developemental biology6 fertilization 2

7/31/2019 Developemental Biology6 FERTILIZATION 2

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Developmental Biology

LS 311/ 411

External Fertilization



Oogenesis: Aim to store massive amountsof cytoplasmic material

1. The egg must contain all the nutrients to support the entire process of

embryonic development2. In humans, the placenta nourishes development, thus the egg doesn’t

need to store cytoplasmic material.

In some organisms, such as frogs, oocytes grow to extremely large size.

During the diplotene stage, certain chromosomes stretch out as large loopsresembling brushes (Lampbrush chromosomes).

They have very active chromosomes that synthesize large amounts of RNA(mRNA, tRNA, rRNA).

Lampbrushchromosomes(ref. gilbert images)



Meroistic Oogenesis

• A type of oogenesis where cytoplasmic

connection remains between the cells

produced by the oogonium.

• Unlike amphibians, the oocyte of insects don’t

pass through transcriptionally active stage.

• Rather RNA synthesis is confined to other

supporting cells and transported to the

oocyte.



In Drosophila melanogaster , the oogonia are called

cytoblasts, and they undergo an unusualspecialization

They undergo multiple mitotic divisions, but fail toundergo cytokinesis (cell division). Thus, they allremain connected to the original cell as cytocytes

One of the cytocysts becomes the oocyte

The other 15 become nurse cells. They make largeamounts of RNA and nutrients but they send it all tothe oocyte. This allows the oocyte to accumulatemassive amounts of cytoplasm to supportdevelopment (15 nuclei instead of 1).



Structure of Drosophila ovary

Ref. googleimages



Circle of Life: The Stages of Animal Development

Frog as an example

Gilbert’s Book



Structure of Spermatozoon

Head

• –Acrosome

• –Nucleus

Middle Piece

• -Mitochondria

Flagellum

• –Axoneme



Structure of Sea urchin egg at fertilization.

Relative size of egg tosperm is 10,000X



Sperm Attraction

• Species specific sperm attraction has beendocumented in some invertebrates

• In Echinoderms a small peptide, Resact,

secreted from the egg jelly attracts the sperms

“Assymetric flagellar

movement”

Increase in cGMP and Ca2+

activates mitochondrial ATP

generation and dynein ATPase

stimulating flagellar movement

in sperm.



• Sea urchin (Arabica punctulata) egg jelly

release Resact (CVTGAPGCVGGRL). Resact also

acts as sperm activating peptide (SAP).

• Speract (GFDLNGGGVG) is a related SAP that

is released by eggs of another sea urchin,

Strongylocentrotus purpuratus

Is the nature of chemoattractant species specific ?



Acrosome Reaction

Acrosome reaction has two components:

1. Exocytosis of the acrosomal contents

2. Extension of acrosomal process

Acrosomal reaction is initiated by the specific sugars present in the egg jelly.



What initiates the Acrosome

Reaction?• Acrosome reaction is initiated by the complex sugars

in the egg jelly that bind to specific receptor on the

sperm surface

• In purple sea urchin, polymer of fucose sulfate bindsto it’s receptor and activate 3 membrane proteins

a. Ca2+ channel

b. Na

+

/H

+

exchangerc. PLC(phospholipase C) triggering IP3 production



Elevation of intracellular Ca2+

a. Triggers fusion of acrosomal membrane

with adjacent sperm membraneb. Activates acrosomal process formation

by activating Rho B that stimulates

polymerization of globular actin to actinfilaments



Role of the Acrosome

• Acrosomal enzymes digest a path through the

egg jelly to egg surface for the sperm

• Acrosomal reaction also involves the

extension of acrosomal process formed by the

polymerization of the globular actin.



Fast block to Polyspermy

• Sperm binding depolarizes the egg membrane,which transiently blocks binding of additional

sperm

• Resting membrane potential is -70mV, highNa+ outside and low Na+ inside the cell

• Binding of sperm results in Na+ influx resulting

in depolarization of the membrane (20m V)• Sperms can’t bind to depolarized membrane



Postulated pathway of egg activation in the sea

urchin

Ref. gilbert .



Fusion of genetic material

• Sperm nucleus decondenses.

• Sperm enters the egg and undergoes a 1800

rotation so that the centriole is between

female and male pronucleus

• Microtubule asters are formed, two pronuclei

migrate , fuse and form the diploid zygote.



Capacitation

• Cholesterol efflux from the membrane

• Particular proteins or carbohydrates that blockthe recognition sites for sperm and zonainteraction are removed

• Membrane potential of the sperm changesprobably facilitating the acrosomal reaction

• Protein phosphorylation events that mediatesperm and zona pellucida interaction

• Changes in outer acrosomal membrane thatprepares the fusion of sperm to egg.



Hypothetical model for

Capacitation

Proposed model for the involvement of tyrosine-phosphorylated molecular chaperones in the



Asquith, K. L. et al. J Cell Sci 2004;117:3645-3657

Proposed model for the involvement of tyrosine-phosphorylated molecular chaperones in theacquisition of mammalian sperm fertilizing ability



Prevention of Polyspermy

Polyspermy is not a problem when the

fertilization is internal

• In mammals the cortical granule reaction

doesn’t produce a fertilization envelope, but

releases enzymes (like N-

acetylglucosaminidase) that modify the Zona

pellucida so that they no longer bind sperm.



Fertilization: Beginning of a new

organism

Fertilization is the process whereby the sperm and theegg –collectively called the gametes-fuse together tobegin the creation of a new individual whose genome

is derived from both parents



Zona reaction in mammals• In mammals, the cortical granule reaction does not create a fertilization

envelope, but its ultimate effect is the same. Released enzymes modifythe zona pellucida sperm receptors such that they can no longer bind

sperm .

• During this process, called the zona reaction, both ZP3 and ZP2 are

modified.

• The cortical granules of mouse eggs contain an enzyme that clips off the

terminal sugar residues of ZP3, thereby releasing bound sperm from the

zona and preventing the attachment of other sperm.

• Cortical granules of mouse eggs have been found to contain N-

acetylglucosaminidase enzymes capable of cleaving N-acetylglucosamine

from ZP3 carbohydrate chains.



Summary of events leading to the fusion of egg



Summary of events leading to the fusion of egg

and sperm plasma membranesExternal Fertilization Internal Fertilization



Postulated pathway of egg activation in the sea

urchin



l



Post Fertilization Events

Throughout the animal kingdom, an incredible variety of embryonic types exist, but

most patterns of embryogenesis are variations on five themes:

• 1. Immediately following fertilization, cleavage occurs.

– Cleavage is a series of extremely rapid mitotic divisions wherein the

enormous volume of zygote cytoplasm is divided into numerous smaller

cells. These cells are called blastomeres, and by the end of cleavage,they generally form a sphere known as a blastula.

• 2. After the rate of mitotic division has slowed down, the blastomeres undergo

dramatic movements wherein they change their positions relative to one another.

This series of extensive cell rearrangements is called gastrulation, and the embryo issaid to be in the gastrula stage.

• 3. As a result of gastrulation, the embryo contains three germ layers: the ectoderm,

the endoderm, and the mesoderm.

G bi di d i i i



Gamete binding and recognition in

mammals • ZP3: the sperm-binding protein of the mouse

zona pellucida. The zona pellucida in mammals

plays a role analogous to that of the vitelline

envelope in invertebrates.

• This glycoprotein matrix, which is synthesized

and secreted by the growing oocyte, plays twomajor roles during fertilization: it binds the

sperm, and it initiates the acrosomal reaction

after the sperm is bound.

• The binding of sperm to the zona is relatively,

but not absolutely, species-specific. (Speciesspecific gamete recognition is not a major

problem when fertilization occurs internally.)

developemental biology6 fertilization 2

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