Embryology II PREIMPLANTATION DEVELOPMENT spring 2025 Zuzana Holubcová Department of Histology and Embryology zholub@med.muni.cz Egg activation and parthenogenesis Egg activation (1) release from MII arrest (2) polyspermy block (3) change in mRNA synthesis and protein expression gamete-to-embryo transition (A) Physiological stimulus - induced by sperm entry (B) Arteficial - physical treatment - chemical treatment - modulation of key proteins FERTILIZATION PARTHENOGENESIS - development of haploid embryo without fertlization (C) Spontaneous - no stimulus Calcium signalling ❖Ca2+ oscillations - series of measurable Ca2+ spikes - periodic increase and fall of cytosolic [Ca2+] Ca2+ signalling „toolkit“ ➢ Ca2+ mobilizing signals ➢ i.c. stores - ER, Mt, lysosomes ➢ Ca2+ influx channels ➢ pumps and exchanges removing Ca2+ from cytoplasm downstream effectors of Ca2+ signalling pathway ↑ i.c [Ca2+] Extracellular (e.c.) Intracellular (i.c.) [Ca2+] gradient 2mM 100nM Calcium signalling at fertilization Stricker at al. 1999 - universal hallmark of egg activation in all sexually reproducing animal species (and flowering plants) Calcium signalling at fertilization - in mammals, species-specific differences in latency, duration, frequency and amplitude of Ca2+ oscillations Carrol et al. 1995 - Ca2+ oscillation pattern related to egg/embryo developmental competence Calcium signalling at fertilization Haverfiled et al. 2016 - age-related alterations of oscilation patttern Calcium signalling at fertilization - Ca2+ oscillations triggered by fertilizing sperm - lasts several hours until PN formation - first Ca2+ wave begins at sperm entry site and propagades radially to opposite pole -directional -faster, nondirectional → GCs exocytosis → release from MII arrest → cytoplasmic movements → PN formation - Ca2+ oscillations triggered also by ICSI and sperm extract injection → role of soluble spermdelivered oocyte activation factor (SOAF) Quest for SOAF Zafar et al. 2021 - testis-specific isoform of phosfolipase C - delivered to the oocyte by fertilizing sperm - localized to post-acrosomal region of sperm head where gamete fusion starts Saunders et al 2002 - after sperm penetration, PLCζ diffuses into the ooplasm, binds to i.c. vesicles containing PIP2 and hydrolyses PIP2 to IP3 and DAG - generated IP3 then binds to IP3 receptor, which forms a Ca2+ channel in ER membrane - the openning of the channel release Ca2+ from i.c. store to cytosol initiating Ca2+ oscillations Egg activation Calcium oscillations Unnikrishnan et al 2021 PIP2 -phosphatidylinositol 4,5-bisphosphate IP3- 1,4,5-trisphosphate DAG - dyacyl glycerol PLC zeta (PLCζ) Hamada and Mikoshiba, 2019 ❑ return to baseline level of i.c. [Ca2+] ←removal from the cell PMCA (plasma membrane Ca2+ pump) NCX (Na/Ca2+ exchanger) ← uptake into i.c. Stores SERCA (sarcoendoplasmic reticulum Ca2+ATPase) Calcium signaling homeostasis ❑ elevation of i.c. [Ca2+] ← Ca2+ release from ER stores IP3-mediated Ca2+ release and subsequent Ca2+- mediated Ca2+ release through a Ca2+ sensitive IP3 receptor channel (allosterically modulated by Zn) ← Ca2+ influx accross plasma membrane SOCE (store operated Ca2+ entry) SER-Mt „necklace“ complexes Cycle of Ca2+ transient generation Stein et al 2020 Ca2+ stimulates mitochondrial ATP production; ATP is required for SERCA pump activity. Ca2+ is pumped back into the ER through SERCA pumps and out of the egg through PMCA pumps and NCX Sperm PLCζ acts on PIP2 in intracellular vesicles to generate IP3, which stimulates IP3R-mediated Ca2+ release and subsequent Ca2+induced Ca2+ release Ca2+ flows into the cytoplasm through TRMP7, CaV3.2 and TRPV3 channels and is then available for SERCA pumps to replenish ER Ca2+ stores in preparation for the next Ca2+ release event - ↑[Ca2+] → modulation of activity of Ca2+-sensitive enzymes ❖ CaMKII (calmodulin-dependent protein kinase II) - phosphorylates Emi2 promoting its interaction with Plk1 - Plk1-induced phosphorylation of Emi2 leads to its ubiquitination and destruction by proteasome - degradation of Emi2 releases a brake on the APC - APC activity degrades cycB exit form MII arrest ❖ MLCK (myosin light chain kinase) → exocytosis of CGs → polyspermy block → cytoplasmic movements ❖ PKC (protein kinase C) - DAG-induced relocation to plasma membrane - regulation of Ca2+ influx - phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) → actin reorganization – GCs exocytosis ❖ TCA enzymes → activation of mitochondrial metabolism → ↑ATP supply → maintainance of Ca2+ waves ❖ PLCζ - positive feedback loop Calcium signaling effectors Emi2 Ajduk et al 2011 - rhythmic cytoplasmic movement triggered by Ca2+ oscilations - actomyosin-mediated spasms detectable by particle image vector analysis - non-invasive prediction of embryo viability? fertilized egg unfertilized egg Fertilized human oocytes exhibit a coordinated cyclic movement of cytoplasm („swirl“, „twirl“) before PBE2 is visible in transmitted light Activation-induced cytoplasmic movements - PLCζ active only in oocytes - existence of oocyte-specific activator? - human PLCζ has higher Ca2+ signalling potency than mouse PLCζ Nomikos et al. 2010 PLCζ activity Ca2+ binding Catalytic binding Lipid binding NLS Nuclear Localization Signal PLCζ activity Location of PLCζ - in mouse, Ca2+ oscillations temporarily ceases with PLCζ being sequestered into newly formed pronuclei - single Ca2+ peak observed shortly before every mitotic division - sinusoidal fluctuations disappear progressively in arrested human embryos - mitosis can be stopped using Ca2+ chelators Larman et al., 2004 Clinical implications ❖Total fertilization failure (TFF) - unexpected low fertilization efficiency (<25% FR) - normal spermiogram and apparently normal oocyte morphology - oocyte activation deficiency (OAD) - failure of sperm chromatin decondensation - conventional IVF failure can be bypassed by ICSI - rare (1-3%) in ICSI cycles - recurrence 30-50% - premature termination of treatment - distressful for patients and personell TFF Oocyte deficiency Sperm deficiency ICSI technique Campos et al. 2023Xue et al 2022 Clinical implications - mutation/impaired expression of PLCζ is related to recurrent fertilization failure and male/idiopathic infertility - PLC ζ deficiency in sperm correlates with altered Ca2+ oscillation pattern - diagnostical screening for PLCζ genetic profile and/or immunostaining pattern in infertile patients for tailored treatment and informed consulting Ramadan et al 2012 • PLCζ fertileinfertile Clinical implications - rescue by (co)injection of PLCζ along with defective sperm? Yamaguchi et al 2017 - injection of mouse/human PLCζ cRNA/protein induces Ca2+ oscillation similar to those seen during fertilization and triggers parthenogenesis Swann et al 2006 • PLCζ Nokimos et al 2013 (mouse) McCarter et al, ESHRE 2024, conference abstract (human) Clinical implications • Oocyte deficiency Increase in the number of IP3R1 and clustering Increase in IP3R1 sensitivity Increase in the amount of calcium ions stored Reorganization of ER, CGs, Mt and cytoskeleton Protein synthesis GV Immature oocyte MII Mature oocyte - deficiency in PLC ζ-triggered activation signalling cascade - activation capacity and Ca2+ oscillation adversely affected by - incomplete/aberrant cytoplasmic maturation - oocyte in vitro maturation - cryopreservation - reproductive aging - in vitro postovulatory aging sER+ oocytes - release more Ca2+ over a period of time? - abnormal disribution of IP3 receptors at SER Clinical implications Aspiration Deposition Puncture • ICSI technique Clinical implications • Mouse oocyte activation test (MOAT) - heterologous ICSI assay (human sperm + mouse egg) - evaluation of fertilization outcome Björn Heindryckx The potency of PLCζ : human > mouse The oocyte volume: human > mouse ❖Diagnosis of activation problems Barberán et al 2020 Is MOAT sensitive enough to reveal activation deficiencies? ICSI ICSI ICSI Clinical implications • Human oocyte activation test (HOCA) - homologous ICSI assay (human sperm + human egg) - analysis of Ca2+ oscillations Björn Heindryckx ❖Diagnosis of activation problems MOAT group 1 MOAT group 3MOAT group 2 Courtesy of B. Heindryckx • Mouse oocyte calcium analysis (MOCA) - heterologous ICSI assay (human sperm + mouse egg) - measurement of Ca2+ oscillations amplitude and frequency Interpretation of results: MOCA < 9MOCA > 9 Group BGroup A Normal sperm factor activity Possible sperm factor deficiency - more sensitive than MOAT but can´t be directly extrapolated to human model Clinical implications ❖Artificial oocyte activation (AOA) Clinical implications • Mechanical activation - hypo/hypertonic solution - hydrostatic pressure - modified ICSI technique ❖Artificial oocyte activation (AOA) - ~50% FR BUT no improvement if used as a routine technique! Thomas Ebner Clinical implications • Electrostimulation ❖Artificial oocyte activation (AOA) ✓ Single pulse of 1.5 kV/cm for 100 µs - direct current voltage causes rearrangement of cell membrane proteins leading to the formation of pores allowing for the influx of Ca2+ ions - ms/µs/ns pulses - risk of large pore opening leading to cell death ! ✓ nanosecond pulsed electric fields (nsPEFs) ✓ 10 ns stimulation - stimulate Ca2+ eflux from ER and spontaneous Ca2+ oscillations while maintaining plasma membrane integrity - high activation rate - not tested in humans Clinical implications • Chemical activation ❖Artificial oocyte activation (AOA) ✓ Strontium chloride (SrCl2)* ✓ Phorbol esters ✓ Thiomersal** ✓ Ethanol ✓ Puromycin ✓ Calcium ionophore − ionomycin − calcimycin (A23187) • Molecular targetting - injection of calcium (0.1 M CaCl2) - injection of sperm extract*** - PLCζ cRNA/recombinant protein – experimental*** Single peak Multiple peaks * Work only in rhodents! ** Impairs spindle *** Regulatory issues ❖Artificial oocyte activation (AOA) • Calcium ionophores - highly selection ion carriers that form stable complex with Ca2+ and pass through the cell membrane - causes transient release of Ca2+ from i.c. stores and enhance Ca2+ influx Ramadan et al., 2012 Mammalian oocytes appear to be tolerant to pertubations in Ca2+ oscillation pattern as long as the total amount of Ca2+ release is uncompromised and passes a critical threshold -different Ca2+ oscillation pattern than IVF/ICSI/P LCζ! different Ca2+ oscillation pattern than IVF/ICSI/PLCζ! (Markoulaki et al., 2003, Johnson et al., 1998, Nikiforaki et al., 2014, Ebner and Montag 2016) Clinical implications - short-term effect - stops after drug washout ↑ i.c [Ca2+] Clinical implications ❖Artificial oocyte activation (AOA) Vanden Meerschaurt et al 2014 - Calcium ionophore treatment - occasional clinical use in specific cases - nonstandartized - supplementation of culture media with chemical compounds OR commertially available ready-to-use medium GM508 CultActive containing calcimycin 15 min (Nikiforaki et al 2016) - ionomycin appears to be more efficient than calcimycin in both mouse and human eggs Clinical implications ❖Artificial oocyte activation (AOA) • Indications - not beneficial for all patients! - experimental, considered as an „adds-on treatment“ by ESHRE ✓ Complete fertilization failure in previous cycle (Ebner & Montag, 2012) ✓ Less than 30% fertilization rate in previous cycle (Montag et al., 2012) ✓ Severe male factor (TESE, PESA, globozospermia) ✓ Developmental problems in previous cycle (Ebner et al., 2015) ✓ Low blastulation rate (<15%) ✓ Developmental arrest ✓ Developmental delay (-24h) ✓ Arrest at 2Pn stage (Darwish & Magdi, 2015) ✓ SER+ eggs ? ✓ Immature eggs? • Contraindication Clinical implications ❖Artificial oocyte activation (AOA) • Safety anal atresia Ebner et al., RBM 2015 - no increase in meiosis II segragation errors - healthy children born from AOA cycles - appears to be safe BUT lack of data ✓ Language skills ✓ Motor skills ✓ Cognition were found to be within expected ranges Clinical implications ❖Management of fertilization failure - adequate patient selection - short gamete co-incubation (1h) - increase sperm number - disrupt COC Courtesy of T. Ebner Egg activation and SCNT AOA 2018 Ian Wilmut Dolly 1996 John Gurdon 1962 2024 ReTro Parthenogenesis - asexual reproduction - „virgin birth“ - an egg can develop without being fertilized by a sperm - occasional reproduction manner in lower species - in mammals, parthenogensis can be induced by AOA - parthenotes can develop to different stages but not to term - all female offsprings (XY determination) OR all male offsprings (ZW determination) Parthenogenesis Induction of parthenogenesis in vitro Meoisis I Cytochalasin B/D Meoisis II PBE1 block Ionophore PBE1 Ionophore activation Cytochalasin B/D Ionophore PBE2 activation PBE2 activation PBE2 block 1n 1n 2n 2n 2n 2n 2 n Diploid heterozygous parthenote 2 n Diploid homozygous parthenote 1 n haploid parthenote 2 cell stage Parthenotes vs. embryos Imprinting perturbations - uniparental origin unphysiological reconstruction of centrosome - Imprinting Control Region - Differentially Methylated Region Rodents less sensitive to the lack of parental centriole! - genetic instability and aneuploidy in pathenotes Parental imprinting in mammals imprinting-mediated balance between paternal and maternal genomes is critical for mammalian development „parental conflict“ fetal growth Igf2H19 Parthenogenesis in mammals - AOA of oocyte containing two sets of maternal genome reconstructed by series of nuclear transfers - one allele derived from mouse with deleted H19 gene (loss of maternal imprints) - the mutated parthenote developed to adulthood with the ability to reproduce Kono et al 2004 genomic imprinting is a barrier for parthenogenesis in mammals Kaguya Parthenogenesis in mammals Wei et al. 2022 - Targeted epigenetic editing of imprinting control regions - oocyte injection with single-guide RNAs with protospacer adjacent motif (PAM) sequences matching one allele but not the other - DNA (de)methylation modification by catalytically inactive 9 (dCas9)-Dnmt3a or dCpf1-Tet1 - transfer of mouse genome-edited parthenogenetic blastocysts to foster mothers - significantly extended development, and viable full-term offspring - low efficiency due to insufficient methylation or loss of imprinting Zhen-Ao Zhao Parthenogenetic stem cells (PESCs) 1n PESCs - spontaneous diploidisation 2n PESCs - trophoblast underdeveloped - little extraembryonic tissue - abnormal genetic imprinting lost during culture - study of imprinting and inheritance mechanisms Supernumerary centrioles hPESCs Autophagy vacuolesBrevini et al 2009 Parthenogenotes and partheno-embryo chimeras ❖ Parthenotes - developmental arrest - physiologically unable to develop to term - reproductively and in vitro aged eggs prone to spontaneous activation and short-term partnogenetic development in ART ! ❖ Partheno-embryo chimeras - Generated by - can develop to term but delayed development - parthenote cells fail to contribute to tissues of mesoderm and endodermal origin (b) injection of PESCs to morula/ blastocyst PESCs (a) combination of embryo and parthenote blastomeres Parthenogenesis in humans - rare event but not impossible Strain et al 1995 - phenotypic male, blood cells XX karyotype - 46,XY/46,XX mosaic - complete maternal isodisomy in blood cells and fibroblasts - Sex reversal, facial asymmetry, mild festures - Proposed speculative mechanism: diploidisation and spontaneous activation before syngamy Parthenogenesis in humans • Implicated in infertility in recurrent miscarriage and pregnancy failure ? Hegazy et al 2023 - reports of ART cases in which cytogenetic analyses of conceptus biopsy samples found match to maternal side exclusively • Role in occurrence of idiopathic ovarian teratoma ? - Slow-growing benign germ cell tumour but can be coverted to malignant - Composed of derivatives of all 3 germ layers - Perinatal teratomas believed to originate from mislocalized PGCs but cystic ovarian teratoma mostly found in women of reproductive age Same sex parent offsprings? - deletion of 20 imprinted regions + haploid hESC generation +SCNT