmly in human oocytes Holubcova et al. Science 2015. Acentrosomal spindle assembly in human oocytes Holubcova et al. Science 2015. - human oocytes assemble a meiotic spindle independently of either centrosomes or other MTOCs - spindle assembly is mediated by chromosomes and the small guanosine triphosphatase Ran - spindle assembly is unusually long,requiring ~16 hours How do oocytes assemble an acentrosomal spindle? Centrosomal Spindle Mitotic cells Acentrosomal Spindle Mouse Oocyte Schuh and Ellenberg, Cell 2007. Acentrosomal Spindle Human oocytes (and plant cells) Nucleation on chromosomes Holubcova et al., Science 2015. Spindle assembly strategies Ran GTP Ran GTPaMTCOs Chromosomes (H2B-mRFP) Microtubules (MAP4-EGFP) Moderate spindle instability Human oocyte spindle is instable Holubcova et al. Science 2015. Human oocyte spindle is unstable Severe spindle instability Chromosomes (H2B-mRFP) Microtubules (MAP4-EGFP) Holubcova et al. Science 2015. Human oocyte spindle is unstable Human oocyte spindle is unstable Scale bar, 10 mm *Surplus oocytes from stimulated IVF cycles matured in vitro ! ❖ Prolonged spindle instability was observed in ~80% of human oocytes* but no mouse oocytes Holubcova et al. Science 2015. ❖ Majority of human oocytes recovered from spindle instability before anaphase and extruded a polar body Human oocyte spindle is unstable Holubcova et al. Science 2015. ❖ Spindle instability correlates with chromosome segregation errors Scale bar, 10 mm Holubcova et al. Science 2015. Spindle instability favours chromosome missegregation ❖ Correction of kinetochore-microtubule attachments is incomplete close to anaphase Scale bar, 5 mm Holubcova et al. Science 2015. Spindle instability favours chromosome missegregation ❖ at the absence of centrosomes, human oocytes rely on MT nucleation from chromatin Holubcova et al. Science 2015. Spindle instability favours chromosome missegregation ❖ chromosome-mediated spindle assembly is slow process and formed spindle is inherently unstable ❖ improper microtubule-kinetochore attachments established during spindle build-up and remodelling persist to anaphase causing chromosome lagging that is likely to result in aneuploidy Zielinska et al. eLife 2015. Acentrosomal spindle assembly - molecular composition of human oocyte spindle - MT nucleation initiated at kinetochores Wu et al., Science 2022 Wu et al., Science 2024 - putative human specific MT nucleators (huMTOC)? - nascent MT (-)ends coalesce into minor spindle poles which later aggregate to generate opposite spindle poles - MT amplification, cross-linking and sliding that is required for spindle elongation and bipolarization * * mutations in IVF patients Zielinska et al. eLife 2015. ❖ Established spindle poles in human oocytes are prone to loosening and disintegration Focused spindle poles Broad spindle poles Loosen spindle poles Disintegrated spindle pole Scale bar, 5 mm. Unpublished. How are spindle poles organized at the absence of centrosomes? Human oocyte spindle poles are not stabilized Why is incidence of unstable spindles so different ? 6% 82 % 4.4 %0 % How are spindles in non-human mammalian oocytes stabilized Incidence of unstable acentrosomal spindles ❖ Liquid-like meiotic spindle domain (LISD) - localized at poles and permeates the MT mass of mammalian oocytes - selectively concentrates multiple centrosomal and MT-associated proteins - allows rapid diffision within the spindle volume - disruption of the LISD disperses spindle regulatory factors and leads to severe spindle assembly defects So et al., Science 2019 Melina SchuhChuh So stable spindles Mammalian oocyte spindle pole organization Human oocyte spindle pole organization ❖ NuMA decorates spindle poles in mammalian oocytes So et al., Science 2022 Melina SchuhChuh So - NuMA is required for MT focusing at spindle poles in human oocytes Human oocyte spindle pole organization ❖ NuMA decorates spindle poles in mammalian oocytes So et al., Science 2022 Melina SchuhChuh So - acute depletion of NuMA → NuMA is required for MT focusing at spindle poles in human oocytes Misalignment of microtubules in central region of human oocyte spindle Human oocytes must lack a stabilizing protein that protects mouse, porcine and bovine oocytes from spindle instability Search for oocyte spindle stabilizing factor So et al., Science 2022 si RNA screen co-depletion of NuMA and candidate spindle- associated proteins phenotypes analyzed Search for oocyte spindle stabilizing factor So et al., Science 2022 Trim-Away So et al., Science 2022 Anti-KIF-CControl Depletion of KIFC1/HSET induces spindle instability and promotes aneuploidy in bovine oocytes Search for oocyte spindle stabilizing factor So et al., Science 2022 ❖ Exogenous KIFC1 rescues spindle instability in human oocytes ❖ Human oocytes are deficient in KIFC1 Chromosomes Microtubules Search for oocyte spindle stabilizing factor Prevention of oocyte spindle instability So et al., Science 2022 ❖ KIFC1 ensures the spindle stability and prevents fragmentation of spindle poles by ensuring alignment of MT at central region and crosslinking MT minus ends at spindle poles ❖ NuMa organize acentrosomal spindle poles by ensuring coalescence of crosslinked MT-minus ends Holubcová et al Science 2015 ❖ Mature egg = metaphase II arrested oocytes with PB extruded and chromosmes aligned in MII spindle DNA, microtubules Egg maturity In IVF practice, all PB-displaying oocytes are regarded as MIIs and subjected to ICSI Oocyte maturity IN VIVO PREOVULATORY FOLLICLES OVULATION MULTIPLE COCS MATURE + IMMATURE OOCYTES SINGLE COC MATURE EGG NATURAL CYCLE CONTGROLLED OVARIAN STIMULATION IN VITRO Holubcová et al Science 2015 MI to MII transition and MII spindle assembly - asynchrony between PB extrusion and MII arrest ! Chromosomes (H2B-mRFP) Microtubules MAP4-EGFP) - MII spindle formation is rapid compared to MI - Emergence of PB precedes MII arrest MI MII MII MII MII MII„MII“„MII“ „MII“ MI to MII transition and MII spindle assembly → risk of untimely fertilization (ICSI) Non-invasive spindle visualization ❖ Polarized Light Microscopy (PLM) - based on interference of polarized light with anisotropic substances e.g. axial crystals, liquid crystals and oriented (bio)polymers BIREFRINGENCE - property of certain materials to split a light beam to two rays (ordinary/extraordinary) - polarized light is refracted by these anizotropic materials and divided to separate components vibrating perpendicularly - both polarized light ray then pass through the analyzer and the relative retardance of one ray to the other is calculated https://www.microscopyu.com/techniques/polarized-light/principles-of-birefringence Non-invasive spindle visualization ❖ Polarized Light Microscopy (PLM) - presence and positioning of MII spindle - pattern of zona pellucida - presence of PLM-detectable MII spindle is a positive marker of egg´s fertilization and developmental competence https://www.youtube.com/watch?v=tlKo9nqmqGY - enables non-invasive imaging of birefringent structures in living cells Non-invasive spindle visualization ❖ Polarized Light Microscopy (PLM) - PLM signal is orientation-dependent - spindle imaging requires oocyte orientation Holubcová et al, JoVE 2019 Non-invasive spindle visualization ❖ Polarized Light Microscopy (PLM) Holubcová et al, JoVE 2019 PM.... Plasma mebrane ZP......Zona pellucida RB.....Refractile body V……..Vacuole - birefringent structures in human oocytes - relative position of spindle and PB Non-invasive spindle visualization ❖ Polarized Light Microscopy (PLM) - the strengh of the signal reflect the material ordering - suffient mass of paralelely oriented MT required to produce noticible signal Holubcová et al, JoVE 2019 Non-invasive spindle visualization ❖ Polarized Light Microscopy (PLM) - enables monitoring of MI/MII transition and ICSI time optimisation in clinical practice Holubcová et al, JoVE 2019 Non-invasive spindle visualization ❖ Polarized Light Microscopy (PLM) - enables monitoring of MI/MII transition and ICSI time optimisation in clinical practice timePostponing ICSI time optimal fertilization window METAPHASE IIANAPHASE I INTERKINESIS EGG IN VITRO AGING Fertilization capacity Montag RBM Online 2006 Montag and van der Ven, RBM Online 2008 Holubcova et al, JARG 2019 Factor affecting human oocyte spindle stability in vitro ❖ MII spindle is sensitive 10 min RT ➢ temperature ➢ pH fluctulation avoid excessive manipulation ! ➢ osmolarity alterations - MOPS/HEPES buffered medium for work in ambient conditions - avoid evaporation - parafine/mineral oil overlay - humid conditions - optimal 37oC OVERHEATING → irreversible denaturation COOLING → spindle desintegration Actin network - actin network consists of F-actin fibers (microfilaments), formed by dynamic (de)polymerization of globular G-actin monomers - in association with its binding proteins play versatile roles (e.g. mechanical support, migration, signalling, trafficing, adhesion, division,contraction,...) Oocyte actin network - large oocyte cytoplasm contains network of longed branched microfilaments and cortical actin ❑ Roles of actin: - mechanical support - vesicular trafficing - arrangement of cytoplasmic organelles - spindle formation (with MTs) - spindle migration - chromosome alignment promotion - PB extrusion - MII spindle anchorage - membrane polarization Large volume Highly asymmetric cell division 110-120 um70-80 um Actin cap Spindle migration for asymmetric spindle positioning - in mouse, bipolar spindle is formed centrally - spindle relocation to cortex ensures high asymmetry of female meiotic division Spindle relocation Spindle relocation failure Schuh and Ellenberg, 2008 - spindle migrates along its long axis towards the closest cortex - spindle accelerates during migration Actin dynamics is required for asymmetric spindle positioning - spindle relocating to the cortex is driven by actin Schuh and Ellenberg, 2008 Pfender et al , 2011 - Active actin nucleation is required for spindle relocation - spindle pole-associated myosin II pulls the actin filaments against the cortex Actin dynamics is required for asymmetric spindle positioning - vesicle-mediated actin network dynamics and myosin force are required for spindle migration to the cortex Holubcová et al, 2013 - density and outward directed dynamics of actin network is regulated by size of vesicles sequestering actin nucleators Actin dynamics is required for asymmetric spindle positioning Londono-Vasques et al. 2022 - identification of subset of metaphase cytoplasmic MTOCs (mcMTOCs) that do not contribute to spindle assembly and localize opposite to PBE side - actin-mediated movement of the meiotic spindle to the cortex is balanced by forces exerted from mcMTOCs to ensure the timely migration and asymmetric division - mcMTOCs are interconnected with polar MTOCs and regulate spindle positioning by anchoring the spindle to the oocyte cortex Spindle actin promote chromosome alignment Mogessie and Schuh, Science 2017 Melina SchuhBinyam Mogessie - actin filaments permeate meiotic spindle in mammalian oocytes - dynamic actin promotes formation of kinetochore fibers - actin prevents lagging chromosomes and promotes chromosome congression and alignment Actin and microtubule cooperate to insure spindle integrity Roeles and Tsiavaliaris, 2019 - actin fibers permeate spindle and structural integrity of actin spindle is dependent on microtubules - actin and microtubules co-operate to assemble functional spindle and and help to align chromosomes in human oocytes PB extrusion (1) cortical membrane protrusion (actin cap) (2) spindle midzone induced membrane furrowing (3) actomyosin ring constriction (4) abscission - Furrow induction by spindle midzone is distance-dependent Deng et al 2007 * Injection of DNA beads to MII oocytes Wang et al 2011 in mice PBE requires spindle rotation Wang et al 2011 MII spindle anchorage - Arp2/3 complex localization - promotes local actin nucleation → actin cap - ensures spindle anchoring during prolonged MII stage local activation of N-WASP N-WASP Ran gradient - Arp2/3 nucleation activity initiates retrograde flow of F-actin provoking cytoplasmic streaming that further pushes on spindle to maintain its cortical position Rong Li Yi et al. 2011 MII spindle anchorage (unpublished) Actin polarization - actin thickeing - „actin cap“ - region overlying MII spindle - actin-enriched microvilli-free zone devoid of cortical granules - induced by spindle chromatin underneath the oolema Actin cap Longo and Chen, 1985; Maro et al., 1986 - prominent in mouse but not human oocytes! Actin polarization - etopic actin polarization - induced artificially by DNA beads or by sperm chromatin during ICSI Deng and Li 2009 „fertilization cone“ Transient phenomenon or can lead to „3rd PB extrusion“