University:
Amherst College
Course:
BIOL 220 | Developmental Biology
Academic year:

2021
Views:

249

Pages:

21
Author:

Braxton Mcdowell

Fertilization When Can Eggs Be Fertilized? • Meiotic State of the Egg – – – – – Young primary oocyte Fully grown oocyte Metaphase I Metaphase II Pronucleus Stage When Are Sperm Capable of Fertilization? • State of Sperm – Meiosis completed – Competent in testis (most animals) – Finish maturation in male ducts and capacitate in female tract (mammals) – Fraser Molec Reprod Devel 77: 197-208 (2010) What Are Barriers to the Sperm? • Egg Vestments – Coats made during oogenesis by follicles – Potential Barriers • tough keratinaceous shells (insects, fish, cephalopods) – micropyle • soft gelatinous shells (sea urchin, frog, mammals) – sperm acrosomal enzymes Sperm Competition I Sperm Competition II Most wily? Fastest? Success! Some Membrane Fusions • Fusion of Gametes – in most species triggers completion of meiosis – result: haploid male and female (pro)nuclei in common cytoplasm • fusion of nuclear membranes (syngamy) – mixing of chromosomes in new nuclear envelope = zygote nucleus Events at the Egg Surface How is the Sperm Activated? • I. Metabolic Activation – sea urchin from pH 7.2 testis to pH 8 sea water – H+ out/Na+ in • increases internal pH to 7.6 – stimulates motility and respiration • pH sensitive dynein • uses up ATP to swim • activates mitochondria to produce more ATP through respiration (oxidative phosphorylation) How is the Sperm Activated? • II. Acrosomal Reaction – induced by egg jelly – influx of Ca++ triggers two things • fusion of granule with p.m. secreting proteases • actin polymerization from behind the granule to make acrosomal filament – plasma membrane covered filament contacts the egg and fuses Acrosomal Reaction How Do Sperm Recognize Eggs? • Sperm – species specific binding to egg vitelline by tip of the acrosomal filament – expt: isolate acrosomal vesicles • major protein: 30,000 MW • make antibody which shows where it is and blocks binding of sperm to egg • protein is called bindin – pure bindin agglutinates eggs, species specific – important barrier to mixing of species • Very rapidly evolves Bindin Localization Agglutination How Does Egg Bind Sperm? • Egg – sperm binding sites can be detached with proteases – ~1500 per egg – detach 70 kDa fragment and purify and make antibody • Antibody blocks gamete binding – recognizes 350 kDa protein on blot – sperm receptor is integral membrane protein What Does Egg Do When Sperm Fuses? Release of Free Ca++ What Does Ca++ Trigger? Raising of vitelline layer – conversion into fertilization envelope Secretion of hyaline of cortical granules How Does Egg Prevent Multiple Fertilizations? • Polyspermy lethal in most species • Two levels of control in sea urchins – slow: physical – fast: electrical How Does the Slow Block Work? • Slow Block – secretion of cortical granules converts vitelline to fertilization membrane (f.m.) or envelope – sperm pushed from surface by f.m. -> barrier • expt: remove f.m., re-fertilize -> polyspermy – isolate c.g. products • remove vitelline, fertilize and collect in sea water • find trypsin-like proteases What Is the Role of Cortical Proteases? • cortical proteases have two roles – 1. Elevation of f.m. (delaminase) • eggs + sperm or parthenogenetic (chemical) agent -> f.m. • eggs + sperm and SBTI (inhibitor) -> rosettes • eggs + cortical proteases, then SBTI and parthenogenetic -> f.m. • Interpretation: cortical proteases sever p.m.v.l. bonds which permits lifting of f.m. What Is the Role of Cortical Proteases? • cortical proteases have two roles – 2. Detachment of sperm (receptor hydrolase) • eggs + cortical proteases, then sperm -/-> f.m. – sperm don’t attach to vitelline • eggs - v.l., then sperm -> polyspermy • eggs + sperm and SBTI -> rosettes and sperm remains on surface of f.m. • Interpretation: cortical proteases detach sperm binding sites from v.l. releasing excess sperm

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