Sea urchin -lll

Sea urchin -lll

PLC-gamma inhibition circumvented by:

Fertilization calcium signaling requires phospholipase C-gamma to generate second messenger IP3 that opens ER calcium channels. Upon Src tyrosine kinase activation at sperm entry site, PLC-gamma hydrolyzes PIP2 producing IP3 binding ER receptors releasing calcium globally as wave. If PLC-gamma inhibited pharmacologically with U73122 inhibitor, calcium wave fails and cortical granule exocytosis and activation do not occur. Direct microinjection of IP3 downstream restores calcium release even when PLC blocked, because IP3 directly opens ER channels bypassing lipid hydrolysis step. Calcium injection also triggers release but less faithfully. ATP and Bindin cannot substitute.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 7: PLC-gamma inhibition rescued by IP3 microinjection calcium release.

View Details

Sea urchin -lll

Micromere signaling induces adjacent cells via:

Adjacent to micromeres, macromeres and large micromeres receive inductive signal mediated by direct cell contact requiring juxtacrine interaction. Micromeres express transmembrane ligand Delta following derepression of HesC by Pmar1. Neighboring cells express Notch receptor; Delta binding triggers gamma-secretase cleavage releasing Notch intracellular domain NICD that translocates to nucleus activating transcription of secondary mesenchyme genes gcm, gataE, foxA for pigment and blastocoelar cells and endoderm. This Delta-Notch induction specifies non-skeletogenic mesoderm. Blocking Notch with DAPT inhibitor eliminates secondary mesenchyme, while Delta misexpression induces ectopic mesoderm, proving essential vegetal patterning mechanism.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 10: Micromere Delta-Notch signaling induces adjacent cells mesoderm.

View Details

Sea urchin -lll

Blocking β-catenin nuclear entry forms:

Beta-catenin nuclear entry drives vegetal gene regulatory network including Pmar1, Wnt8, blimp1b required for endomesoderm specification. Blocking nuclear entry via overexpression of cadherin cytoplasmic tail that sequesters beta-catenin, or dominant-negative TCF that occupies promoters without activation, or overactive GSK-three, eliminates endoderm and mesenchyme specification completely. Resulting embryos lack archenteron and skeleton, becoming permanent blastulae covered with long cilia called ciliated ectodermal balls, expressing only animal ectoderm markers like spec1. This animalized phenotype shows beta-catenin necessity for vegetal fates, producing ectodermal ball when blocked, opposite of vegetalization induced by GSK-three inhibition.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 10: Beta-catenin blocking forms animalized ciliated ectodermal ball.

View Details

Sea urchin -lll

Cortical reaction prevents polyspermy by:

Cortical reaction and fast electrical block together prevent polyspermy, but initial rapid depolarization provides immediate transient protection. Upon first sperm fusion, sodium influx depolarizes egg plasma membrane from negative to positive within seconds, making membrane refractory to additional sperm fusions that require negative potential. This electrical change precedes cortical granule exocytosis by about half minute. Although some textbooks attribute depolarization to cortical granule release, it represents initial phase of reaction sequence. Membrane depolarization thus serves as fast mechanism within cortical reaction leading to polyspermy prevention before permanent fertilization envelope elevation completes protection of embryo.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 7: Cortical reaction depolarization fast block polyspermy prevention.

View Details

Sea urchin -lll

Micromere specification involves:

Specification of micromeres depends on intricate gene regulatory network based on activation of Pmar1 by nuclear beta-catenin localized vegetally. Pmar1 encodes transcriptional repressor that specifically inhibits transcription of HesC, itself a global repressor of micromere fate genes. This double-negative gate Pmar1 represses HesC, HesC represses Delta, alx1, ets1, tbr, tel, dri, allows micromere program expression exclusively where Pmar1 present in large micromeres. Therefore micromere specification fundamentally involves Pmar1 and HesC interaction forming logic gate. Beta-catenin and Delta act upstream and downstream respectively, but core gate is Pmar1-HesC, while Frizzled and Dishevelled regulate beta-catenin earlier.

Ref: Oliveri et al., Development 2008: Micromere specification via Pmar1-HesC double-negative gate regulatory.

View Details

Sea urchin -lll

Vegetal cortex localization of Disheveled occurs:

Dishevelled protein required for vegetal beta-catenin stabilization shows precise temporal localization pattern. Maternal Dishevelled protein and mRNA are deposited and anchored to vegetal cortex during oogenesis, well before fertilization occurs, through actin and microtubule dependent transport mechanisms. In full-grown oocytes, in situ hybridization and immunostaining reveal vegetal cortical enrichment. Fertilization does not induce this localization; it is inherited from oogenesis. This pre-fertilization placement ensures that by fourth cleavage, micromeres inherit high Dishevelled and therefore high nuclear beta-catenin, permitting autonomous specification. Relocalization after fertilization cannot fully rescue patterning, indicating temporal requirement.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 10: Vegetal Dishevelled localization occurs before fertilization maternal.

View Details

Sea urchin -lll

Removal of cortical granules prevents:

Cortical granules lie beneath egg plasma membrane and upon calcium rise fuse releasing contents that transform vitelline membrane into fertilization envelope constituting slow, permanent block to polyspermy. If granules are removed by mild centrifugation that stratifies them away or exocytosis blocked by inhibitors, envelope elevation fails despite normal sperm fusion. Fast electrical block still occurs but membrane repolarizes within minute, allowing supernumerary sperm to fuse causing polyspermy, multipolar mitoses, and lethal aneuploidy. Therefore granule removal prevents polyspermy prevention, while sperm attraction, entry, and cleavage initiation can still initially occur until developmental defects manifest and embryo arrests.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 7: Cortical granule removal prevents polyspermy slow block.

View Details

Sea urchin -lll

Respiratory burst involves:

Following fertilization calcium transient, sea urchin eggs increase oxygen consumption several-fold within minutes, termed respiratory burst. Calcium activates NADPH-dependent oxidase complex in cortex producing superoxide converted to hydrogen peroxide. Hydrogen peroxide serves as substrate for ovoperoxidase released from cortical granules to catalyze di-tyrosine cross-links hardening fertilization envelope. Burst is calcium dependent, inhibited by calcium chelators BAPTA, and occurs concurrently with envelope elevation and cortical reaction. It parallels NADPH oxidase activity in phagocytes but here functions developmentally for extracellular matrix stabilization rather than pathogen killing, not for immediate ATP synthesis or meiotic regulation.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 7: Respiratory burst calcium dependent oxygen increase fertilization.

View Details

Sea urchin -lll

Resact specifically attracts:

Resact is fourteen amino acid peptide purified from egg jelly of Arbacia punctulata that guides sperm chemotaxis toward egg. It binds high-affinity receptors on sperm flagellum coupled to guanylate cyclase, increasing cGMP and calcium oscillations altering flagellar waveform and steering toward gradient source. Chemotactic response is highly selective; Arbacia sperm respond strongly to Resact at picomolar concentrations, while Strongylocentrotus sperm show minimal response, preferring Speract. This species specificity enhances conspecific fertilization assurance when multiple species spawn simultaneously, contributing to reproductive isolation and preventing hybridization in broadcast spawning marine environment, improving fertilization success significantly.

Ref: Ward et al., Chemotaxis: Resact species-specific attraction conspecific sperm sea urchin reproductive isolation.

View Details

Sea urchin -lll

Cortical granules convert:

Unfertilized sea urchin egg vitelline membrane lies closely apposed to plasma membrane with little perivitelline space. Upon calcium-dependent cortical granule exocytosis, contents including hyalin, proteases, sulfated mucopolysaccharides, and ovoperoxidase are released. Proteases cleave linkers between vitelline membrane and plasma membrane, osmotic swelling by glycosaminoglycans elevates membrane outward, and cross-linking enzymes harden it via di-tyrosine bonds. Modified vitelline membrane becomes fertilization envelope raised above egg surface with perivitelline space between. This conversion changes soft vitelline layer into tough barrier preventing supernumerary sperm entry and protecting embryo before hatching occurs.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 7: Cortical granules convert vitelline membrane to fertilization envelope.

View Details

Sea urchin -lll

HesC expression represses:

HesC is global transcriptional repressor containing basic helix-loop-helix Orange domain expressed ubiquitously early in sea urchin. It binds E-boxes in promoters of micromere-specific genes including Delta ligand and skeletogenic transcription factors alx1, ets1, tbr, dri. Repression keeps Delta silent outside micromeres. In micromeres, Pmar1 represses hesC gene, derepressing targets and allowing Delta-Notch signaling. Therefore HesC expression represses Delta expression, ensuring spatially restricted signaling. Overexpression of HesC eliminates Delta and skeleton, while HesC morpholino knockdown causes ectopic Delta throughout embryo, confirming direct negative regulation rather than regulation of beta-catenin or micromere formation itself.

Ref: Davidson Lab, Sea Urchin GRN: HesC represses Delta expression non-micromere lineages double-negative gate.

View Details

Sea urchin -lll

Egg membrane depolarizes within:

Upon sperm-egg fusion in sea urchin, sodium channels open causing rapid influx of sodium ions into egg cytoplasm raising positive charge. Membrane potential shifts from resting about minus seventy millivolts to positive twenty millivolts within one to three seconds, constituting fast electrical block to polyspermy. Depolarized membrane is refractory to additional sperm fusions because fusion machinery requires negative potential for electrostatic interaction. This transient block lasts about minute until cortical granule-derived fertilization envelope elevates permanently. Later cortical reaction provides slow block, while ATP synthesis and meiosis completion occur after electrical block, preventing lethal polyspermy during spawning.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 7: Membrane depolarization within 1-3 seconds blocks polyspermy.

View Details