Developmental Biology

LS 311/ 411 External Fertilization

Oogenesis: Aim to store massive amounts of cytoplasmic material

1. The egg must contain all the nutrients to support the entire process of embryonic development
2. In humans, the placenta nourishes development, thus the egg doesnt 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 loops resembling brushes (Lampbrush chromosomes).

Lampbrush chromosomes
(ref. gilbert images)

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

Meroistic Oogenesis
A type of oogenesis where cytoplasmic connection remains between the cells produced by the oogonium. Unlike amphibians, the oocyte of insects dont 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 unusual specialization They undergo multiple mitotic divisions, but fail to undergo cytokinesis (cell division). Thus, they all remain connected to the original cell as cytocytes One of the cytocysts becomes the oocyte

The other 15 become nurse cells. They make large amounts of RNA and nutrients but they send it all to the oocyte. This allows the oocyte to accumulate massive amounts of cytoplasm to support development (15 nuclei instead of 1).

Structure of Drosophila ovary

Ref. google images

Circle of Life: The Stages of Animal Development Frog as an example

Gilberts Book

Structure of Spermatozoon
Head Acrosome Nucleus Middle Piece -Mitochondria Flagellum Axoneme

Structure of Sea urchin egg at fertilization.

Relative size of egg to sperm is 10,000X

One of the most fundamental questions of external fertilization is how do sperm locate eggs? Chemo-attractant source mediates attraction to the egg How can sperm be prevented from trying to fertilize eggs of another species? Species-specific recognition

Sperm Attraction
Species specific sperm attraction has been documented in some invertebrates In Echinoderms a small peptide, Resact, secreted from the egg jelly attracts the sperms
Increase in cGMP and Ca2+ activates mitochondrial ATP generation and dynein ATPase stimulating flagellar movement in sperm.

Assymetric flagellar movement

Is the nature of chemoattractant species specific ?

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

How is the Species specific recognition maintained?

1. Acrosome Reaction

2. Species specific proteins on the acrosomal process

Acrosome Reaction

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

Acrosome reaction has two components: 1. Exocytosis of the acrosomal contents 2. Extension of acrosomal process

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 binds to its receptor and activate 3 membrane proteins a. Ca2+ channel b. Na+/H+ exchanger c. PLC(phospholipase C) triggering IP3 production

Elevation of intracellular Ca2+ a. Triggers fusion of acrosomal membrane with adjacent sperm membrane b. Activates acrosomal process formation by activating Rho B that stimulates polymerization of globular actin to actin filaments

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.

Species-Specific Recognition
Sperm attraction is species specific Acrosomal reaction is species specific Acrosomal protein like Bindin, mediates the species specific interaction of sperm with the vitelline envelope.
Species-specific bindin isolated from the acrosomes of Strongylocentrotus purpuratus binds to its own dejellied eggs, but not to those of Arbacia

punctulata.

Summary
1. Egg secrete soluble molecules for chemoattraction of sperms. 2. The exocytosis of the acrosomal vesicle to release its enzymes 3. Binding of sperm to the external envelope of the egg 4. Passage of sperm through the extracellular envelope 5. Fusion of egg and sperm cell membranes

Polyspermy: Entrance of Multiple sperms

A. If a single egg is fertilized by multiple sperm (polyspermy), additional chromosomes and centrioles are brought inside the egg. B. This causes defects at the first cleavage division as the chromosomes are segregated randomly between multiple spindles

Prevention of Polyspermy in Sea urchins

Fast block to polyspermy mediated by potential change across the egg membrane Slow block to polyspermy is mediated by cortical granule reaction, wherein the extracellular matrix is modified such that further sperms are unable to reach the egg membrane.

Fast block to Polyspermy

Sperm binding depolarizes the egg membrane, which transiently blocks binding of additional sperm Resting membrane potential is -70mV, high Na+ outside and low Na+ inside the cell Binding of sperm results in Na+ influx resulting in depolarization of the membrane (20m V) Sperms cant bind to depolarized membrane

Slow block to Polyspermy

Mediated by the exocytosis of cortical granule contents in the space between egg membrane and vitelline envelope Serine protease dissolves the protein posts connecting the vitelline envelope proteins to egg membrane and clips off the bindin receptor and any attached sperm Fertilization envelope is formed as cortical granule contents fuse with vitelline envelope Mucopolysaccharides produce an osmotic gradient Due to the osmotic gradient water rushes in and fills the space between vitelline envelope and egg membrane. Hyalin forms a coating around the egg (hyalin layer), provides support during the cleavage.

Calcium as the initiator of cortical granule reaction

Upon fertilization the concentration of Free Calcium in the egg cytoplasm increases. In high calcium environment, the cortical granule fuses with the egg membrane to release their contents.

The calcium is released from the Endoplasmic reticulum.

Activation of Egg Metabolism

Fertilization is not only important for merging of two genetic material but also plays a role in initiating the development process. The responses of egg to the sperm 1. Early responses 2. Late responses

Early responses:
Fast block initiated by influx of Na+ Slow block initiated by intracellular release of Ca 2+ leading to cortical granule reaction Ca 2+ release activates a whole of series of metabolic reactions a. Affecting lipid biosynthesis (new membranes are formed) b. Leads to respiratory burst crosslinking of fertilization envelope (peroxidase) c. Antioxidants like glutathione and ovothiols are generated that prevent damage to DNA

Late Responses:
Activation of DNA & protein synthesis. Protein synthesis is dependent on the stored messages in the egg and mediated probably by release of inhibitors from the mRNAs. In sea urchin egg translational initiation factor eIF4E in inhibited Post fertilization, the inhibitor is degraded and mRNAs are freed for translation. Cyclin B is one the cell cycle regulator is synthesized, creates Mitosis promoting factor (MPF), required to initiated cell division.

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.

Mammalian fertilization
(Internal Fertilization)

Differences between the female eggs

Hyaluronidase potentiates penetration of mammalian sperm through Cumulus

Gamete translocation & maturation

The female reproductive tract actively regulates the transport and maturity of both gametes. The male gamete undergoes capacitation rendering it competent to fertilize the egg In some species the female reproductive tract hyperactivates the sperm. Experimental evidences suggest that the follicle cells and oocyte mediate chemotaxis of sperms

Capacitation
Cholesterol efflux from the membrane Particular proteins or carbohydrates that block the recognition sites for sperm and zona interaction are removed Membrane potential of the sperm changes probably facilitating the acrosomal reaction Protein phosphorylation events that mediate sperm and zona pellucida interaction Changes in outer acrosomal membrane that prepares the fusion of sperm to egg.

Hypothetical model for Capacitation

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

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

Recognition at Zona Pellucida

Zona pellucida analogous to vitelline envelope consists of 3 major glycoproteins (ZP1, ZP2 &ZP3) Sperms SED protein interacts with Zona protein complex Binding to ZP3 initiates the acrosomal reaction

Carbohydrate moieties of ZP3 critical for sperm attachment

GalT- sperm galactosyltransferase

Intial tethering of sperm and egg accomplished SED1 sperm cell surface adhesion protein binds to zona protein complex GalT on sperm surface recognizes Nacetylglucosamine moieties on ZP3

Gamete Fusion
Unlike the sea urchins, mammalian sperm contacts the egg at it sides and not at the tip It is believed that CD9 (in the egg) and immunoglobilin like protein Izumo (in sperm) mediate fusion of sperm to egg membrane.

Prevention of Polyspermy
Polyspermy is not a problem when the fertilization is internal In mammals the cortical granule reaction doesnt produce a fertilization envelope, but releases enzymes (like Nacetylglucosaminidase) that modify the Zona pellucida so that they no longer bind sperm.

Fusion of genetic material

DNA of the sperm decondenses Entry of sperms potentiates the oocyte nucleus to finish meiosis II The centriole accompanying the sperm produces the asters & the two pronuclei migrate towards the each other Instead of forming the common zygote, the chromatin condenses and their chromosomes gather around a common metaphase plate Thus a true diploid nucleus in mammals is seen only at two cell stage

Fertilization: Beginning of a new organism

Fertilization is the process whereby the sperm and the egg collectively called the gametes-fuse together to begin 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 modify the 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 Nacetylglucosaminidase enzymes capable of cleaving N-acetylglucosamine from ZP3 carbohydrate chains.

Major events of Fertilization

Four major events:
1. Contact and recognition between sperm and egg. In most cases, this ensures that the sperm and egg are of the same species. 2. Regulation of sperm entry into the egg. Only one sperm can ultimately fertilize the egg. This is usually accomplished by allowing only one sperm to enter the egg and inhibiting any others from entering. 3. Fusion of the genetic material of sperm and egg. 4. Activation of egg metabolism to start development.

Summary of events leading to the fusion of egg and sperm plasma membranes
External Fertilization Internal Fertilization

Postulated pathway of egg activation in the sea urchin

 A. punctulata eggs release resact (CVTGAPGCVGGGRL), Speract (GFDLNGGGVG) is a related SAP that is released by eggs of another sea urchin, Strongylocentrotus purpuratus

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 is said 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.

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 two major 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. (Species specific gamete recognition is not a major problem when fertilization occurs internally.)