Fertilization I

Gamete Structure & Recognition of Egg and Sperm

Major Events of Fertilization

1. Contact & recognition between sperm and egg

2. Regulation of sperm entry into the egg. Only one can enter - others inhibited from entering

3. Fusion of genetic material

4. Activation of egg metabolism to start development

Discovery of Sperm

• Leewenhoek (co-discoverer) originally thought them to be parasitic animals within the semen

– Called spermatozoa - sperm animals

– Attempted to find a whole preformed embryo within spermatozoa

• Nicholas Hartsoeker - co-discoverer of sperm drew a picture of what they expected to find - a preformed human called a homunculus

Homunculus

Sperm as the Agent of Fertilization

• In 1824 - J.L. Prevost and J.B. Dumas claimed that sperm were the agents of fertilization

– But disregarded by most

• In 1876 - Oscar Hertwig and Herman Fol independently demonstrated sperm entry into the egg and the union of the two cell’s nuclei

Structure of Sperm

• Consists of haploid nucleus, a propulsion system and sac of enzymes that enable the nucleus to enter the egg

• The sperm cell has very little cytoplasm and very compact DNA

• In the front is the acrosomal vesicle or acrosome - from golgi - contains the enzymes to enable the nucleus to enter the egg

Structure of Sperm

• The sperm head - nucleus + the acrosome

• Recognition of egg & sperm involve molecules of the acrosomal process (often finger-like proteins between the nucleus & acrosome

Germ Cell to Sperm

Sperm Propulsion

• Ascaris - amoeboid motion

• Most use - flagellum

• Motor portion - axoneme - composed of microtubules (9 + 2 arrangement)

• Propulsion via dynein - protein attached to the microtubules

– The energy of ATP allows active sliding of the outer doublet microtubules - causing the flagellum to bend

Mammalian Sperm

• After being expelled from the seminiferous tubules - sperm is stored in the epididymis

• In the epididymis they gain the ability to move

– Perhaps through modification of dynein

• When initially ejaculated, sperm do not have the ability to bind and fertilize the egg

• This final phase of maturation is called capacitation

The Egg

• Stores all the material necessary for the beginning of growth and development

• Mature egg - called an ovum (not commonly used) - also mature oocyte

• Developing egg - oocyte (before it reaches the stage of meiosis where fertilization can occur)

Common Egg Contents

• Proteins

– For supply of energy and amino acids - may be by accumulation of yolk (typically made elsewhere - liver & hat body)

• Ribosomes & tRNA

– Needs to make many proteins - may be burst of protein synthesis after fertilization - therefore need ribosomes & t-RNAs

• Messenger RNA

– mRNAs for early development usually already present (dormant) in the egg

– In sea urchins eggs- 25,000 to 50,000 different types of mRNA

Common Egg Contents

• Morphogenic factors

– Molecules that direct the differentiation of cells into certain cell types are present

– Localized to different regions - therefore become segregated into different cells during cleavage

• Protective chemicals

– Many eggs have UV filters & DNA repair enzymes

– Some have molecules that predators find distasteful - some birds have yolks with antibodies

Egg Structure

• Large nucleus - may be haploid at fertilization (e.g. sea urchins) - in many others such as worms & mammals its diploid

• Outside plasma membrane - vitelline envelope - a fibrous mat around the egg

• Made of 8 or more glycoproteins - can be involved in sperm-egg recognition

– Essential for species-specific binding of the sperm

– Called the zona pellucida in mammals

Egg Structure

• Cumulus - layer of cells around the egg - follicular cells that were nurturing the egg at the time of its release - innermost layer - corona radiata

• Cortex - layer of cytoplasm just below - stiffer - high concentration of globular actin - polymerize during fertilization into microfilaments - necessary for cell division & also extend into microvillae - may aid sperm entry

Egg Structure

• Cortical granules - membrane bound - homologous to acrosomal vesicle on sperm - 15,000 in sea urchins cortical cytoplasm

– Contain, digestive enzymes, mucopolysaccharides, adhesive glycoproteins & hyalin protein

– Digestive enzymes & mucopolysaccharides - help keep other sperm out

– Adhesive glycoproteins & hyalin protein - surround the new embryo & act as support during cleavage

• Egg Jelly - outside vitelline membrane - glycoprotein meshwork - may attract or activate sperm

Sea Urchin Egg

Stages of Egg Maturation at Sperm Entry

Hamster & Mouse Eggs

Interaction of Sperm and Egg

1. Chemoattraction of sperm to egg

2. Exocytosis of acrosomal vesicle to release contents

3. Sperm binds extracellular envelope - vitelline layer or zona pellucida

4. Sperm passes through extracellular envelope

5. Fusion of eggs and sperm plasma membranes

• Sometimes 2 & 3 are reversed - in mammals

• After the above - haploid nuclei meet & developmental reactions begin

Fusion of Sperm & Egg Membranes - Sea Urchin & Mouse

Sperm Attraction

• Chemotaxis - sperm follow a gradient of a chemical secreted by the egg

• Also control time of release - after 2nd meiotic division in certain cnidarians

• Resact - 14 amino acids - from jelly of sea urchin

– Also acts as a sperm-activating peptide - cause dramatic & immediate increase in mitochondrial respiration & sperm motility

Acrosomal Reaction in Sea Urchins

• 1. Fusion of the acrosomal vesicle with sperm plasma membrane - content release

• 2. Extension of acrosomal process

Acrosomal Reaction in Sea Urchins

Translocation of Sperm in Mammals

• Sperm are found in the oviducts of mice, hamsters, guinea pigs, cows & humans within 30 minutes

• This is too short a time to swim there - appear to be transported to oviduct by muscular activity of the uterus

Capacitation of Sperm in Mammals

• Set of physiological changes that allow the sperm to be able to fertilize the egg

• Can be induced if sperm are incubated with Ca⁺⁺, bicarbonate, serum albumin or in fluid from oviducts

• Sperm not capacitated are held up in the cumulus - do not reach the egg

Model of Mammalian Capacitation

Species-Specific Recognition

• When the sperm reaches the eggs membrane - the acrosomal recognition protein is bindin

• Bindins from even closely related sea urchin species are different - thus there must be species-specific bindin receptors on the egg

– Bindin receptors thought to be aggregated into complexes on the egg cell surface

Species-Specific Binding of Acrosomal Process to Egg

ZP3 - Sperm Binding Protein

• Found in the zona pellucida

• Relatively but not absolutely species-specific (not a major problem with internal fertilization)

• It initiates the acrosomal reaction after sperm have bound to it

• Mechanism under study - probably a set of proteins on the sperm that can recognize specific carbohydrate regions of ZP3

Induction of Acrosomal Reaction by ZP3

• After sperm has bound to the zona pellucida

• Induced by the crosslinking of ZP3 with receptors for it on the sperm membrane

• The crosslinking opens calcium channels - inc. in Ca⁺⁺ inside the sperm

• Subsequent to this is exocytosis of the acrosome

Secondary Binding to the Zona Pellucida

• During the acrosomal reaction - anterior portion of the sperm plasma membrane is shed (where ZP3-binding proteins are located

• In the mouse secondary binding involve ZP2 and the inner acrosomal membrane