Embryology II PREIMPLANTATION DEVELOPMENT spring 2025 Zuzana Holubcová Department of Histology and Embryology zholub@med.muni.cz Fertilization Preparation for fertilization SPERMATOGENESISOOGENESIS - entry to meiosis - follicle formation - oocyte and follicle growth - dominant follicle selection - oocyte maturation - ovulation - completion of meiosis - morphological transformation - sperm maturation (forming transient sperm reservoirs) - capacitation - hyperactivation - acrosomal reaction - cumulus and zona penetration INTERNAL FERTILIZATION Preparation for fertilization ❖Capacitation - series of biochemical and physiological changes in the sperm cell - takes place inside the female reproductive tract - can be achieve in vitro in media containing appropriate compound and with high pH - critical for sperm ability to reach fertilization site and interact with the oocyte - changes in (1) membrane properties (fluidization) (2) intracellular ion concentration (3) activities of enzymes Preparation for fertilization ❖Capacitation Jin and Yang, 2017*NBC = Na+/HCO3 co-transporter ROS H+ motility - removal of cholesterol increases sperm membrane permeability - leak of H+ → rise of pHi (alkalization) - Ca2+ influx → activation of soluble adenylate cyclase (sAC) → Tyr-kinase phosphorylation - physiological ROS generation Preparation for fertilization ❖Sperm transport https://clinicalgate.com/transport-of-gametes-and-fertilization/ Preparation for fertilization ❖Sperm reservoirs Thys et al 2009 - sperm deposition sites in lower oviduct (isthmus) - adhesion to oviductal epithelium - sperm membrane proteins bind to carbohydrate moities of oviduct epithelial cells - storage of functional sperm before ovulation, delay of capacitation, maintenance of sperm cell viability Suarez et al 2005Ellington 1998 Preparation for fertilization ❖Hyperactivation https://www.youtube.com/watch?v=PFPNv2LX3XY - change from basal to hyperactivated motility - symmetric flagellar bending with low frequency → asymmetring beating - critical for sperm release from reservoir, propulsive movement in viscous elastic fluid, penetration through extracellular matrix of COC, and for drilling zona pellucida Basal movement Hyperactivated movement Preparation for fertilization ❖Hyperactivation - caused by rapid rise of intracellular Ca2+ ([Ca2+]i) - Ca2+ serves as a second messenger in cell signalling and directly enhaces dynamics of cytoskeletal components in sperm flagellum - primed by alkalic environment - multiple (voltage-dependent/pH-sensitive/ligand-activated) ion chanells in sperm midpiece and tail - H+ efflux increase rise of intracellular pH required for capacitation - oviduct lumen pH increases after ovulation providing favourable conditions for hyperactivation - ion movements accross membrane manifest as membrane depolarization „patch-clamp“ technique Preparation for fertilization ❖Hyperactivation CATSPER channel Hwang et al, Cell 2019 principal piece of the sperm tail - pH sensitive ion channel - testis-specific expression - its opening causes Ca2+ influx resulting in rapid rise of [Ca2+]i -/- normal mating, sperm count and morphology - impaired motility, incapable to penetrate - sterile - mutation and altered experession linked to asthenospermia and male infertility Ca2+ - Preparation for fertilization PROGESTERONE = chemoattractant and hyperactivation-inducing factor Nature 2011❖Hyperactivation Miller et al Science 2016 - sperm-specific Na/H+ exchanger SLC9C1 mediates i.c. alkalization - ↑i.c. pH → conformational change of the blocker molecule EFCAB9 at CatSper pore and its opening Polina Lishko - CatSper openning results in Ca2+ influx and triggers sperm hyperactivation - binds to ABHD2 - α/β hydrolase domain-containing protein 2 which serves as a non-genomic progesterone receptor on the extracellular side of sperm membrane - after progesterone binding lipid hydrolase ABHD2 degrades endocannabinoid 2arachidonoylglycerol (2AG) enriched in the sperm membrane - the replenishment of AG2 leads to the CatSper full opening and triggers sperm hyperactivation ← alkalization ← progesteron Trebichalska and Holubcova, JARG 2020 Role of PGE1? Direct competition with 2AG? Preparation for fertilization ❖Hyperactivation Trebichalska and Holubcova, JARG 2020 - ligand-sensitive nature of CatSper channel „promiscuous“ channel - opportunity for development of „male/unisex“ contraception based on prevention of hyperactivation Preparation for fertilization ❖Hyperactivation Ca2+ influx through CatSper + Internal Ca2+ stores mobilization Preparation for fertilization ❖Hyperactivation - Acrosome - Mitochondria in the midpiece - Redundant nuclear envelope (RNE) in the connecting piece *calcium-induced calcium release * - CatSperm mediated elevation of flagellar [Ca2+]i spreads forward and stimulates secondary Ca2+ release from intracellular stores in sperm midpiece and head region Preparation for fertilization ❖Hyperactivation Acrosomal Ca2+ store Neck/midpiece Ca2+ store Costello et al 2009, modified ❖Passage through cumulus Preparation for fertilization ▪ NYD-SP8 and PACAP - sperm –derived surface factors - activate Ca2+ signalling in cumulus cells → ↑ progesterone release → acrosomal reaction ▪ PH-20 and Hyal5 - glycosylphosphatidylinositol (GPI)-anchored proteins - localized on head of both human and mouse sperm - hyaluronidase activity - hydrolyses HA-rich cumulus matrix 1) 2) SP8, PACAP Sun et al 2011Salustri et al 1999 Preparation for fertilization ❖Acrosome reaction - membrane fusion and lytic enzyme release - enables sperm to penetrate cumulus and zona pellucida surrounding the egg - exposition of new set of surface antigens - release of hyaluronidase and acrosin Preparation for fertilization ❖Acrosome reaction Kateřina Komrsková (Dvořáková/Hortová) Frolikova et al 2016 CD46 b1 integrin Baibakov et al 2012 Jurrien Dean huZP1 rescue huZP2 rescue huZP3 rescue huZP4 rescue - human sperm binds to human ZP2 ❖Zona binding - transgenic mice expressing human ZP proteins - coincubation with human sperm Preparation for fertilization zona pellucida = meshwork of interconnected filament - glycoproteins ZP1-4 - outer porous region (~25%) - inner compact region (~75%) - thickening from inside to outside - -mutations of ZP genes linked to conventional fertilization failure and EFS zona pellucida oolema Mio et al 2008 Sperm adhesion to microvilli Preparation for fertilization ❖Zona penetration - hydrolysis of hyaluronan by acrosome-released proteases hyaluronidase and acrosin (1) INITIAL APPROACH (2) MOLECULAR RECOGNITION (3) MEMBRANE APPOSITION (4) MEMBRANE FUSION Gamete interaction -/- from a number of genes shown to play a role in sperm-egg interaction only a few have been proven essential for fertilization in mouse model - normal mating behaviour and gamete number and morphology - BUT sex-specific reduction of fertility resulting from failed sperm-egg fusion ❖ CD9 - localized on oolema and required for normal microvili morphology and distribution - organizer of membrane architecture facilitating sperm-egg contact preceeding fusion - antiCD9-antibody inhibits sperm-egg binding in vitro - microvesicles transport CD9 on sperm membrane before fertilization - microvesicle-transported CD9 fragments can restore fertilisation capacity of CD9-/- oocytes Runge et al 2007 Miyado et al 2018 Key molecules of gamete recognition Key molecules of gamete recognition ❖ CD81 - expressed on oocyte surface, associates with CD9 - incorporated to microvesicles, present in PVS - depletion inhibits gamet fusion - CD81-/- subfertile, CD81/CD9 -/- sterile - CD9 microinjection reverse CD81-/- phenotype - synergic affect with CD9? Ohnami et al 2012 - regulation of membrane architecture - rearrangement of sperm membrane in preparation for adhesion/fusion? - expressed on the sperm surface after acrosomal reaction - spermatozoa of -/- mise penetrate ZP but fail to fuse with the egg - fertilization defect can be bypassed by ICSI - antibody against Izumo has contraceptive affect - both Izumo and CD9 are enriched in adhesion area, but NO physical interaction between the two molecules Inoue et al 2005, Satouh et al 2012 Key molecules of gamete recognition ❖ Izumo1 TSSK6 - localized to inner acriosomal membrane and redistributed after acrosomal reaction Izumo= Japanese shrine of marriage - Izumo-complementary receptor expressed on the egg´s surface - Juno-deficient eggs do not fuse with spermatozoa - direct Juno-Izumo interaction is crucial for egg-sperm adhesion - Juno mutations linked to clinical cases of idiopathic fertilization failure Bianchi et al 2014 Key molecules of gamete recognition ❖ Juno Juno = Roman goddess of marriage and childbirth = Folate 4 receptor (Folr4) - ectopic expression of fluorescently tagged Juno/Izumo - firm adhesion but not fusion oocyte Izumo-expressing cell Chalbi et al 2014 JUNO-IZUMO COMPLEX is vital for membrane tethering BUT lacks fusogenic activity further factors must be involved PROPOSED MODEL: Izumo-Juno binding induces accumulation of CD9 at adhesion site thus promoting CD9-mediated clustering of membrane proteins that participate in assembly of the cell fusion machinery Chalbi et al 2014, Bianchi et al 2014 Key molecules of gamete recognition CD81? Key molecules of gamete recognition ❖ MAIA Kateřina Komrsková (Dvořáková/Hortová) - Fc receptor-like 3 - named after fertility goddess - localized on oolema of unfertilized human oocytes - close association with Juno Vondrakova et al 2022 MAIA - transgene coexpression of MAIA+Juno led to sperm binding to primary cultured cells - MAIA facilitates sperm fusion in the presence of Juno Key molecules of gamete recognition ❖ MAIA ? -unknown or CD9 protein Juno/Izumo1 interaction ensures thethering of sperm and egg membranes. Fertilizing sperm display oscillatory motility. Izumo1 dimerization triggers the conformational change of Juno and its shedding from the egg surface. MAIA binds in the emptied Juno/Izomo1 binding pocket. MAIA conformational change to extracellular Fc domains, leading to close membrane proximity enabling gamete fusion with the loss of sperm motility. PROPOSED MODEL OF MAIA/Juno-Izumo1 BINDING - mouse/human sperm expressed surface protein - SPACA6-/- male mice infertile due to sperm inability to fuse with egg - anti-SPACA6 Ab inhibits human IVF - structural similarity with Izumo1 Barbaux et al 2020 Key molecules of gamete recognition ❖ SPACA6 - absence of SPACA6 has no effect on Izumo localization - SPACA6 alone does not induce adhesion! Key molecules of gamete recognition ❖ TMEM95, SOF1, FIMP, DCST1/2 - sperm specific proteins - CRISPR –depletion/gene targetting causes male mice sterility - spermatozoa can not fuse with oolema - no effect on Izumo localization Noda et al 2020 Fujihara et al 2020 - roles in molecular recognition? Lams-Torazo al 2020 Noda et a 2020 Inoue et a 2021 DCST 1/2 Key molecules of gamete recognition Deneke et al. 2024 Elofsson et al 2024 - AlphaFold Multimer ( an extension of AlphaFold) - a deep learning model for predicting the 3D structure of proteins trimeric complex IZUMO1-TMEM81-SPACA6 dimeric complex Juno-CD9 MAIA? Key molecules of gamete recognition MAIA ? TMEM81 Key molecules of gamete recognition Brukman et al. 2023 - Izumo is capable to induce fusion of primary cells via viral-like mechanism - fusogenic activity of Izumo follows adhesion mediated by Juno-Izumo interaction - Juno-binding and fusion are mediated by two differrent Izumo domains Interspecies gamete interaction Naz 2014 Inoue 2013 Bianchi and Wright 2015 Yanagimatchi 1976 prevents cross-species fertilization Mouse Mouse /Human HamsterMouse/Pig/Hamster ZP-free eggs Human Human/Hamster The nature of Juno-Izumo interaction is conserved accross mammalian species Gamete misrecognition Mutation of receptors Idiopathic infertility and fertilization failure Altered expression Interfering agents CLINICAL IMPLICATIONS: Polyspermy - lethal condition when the egg is penetrated by more than one spermatozoa - more then two copies of each gene cause genetic inbalances and resulting embryo is non-viable Polyspermy prevention - In vivo - Fish: the sperm can only enter the egg just via the narrow opening, the micropyle, the rest of the egg being covered by impermeable chorion - Sea urchin: limitation on the number of sperm that are able to penetrate the extracellular coats and fuse with the egg - Mammals - passage through the female reproductive system - limited viability of sperm in oviductal reservoirs - structural organisation and postfertilization modifications of zona pellucida - rearrangement of oolema - In vitro - optimising sperm concentration and coincubation time with the egg - ICSI monospermy polyspermy oolema ❑ CORTICAL REACTION ❑ ZINC SPARK ❑ MEMBRANE DEPOLARIZATION ❑ JUNO REMOVAL MODIFICATION OF OOLEMA MODIFICATION OF ZONA PELLUCIDA Trebichalska and Holubcova, JARG 2020 Polyspermy block overview Mio et al 2012 Polyspermy block on ZP oolema ❖CORTICAL REACTION - slow and permanent block to polyspermy - sperm penetration triggers exocytosis of cortical granules - the content of granules modifies structure of zona pellucida and makes it impermeable for incoming sperms = zona hardening Polyspermy block on ZP oolema ? ? ? ? ❖CORTICAL REACTION De Paola et al 2015 - exocytosis og GCs is mediated by SNARE pathway Disassembly of cis-SNARE complex Assembly of trans-SNARE complex - SNARE association with complexin prevents spontaneous secretion of CGs - exocytosis followed by clatrin dependent endocytosis - premature exocytosis in in vitro matured and vitrified/thawed human oocytes ! SNARE proteins - membrane associated proteins oriented to cytosol - involved in fusion of vesicles with targeted membrane Polyspermy block on ZP ❑ OVASTACIN - belongs to astacin family of metaloproteases - key enzyme of cortical granule content - cleaves ZP2, a building component of ZP and primary sperm-binding ligand - ZP2 destruction establishes definitive post fertilization block ❖CORTICAL REACTION Jurrien Dean Burkart et al 2012 - ovastacine-deficient eggs bind sperm after fertilization - ovastacin is partially active before fertilization-triggered exocytosis and pre-hardens the ZP N-terminal fragment of ZP2 detection - western blot: - ovastacin encoded by Astl gene Astl-/Polyspermy block on ZP oolema ❖CORTICAL REACTION ❑ FETUIN -B - liver-derived plasma protein - antagonize basal activity of constantly leaking ovastacin from unfertilized oocytes - cortical reaction overwhelms fetuin-B buffering capacity thereby intiating Zona hardening Dietzel et al 2013 Cappa et al 2018 - Modulation of female fertility? - Supplementation of IVF medium for prevention of ZP hardening? - serum levels fluctuate during menstrual cycle CLINICAL IMPLICATIONS: Polyspermy block on ZP oolema - Zn accumulation during maturation sustains MII arrest - rapid exocytosis of Zn containing vesicles immediately upon sperm entry - Zn stabilizes ZP proteins and causes its hardening Zinc spark = early hallmark of fertilization Jurrien Dean ❖ZINC SPARK Tokuhiro and Dean, 2018 - quantification of Zn spark after conventional IVF -> best quality embryo selection -> oocytes exerting a low signal -> rescue ICSI? - Zn-specific chelatators for AOA? - Supplementation of IVM medium? - Zn release = fast and transient block to polyspermy CLINICAL IMPLICATIONS: Polyspermy block on ZP oolema (1) (2) Zinc spark = early transient ZP block -strat in 2-3 min Ovastacin cleavage of ZP2 = late permanent ZP block - start in >30 min Tokuhiro and Dean, 2018 Polyspermy block on ZP oolemaBianchi et al 2014 - rapid permanent oolema block - Juno receptor is removed from oolema after normal fertilization (but not activation and ICSI) - absence of Izumo receptor makes the oocyte refractory to late-coming sperms Polyspermy block on oolema ❖JUNO SHEDDING oolema - fast and short-lasting block to polyspermy - transient alteration of electric charges on egg´s surface - discovered and studied in marine animals, but unfounded in mammals Wozniak et al 2018Purves et al 1998 Sea urchin Xenopus laevis Polyspermy block on oolema ❖MEMBRANE DEPOLARIZATION Practical implications ❖ IMPROVED DIAGNOSTICS AND INFORMED CONSULTING ❖ BETTER GAMETE CULTURE AND/OR IN VITRO FERTILIZATION CONDITIONS ❖ BIOMARKER-BASED ASSAY FOR SPERM SELECTION ❖ SHORT-ACTING NON-HORMONAL CONTRACEPTIVE Cat Practical implications CatSperm test 1. Add ejaculate 2. Incubate 3. Determine motility CatFlux bufferTM - low Ca2+ and progesterone CatSperm channel functional → sperm become immotile Conventional IVF vs. ICSI 1992 Gianpiero Palermo - developed for sperm factor infertility - bypassess natural sperm selection and gamete interaction - overused in clinical practice - increased cost vs. effectiveness ICSI (IntraCytoplasmic Sperm Injection) ZP free oocyte ICSI