Embryologie I OOGENESIS autumn 2024 Cytoplasmic factors Zuzana Holubcová Department of Histology and Embryology zholub@med.muni.cz Oogenesis Female gamete must acquire functional competencies 1. Meiotic - capacity to reach the metaphase II arrest 2. Activation - capacity to finish meiosis, block polyspermy, and form pronuclei at fertilisation 3. Developmental - capacity to trigger and support embryonic development Oogenesis ➢ Dormant stage - cellular quiescence (inactivity) ➢ Growth stage - synthesis of RNAs and proteins - intense accumulation of cellular material ➢ Maturation stage ❖ Nuclear maturation - resumption of meiosis (release from prophase arrest) - chromosome segregation - MII arrest - capacity to complete meiosis II after activation ❖ Cytoplasmic maturation - storage of maternal mRNA - modification organelles - global rearrangement of organelles Wright et al 2000 mRNA transcription and translation ❑ Transcription - executed by transcription factors - regulated by epigenetic DNA modification - active during growth phase - silenced during oocyte maturation due to condensed chromatin state ~24 hoursweeks ❑ mRNA processing - spatiotemporal control over activation, storage and degradation of transcripts - translation is mediated by RNA-binding proteins (RBP) that recruit/deter actors of protein synthesis machinery - resumed only if fertization occurs = „embryonic genome activation - EGA“ - polyadenylation of 3´end by Poly(A) Polymerase (PAP) - long poly(A) tail favours efficient translation and protects mRNA from degradation - deadenylation slows translation and initiates mRNA degradation 5´ 3´ 5´ 3´ Polyadenylation signal CBC Cap binding complex CBC PAP mRNA transcription and translation ❑ mRNA processing - the 5′ end of the nascent RNA molecule receives a 7methylguanosine cap and bounds with nuclear cap-binding complex (CBC) - molecular complex containing PAP binds the AAUAAA sequence (polyadenylation signal) - PAP catalyses the addition of adenine bases to the 3′ terminus forming the 3′ poly-A tail of around 200 adenine nucleotides - poly(A)-binding protein (PABP) binds to the poly(A) tail, protecting transcript from degradation and playing a major role in translation initiation - the mature mRNA and its associated proteins, forming a ribonucleoprotein (RNP) complex, are exported from the nucleus to the cytoplasm Poly(A) tail mRNA transcription and translation ❑ mRNA translation - spatially and temporally controlled translation of these stored mRNAs (e.g. meiotic genes silencing in antral stage oocytes) mRNA transcription and translation ❑ mRNA translation Tsukumo et al 2014 - mammalian oocytes and ovarian somatic cells contain large number of very longed-lived proteins - role of chaperones and cellular antioxidants Harasimov et al 2023 -translation driven by mTOR activity mTOR = mammalian target of rapamycin mTORC1/mTORC2 – complex 1/compex 2 Storage of mRNA ❖ Balbiani body Boke et al 2016 - transient membraneless organelle found in immature dormant oocytes of diverse species and typically dispersed during development - adjacent to nucleus facing vegetal pole - giant clump of RNA, proteins and organelles, embedded in a dense network of amyloid fibers - storage of mRNA granules during prolonged development? Rodler and Sinowatz 2013 Storage of mRNA Cheng et al, 2022 ❖ MARDO = mitochondria-associated ribonucleoprotein domain - membrane-less compartment with hydrogel-like properties located around Mts - stores transiently transcriptionally repressed maternal mRNAs - assembly driven by RNA-binding protein ZAR1 which clusters the Mts and protects the mRNAs against degradation - dissolution in mature mammalian eggs ensures timely degradation of maternal mRNAs Mitochondria - semiautonomous double membrane- bound organelle producing energy - production of 90% ATP necessary for cellular function via oxidative phosphorylation (OXPHOS) - endosymbiotic origin - circular mtDNA (16 569 bp encoding 37 genes: 13 polypeptides, 2rRNA, 22tRNAs) - bioenergetic capacity optimized by fusion (enlargement) x fission (fragmentation) Mitochondria Atypical „primitive“ mitochondria Somatic type mitochondria Prigione et al 2015 Mitochondria HUMAN OOCYTEMOUSE LIVER MOUSE HEART HUMAN EMBRYOS Atypical „primitive“ mitochondria - rounded/oval shape - parallel/circular cristae 1 μm Trebichalská, Z. (2020). Analýza ultrastrukturních znaků lidských oocytů. Brno, 2020. Diplomová práce. Masarykova univerzita, Přírodovědecká fakulta. Oocyte mitochondria Oocyte mitochondria Mitochondrion constitute 4-5 % of oocyte volume Mitochondria are the most abundant oocyte organelle Trebichalska et al, 2021 Oocyte mitochondria Kirillova et al 2021 How long-lived oocytes balance mitochondrial activity and long-term ROS* production? indirect quantification of mitochondrial mass highest mtDNA copy number of all cells *ROS (radical oxygen species) - by-products of mitochondrial activity - cause of oxidative damage, mutagenesis and apoptosis Oocyte mitochondria - early oocyte avoid accumulation of ROS by a eliminating complex I of electron transport chain - keeping complex I shut down (but rest of OXPHOS functional) during dormancy enables to avoid ROS built up - functional complex I assembled in final stages of oogenesis Rodriguez-Nuevo et al 2022 Elvan Böke Where do the oocytes get energy? Bahety et al 2024 - the cumulus cells directly surrounding the oocyte supply nutrients and energy substrates such as pyruvate and lactate to the oocyte through gap junctions - pyruvate and lactate supplied by cumulus cells constitute energy source required for oocyte development Oocyte mitochondria Lounas et al 2024 - FSH regulates mitochondrial structure and dynamics in cumulus cells - the mitochondria elongation followed by fragmentation is accompanied by a decrease in mitochondrial activity and a switch to glycolysis Glycolysis OXPHOS → 3-15,7 mtDNA/sperm→ ~ 150,000 mtDNA/oocyte Mitochondrial inheritance Non-Mendelian maternal inheritance Mitochondrial inheritance - Sperm mtDNA quantity associated with semen quality Carlson, B. (2018). Human Embryology and Developmental Biology. Elsevier, 6th edition. Mitochondrial inheritance Sato and Sato, 2013 Failure to eliminate paternal mtDNA after fertilization? Mitochondrial diseases ❖ MELAS syndrome mitochondrial encephalopathy, lactic acidosis and stroke-like episodes ❖ LHON syndrome Leber hereditary optic neuropathy ❖ Leigh syndrome (LS) ←point mutation of mtDNA - maternally inherited DiMauro, 2019. ❖ MERRF disease Myoclonic epilepsy and ragged-red fiber disease psychomotor regression ← mutation of nDNA encoding mitochondrial proteins - mendelian inheritance (+ mtDNA mutations) ➢mtDNA diseases ➢Mitochondrial diseases Mitochondrial diseases - deficient energy production - embryo developmental arrest - „mitochondrial cytopathy“ - multisystem syndrom - clinically heterogenous - rare but often progressive and severe Mitochondrial diseases - clonal expansion of mitochondria with normal/defective mtDNA variants → degree of phenotype severity - heteroplasmy can drastically change during development Treatment mitochondrial diseases Soldatov et al 2022. ❖Mitochondrial replacement techniques (MRT) = micromanupulation procedures designed to prevent maternal transmission of mtDNA diseases • GERMINAL VESICLE TRANSFER • MEIOTIC SPINDLE TRANSFER • PRONUCLEAR TRANSFER • POLAR BODY TRANSFER Avoidance of mitochondrial diseases • CYTOPLASMIC TRANSFER • AUTOLOGOUS GERMLINE MITOCHONDRIAL TRANSFER (AUGMENT TREATMENT) Mitochondrial dysfunction therapies ❖Egg rejuvenation Energizing egg with healthy mitochondria = micromanupulation procedures designed to boost egg fitness and fertilization potential Partial cytoplasm supplementation Total cytoplasm transfer ❑ Germinal vesicle transfer - transplanting the GV from a patient’s immature oocyte to an enucleated oocyte derived from a healthy donor - fusion of karyoplast and cytoplast achieved by electroporation or HVJ-E (Sendai virus extract) - only in animal models - low efficiency Sendra et al 2021 Mitochondrial replacement techniques (MRT) - ❑ Meiotic spindle transfer Mitochondrial replacement techniques (MRT) - transferring the meiotic spindle of a patient’s metaphase II oocyte to a healthy enucleated donor oocyte Sendra et al 2021 - spindle visualized by PLM ❑ Meiotic spindle transfer Mitochondrial replacement techniques (MRT) Mito Tracker - successfully used in primates - low level of mtDNA carry over - 3-year postnatal follow-up Sendra et al 2021 Shoukhrat Mitalipov Tachibana et al., 2009 ❑ Meiotic spindle transfer Mitochondrial replacement techniques (MRT) - 1st baby (Mexico, 2016) - prevention of Leigh syndrome transmission Mitochondrial replacement techniques (MRT) ❑ Pronuclear transfer - the pronuclei of a patient’s oocyte is isolated in a karyoplast and transferred into a donor oocyte without its pronucleus Mery Herbert Hyslop et al. Nature, 2016. Craven et al., Nature 2010 - time-dependent effciency - early (8h post ICSI) better than late PNs transfer (16-20h post ICSI) Mitochondrial replacement techniques (MRT) ❑ Polar body transfer PB1 transfer PB2 transfer Transfer of the 1st PB from a patient’s MII to a donor MII oocyte without meiosis spindle Transfer of the PB2 from a patient’s zygote to a donor zygote whose female pronuclei has been previously removed - Low Mt carry-over! Sendra et al 2021 Mitochondrial replacement techniques (MRT) ▪ Clinical applications ▪ Risk of MRT? Mitochondrial replacement techniques (MRT) Carry-over of defective mtDNA, genetic drift and reversal of maternal phenotype?! Mitochondrial-genome mismatch ?! Reznichenko, et al 2016 Egg rejuvenation ❑ Cytoplasmic transfer - microinjection of patient´s eggs with healthy mitochondria from young egg donor ❑ Autologous germline mitochondrial transfer (Augment treatment/OvaScience) mitigation of ovarian aging - microinjection of patient-matched mitochondria isolate from putative ovarian stem cells Woods and Tilly 2015 Cozzolino, et al 2019 Oocyte cytoplasmic organelles ❖ Endoplasmic reticulum (ER) - smoth ER (sER) - ribosomes not detected - major storage of Ca2+, mediates Ca2+ signalling - perinucler location in GV oocytes, relocation to cortex after GVBD - sER types (1) vesicular (cisternea) (2) tubular (dense arrays and large clusters in the cortex area) - gradual association with mitochondria during maturation → coordination of Ca2+ homeostasis and ATP production Necklace complexes Trebichalska et al, 2021 Oocyte cytoplasmic organelles ❖ Endoplasmic reticulum (ER) - ER clusters around MI spindle - cortical ER clusters in MII - cortical ER clusters in MII - no ER clusters around MI spindle Oocyte cytoplasmic organelles ❖ Golgi apparatus (GA) - modifying, sorting, packing of macromolecules for intracellular trafficking and cell secretion - perinucler location in GV oocytes, fragmentation at GVBD - GA fragmentation generates vesicles taht are relocated to the cortex in actin dependent manner Trebichalska et al, 2021 Oocyte cytoplasmic organelles ❖ Cortical granules (CG) - oocyte-specific secretory vesicles derived from GA - located in suboolema cortex of mature oocytes - critical role in fertilization and prevention of polyspemy - acquire peripheral and cortical position during oocyte maturation - in mouse, CGs-free zone in spindle proximity Trebichalska et al, 2021Rojas et al., 2021 Oocyte cytoplasmic organelles ❖ Cortical granules (CG) - translocated to the cortex during oocyte maturation - myosin-dependent movement along actin filaments, hitchhiking on specific vesicles - anchoring to the cortex is dependent on subcortical maternal complex (SCMC, MATER) - clearance of cortical actin prior CGs exocytosis Cheeseman et al, 2016 Vogt et al, 2019 - no CGs depletion close to spindle pole Organelle rearrangement during maturation Trebichalska et al, 2021 - cortical area populated by organelle after NEBD - SER-Mt association and SER agreggation - GA fragmentation and CGs relocation to cortex Organelle rearrangement during maturation Trebichalska et al, 2021 - cortical area populated by organelle after NEBD - SER-Mt association and SER agreggation - GA fragmentation and CGs relocation to cortex Normal oocyte Abnormal shape Big polar body Abnormal zona pellucida Abnormal perivitelline space Cellular fragments Refractile bodies GranularityVacuoles sER disc Human oocyte dysmorphism For more examples see: aAtlas of Clinical Embryology (MUNI) https://is.muni.cz/do/med/el/ake/index.html Atlas of human embryology (ESHRE) https://atlas.eshre.eu/ Cytoplasmic abnormalities ❖ Vacuoles - membrane-bound, translucent bodies with 3D appearance - persist after fertilization cortical vacuolisation common shortly after thawing Cytoplasmic abnormalities ❖ Vacuoles - membrane- bound, fluid-filled structures - some tend to merge - may contain granular material granular vacuole Tatíčková et al 2023 Cytoplasmic abnormalities ❖ Vacuoles Van Blerkom 1990 - endocytic origin - vacuolisation caused by instability of cell cortex? - contain follicular fluid - persist after fertilization Cytoplasmic abnormalities ❖ sER disc - a smooth plate-like structure - rare feature - dissolutes after fertilization Cytoplasmic abnormalities ❖ sER disc Van Blerkom 1990 - enormous aggregate of tubular sER - decreased fertilisation capacity reported - not recommended to used for ICSI, nevertheless healthy children born Tatíčková et al 2023 Cytoplasmic abnormalities ❖ Cytoplasmic granularity - irregular texture of cytoplasm - crater-like appearence - disappears after fertilization Cytoplasmic abnormalities ❖ Cytoplasmic granularity Tatíčkoví et al 2023 - excessive aggregation of organelles - dysfunction of actin cytoskeleton? Cytoplasmic abnormalities ❖ Refractile bodies - various-sized dark inclusions in the cytoplasm - incidence increases with reproductive aging - persist after fertilization „Bull eye- inclusion*“ Cytoplasmic abnormalities ❖ Refractile bodies Composed of - electron-dense granules - fibrilar material - amorphous substance - membrane remnants - lipid droplets Heterogenous clumps corresponding to tertiary lysosomes Tatíčková et al 2023 Cytoplasmic abnormalities ❖ Refractile bodies Otsuki et al 2017 - birefringent specs (PLM signal) - exhibit autofluorescence characteristic for lipochrome lipofuscin („lipofuscin bodies“) – an insoluble pigment which accumulates in aged terminally differentiated cells - incorporate biological “garbage” and/or sequester xenobiotics? ← impaired protein and/or lipid metabolism? ← oxidative stress? ← reduced intralysozomal degradation? Cytoplasmic abnormalities ❖ Refractile bodies ~ ELVA = EndoLysosomal Vesicular Assemblies - in immature mouse oocytes, ELVAs sequester aggregated proteins and degrade them upon oocyte maturation - ELVAs degradative activity increases upon oocyte maturation promoting healthy embryogenesis - retention of protein aggregates in the embryo leads to early embryonic arrest Zaffagnini et al 2024 - non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a liquid-like protein matrix - Strategy to deep-clean toxic substances and damaged protein and promote logitivity´ Cytoplasmic abnormalities Tatíčková et al 2023 Cytoplasmic abnormalities ❖ Patological mitochondria Trebichalska Z, Diploma thesis, Faculty of Science, Masaryk University 2020 - loss of ΔΨm triggers collapse of mitochondrial structure Cytoplasmic abnormalities Oocyte morphology and IVF outcome - meta-analysis of 14 studies Egg fertilization capacity significantly reduced by: (1) large PB1 (2) large PVS (3) refractile bodies (4) vacuoles Setti et al 2011 Oocyte degeneration - dark, granular cytoplasm videos bursting shrinking vacuolization Lipid droplets Human Rat Cow Sheep Pigstrain-dependent Mouse Rabbit DogCat Dalbies-Tran et al 2020 - the oocytes with high lipid content droplets appear darker - dark, lipid storing inclusions - composed of neutral lipids (triacylglycerides) and cholesterol esters - interspecies variability in amount of lipid droplets Oocyte lipids - balanced amount of saturated and unsaturated fatty in oocyte´s microenvironment is critical for cytoplasmic maturation ❖ ROLES ➢ energy production via mitochondrial b-oxidation and TCA (tricarboxylic acid) cycle ➢ precursors of steroid homones ➢ cellular signalling ➢ membrane components Khan et al 2021 - lipid droplets - phospholipids - free fatty acids - saturated (palmitic C16, stearic C18 acid) - monounsaturated (oleic acid) - polyunsaturated (linoleic, arachidonic acid) Oocyte metabolism Helen Picton - metabolic cooperativity between oocytes and follicular cells - metabolic coupling through gap-junctions - different nutritional needs of these cell types – “nutrient partitioning“ - change of energy source during growth and differentiation - influenced by endocrine environment and oxygen availabilty Diffusion distance for nutritients and O2 Metabolic switch Oocyte metabolism ❖ Glucose (Glc) - uptake by GCs affected by FSH, LH, and insulin - glycolysis in GCs or transfer to oocytes via gap junctions (oocytes have only low glycolytic capacity) - metabolized to Pyr and Lac - Glc consumption in COC increase during follicle growth and peaks before ovulation ❖ Pyruvate (Pyr) - preferred energy source for mammalian oocytes - supplemented by GCs - oxidation through TCA and OXPHOS - acts as free ROS scavanger and buffer ❖ Amino acids - transferred to oocyte from cumulus cells - energy substrates - heavy metal chelatators - pH regulation - elimination of amonia Lactate (Lac) (Pyr) (Glc) ❖ Cholesterol - supplemenated by cumulus cells under oocyte´s influence (i.e. BMP15, GDP9) - transfer through raft structures - oocytes promote steroidogenesis and suppress luiteinisation in cumulus cells Oocyte metabolism Impact of maternal diet on egg quality? - undernutrition ← decresed caloric intake (starving, anorexia, bulimia) ← increased nutritional requirement/loss (athletes, disease) ← impaired ability to absorb or utilize nutritiones (intolerance) - overnutrition - Overweight BMI=25-29.9 - Obese BMI ≥ 30 > 30% world population! - conflicting data from human and animal studies - overnutrition seems to be more detrimental than undernutrition - → lipotoxicity, ER stress, mitochondria alteration, absence of microvilli, ROS production, maturation arrest, inflamation, apoptosis (of GSc)... - high-fat and high-protein diet impair oocyte developmental competence - drastic weight loss before IVF treatment? ❖Malnutrition Oocyte compartmentalization - polarization of ooplasmic determinants and embryonic prepatterning in lower species - marked asymmetric deposition of pigments and yolk in Xenopus eggs animal pole vegetal pole - mammalian oocyte compartmentalization? ▪ actin polarization (thickening of actin = actin cap) ▪ hyperpolarized mitochondria ▪ lipid rafts ▪ localization of maternal factors animal pole vegetal pole AV axis - 5-10 μm zone beneath oolema of mouse oocytes was found to be enriched for mitochondria with higher membrane potential (ΔΨm)* - detected by mitochondria-specific potentiometric fluorescent stain JC-1 * difference across inner and outer Mt membrane (-mV) - low potential – green monomer - high potential → multimerization (J-aggregates) orange-red signal - asymmetric distribution mitochondria of higher functional activity at the periphery? - proximity to 02 source? - lower ATP consumption at the periphery? Oocyte compartmentalization ❖ Hyperpolarized mitochondria in subplasmalemal region Van Blerkom et al 2008 Johnathan van Blerkom - submicron sized oolemal microdomains which serve as concentrating platforms for membrane activities - composed of cholesterol, sphingolipids and protein receptors; enriched for ganglioside GM-1 - organisation influenced by ΔΨm of suboolemmal Mt - disruption of lipid rafts might explain fertilization failures Oocyte compartmentalization ❖ Lipid rafts Van Blerkom and Caltrider 2013 „island“ pattern -associated with fertilization failure „finely punctate“ pattern Oocyte compartmentalization ❖ Localization of maternal factors ▪ Maternal effect genes (MEG) - transcribed during oogenesis but function during fertilization and preimplantation development (e.g., molecular remodelling of paternal genome, degradation of maternal mRNA and proteins, completion of EGA,...) - mutations typically manifest as idiopathic infertility and cleavage arrest in IVF and imprinting disorders SubCortical Maternal Complex (SCMC) Developmentally lethal -/- mutations Maternal factors Jentoft et al. 2023- maternal factors are stored at cytoplasmic lattices - fibers made of short twisted filaments filaments with high surface area - accumulate maternal proteins that are critical for postfertilization epigenetic reprogramming and early embryonic development - separation of maternal factors from cytosol prevents their premature activity and degradation - PADI6 and SCMC (subcortical maternal complex) essential for cytoplasmic lattices formation - embryos from PADI6 and SCMC -/- females (mouse/human!) arrest early in development - discovered using super resolution and cryoelectron microscopy tomography Maternal factors - capable to orchestrate chromatin remodelling and reprogramming (A) sperm-delivered genome during fertilization (B) somatic nucleus after somatic cell nuclear transfer (SCNT) Somatic cell nuclear transfer (SCNT) „for the discovery that mature cells can be reprogrammed to become pluripotent“ 1962 2012 Ooplasm possess factors capable to revert somatic cell genome to undifferentiated state animal cloning Shinya Yamanaka John Gurdon Ian Wilmut Somatic cell nuclear transfer (SCNT) Dolly: the cloned sheep 1996 - low efficiency - epigenetic alterations - cell cycle stage mismatch between cytoplast (MII oocytes) and cell nucleus (G0/G1) ?? Ian WilmutDolly Somatic cell nuclear transfer (SCNT) (Wakayama et al. 1998) SCNT in other species (Kato et al. 1998) (Polejava et al. 2000) (Baguisi et al. 1999) (Chesné et al. 2002) ? (Lee et al. 2005) Somatic cell nuclear transfer (SCNT) Cloned macaque monkeys 2018 - fetal fibroblast nucleus fused with enucleated MII oocyte using HVJ-E virus - artificial activation with ionomycin and protein synthesis inhibition with 6-dimethylaminopurin (I/D) - epigenetic modification (injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at 1cell stage) - genetically uniform non-human primates - live but short-lived Liu et al 2018 Somatic cell nuclear transfer (SCNT) How do SCNT and ICSI embryos differ? - SCNT embryos show hyperplasia and calcification of placenta - SCNT embryos have ↓DNA methylation and loss of maternal gene imprinting Somatic cell nuclear transfer (SCNT) - trophoblast replacement method → healthy adult male monkey = succesful cloning of primates