CMMB 403

DEVELOPMENTAL BIOLOGY

FALL 2001

CURRENT LECTURE NOTES

Fertilization

Two major outcomes:

Events:

    1. Sperm-egg recognition and contact
    2. Prevention of polyspermy
    3. Fusion of pro-nuclei
    4. Activation of development

Sperm-egg recognition and contact:

Different animals – different mechanisms

Big factor: is fertilization internal or external?

Eg. Sea urchin (various species) – external

Eg. Mammals - internal

A. Sea Urchin:

External fertilization

Challenges:

  • get sperm to egg
  • prevent cross-species fertilization (why?)

Strategies:

Species-specific sperm attraction

Species-specific acrosome reaction:

Figure 4.9 (Gilbert 1997)

Figures 7.8 and 7.10 (Gilbert 2000)

Species-specific binding of sperm to egg vitelline envelope

Bindin may also mediate fusion of sperm and egg plasma membranes

Figure 7.17 (Gilbert 2000)

B. Mammals

Internal fertilization

Egg envelope – zona pellucida

- a gel composed of glycoproteins:

ZP1, ZP2, ZP3

Sperm-egg attraction:

  • uterine contractions help sperm get to oviduct
  • substance(s) in female reproductive tract cause capacitation: molecular changes in sperm which are required for fertiliztion
  • egg secretions attract sperm: chemotaxis

Binding of sperm to zona pellucida:

  • relatively species-specific binding of sperm to zona receptor ZP3

How do we know ZP3 is the receptor?

(Bleil and Wassarman, 1980, 1986, 1988)

mouse

Sperm bind intact zona in vitro

Preincubate sperm with zona proteins

Then add to intact zona no binding

Fractionate zona proteins

And assay for that fraction which inhibits binding of sperm to intact zona

That fraction contained ZP3

Figure 4.15 (Gilbert, 1997)

Figure 7.17 (Gilbert, 2000)

After initial binding to ZP3, sperm sheds outer membrane, and attaches to ZP2 via inner membrane.

Sperm receptor?

There may be as many as three

  1. Lectin protein p56
  • isolated by binding to ZP3

(Bookbinder et al, 1995)

2. Galactosyltransferase

  • aggregates form on sperm surface upon binding to zona
  • activates a G protein which may be involved in acrosome reaction

3. Zona receptor kinase

  • tyrosine kinase
  • may be involved in acrosome reaction

Acrosome reaction:

  • occurs only after sperm binds ZP3

ZP3 cross-links receptors in sperm membrane

? Signal transduction

Ca2+ channels open

Ca2+ influx

Exocytosis

 

Sperm-Egg Membrane Fusion:

Figure 4.20 (Gilbert, 1997)

Figure 7.20 (Gilbert, 2000)

(a domain that binds integrins – membrane proteins that are involved in cell adhesion)

Maybe the receptor for sperm in egg membrane is an integrin? (Almeida et al, 1995)

Screened mouse egg surface for integrins using antibodies to known integrins

- found that a 6b 1 and a vb 3 integrins were present on egg surface

Tested for function:

Does fertilin disintegrin domain bind to egg?

Does fertilin disintegrin domain bind to a 6b 1 integrin?

 

Fertilization: Activation of Development

Early Responses:

    1. Sperm-Egg binding
    2. Increase in egg membrane potential (fast block to polyspermy) Sea Urchin and Frogs
    3. Sperm-Egg membrane fusion
    4. Increase in intracellular Ca2+
    5. Exocytosis of cortical vesicles (slow block to polyspermy)
    6. Inactivation of CSF mediated by calmodulin-dependent kinase II and calpain Mammals and Amphibians
    7. Completion of meiosis Mammals and Amphibians
    8. Late Responses:

    9. Efflux of H+ ions Sea Urchin
    10. Increase in intracellular pH Sea Urchin
    11. Protein synthesis activated Sea Urchin
    12. Sperm chromatin decondensation
    13. Pronuclei meet in center of egg
    14. DNA synthesis initiated
    15. First mitosis

Ca2+ Calpain (protease)

                                     |

Calmodulin                |

                                     |

Calmodulin-dependent    |

Kinase II         |

                  

Inactivation of Cytostatic Factor

 

Prevention of Polyspermy

Why?

Fig. 4.21 in Gilbert, 1997

Fig. 7.21 (Gilbert, 2000)

Fast block to polyspermy:

Occurs in sea urchins and frogs, not in mammals

Resting membrane potential of about –70mV

When sperm binds to egg membrane

 

If increase in intracellular Na+ is not sufficient,

Eg. due to low Na+ concentration in environment,

Then polyspermy is not prevented

Membrane potential returns to normal very quickly (1 minute), so a more permanent block is needed.

 

Slow block to polyspermy

Sea urchin

Cortical reaction

Figure 4.24 (Gilbert, 1997)

Figure 7.24 (Gilbert, 2000)

Mammals

Zona reaction

Signal transduction pathway

How do we know Ca2+ is responsible for the cortical reaction?

Add A23187 (Ca2+ ionophore) to unfertilized eggs

+/-  Ca2+ in the medium

Cortical reaction

Ca2+ is released into cytoplasm from internal stores (endoplasmic reticulum)

Ca2+ release occurs in a wave that starts at the point of sperm entry

If a Ca2+ chelator is injected into the egg

eg. EGTA

sperm binding

No egg activation!

What causes Ca2+ release from ER?

What causes increase in IP3?

PIP2 (phosphatidylinositol 4,5-bisphosphate)

membrane phospholipid

phospholipase C enzyme

IP3 and DAG (diacyl glycerol) 2nd messengers

Figure 3.35 (Gilbert, 1997)

Figure 4.27 (Gilbert, 1997)

Figs. 7.27 and 7.28 (Gilbert, 2000)

 

Recent evidence for the TK pathway:

In sea urchin

H+/Na+ exchange increases pH

burst of protein synthesis from stored maternal mRNA’s

Fig. 7.30 (Gilbert, 2000)

 

Meeting of the pronuclei

Sperm membrane fuses with egg membrane

Sperm flagellum and mitochondria are degraded

Sperm pronucleus and centrosome enter cytoplasm

Sperm nuclear envelope breaks down

Sperm-specific DNA-binding proteins are exchanged for somatic histones

Sperm DNA decondenses

Sperm centrosome forms aster

DNA synthesis

Mitosis

In sea urchin - the pronuclei meet to form the zygote nucleus

In mammals:

Rearrangement of egg cytoplasm

Occurs in frog eggs and some other species

Can be easily seen in frog Rana pipiens

Sperm enters somewhere in animal hemisphere

Cortical cytoplasm, which is darkly pigmented, shifts about 30 toward sperm entry point, relative to deep cytoplasm.

How does this happen?

Newly exposed deep cytoplasm, which appears grey, is called the grey crescent, an area of great importance in amphibian development

Unfertilized frog egg – radially symmetrical

Fertilized frog egg – bilaterally symmetrical

Figure 4.33 (Gilbert, 1997)

Figure 7.33 (Gilbert, 2000)

 

Cleavage

First stage in development: egg blastula

Increase in ratio of nuclear/cytoplasmic volume

Increase in length of cell cycle

Transcription from embryonic genome commences

How could the change in nuclear versus cytoplasmic volume exert such an effect?

 

Mechanics of cell division

Timing:

Orientation:

Are karyokinesis and cytokinesis always linked?

Figure 5.43 (Gilbert, 1997)

Figure 8.3 (Gilbert, 2000)

Patterns of Cleavage

Will determine:

Determined by:

An aside on yolk and energy sources during development:

Development of the embryo requires large amounts of energy and raw material. Where does it come from?

Birds

Reptiles

Fish

External Gestation No Larval Stage Large amount of yolk
Sea Urchins

Frogs

External Gestation Self-feeding Larval Stage Small to moderate amount of yolk
Mammals Internal Gestation No Larval Stage Small amount of yolk

Sea Urchin

Yolk – Isolecithal

Cleavage patterm – holoblastic (complete)

Cleavage symmetry – radial

Figure 5.3 (Gilbert, 1997)

Figure 8.8 (Gilbert, 2000)

Micromeres:

After micromere formation, the rate of division of the micromeres decreases, so that by the 128-cell stage, all cells are fairly equal in size.

What might be the point of micromere formation early in cleavage?

 

Blastula stage:

Figure 5.5 (Gilbert, 1997)

Figure 8.16 a-c (Gilbert, 2000)

 

 

 

 

 

Amphibians

Yolk – Mesolecithal (moderately telolecithal)

Cleavage pattern – Holoblastic

Cleavage symmetry – Radial

Figure 5.7 (Gilbert, 1997)

Figure 10.1 (Gilbert, 2000)

Blastula:

eg. EP cadherin

  • oocyte messenger RNA
  • antisense to mRNA reduces adhesion between blastomeres

 

 

 

Snails

Yolk – Isolecithal

Cleavage pattern – Holoblastic

Cleavage symmetry – Spiral

Figure 5.14 (Gilbert, 1997))

Figure 8.28 (Gilbert, 2000)

 

 

Mammals

Yolk – Isolecithal

Cleavage pattern – holoblastic

Cleavage symmetry – rotational

Fertilization occurs in oviduct

Cleavage occurs within 12-24 hours

Figure 5.19 (Gilbert, 1997)

Figure 11.21 (Gilbert, 2000)

 

Morula:

Figs. 5.19 and 5.21 (Gilbert, 1997)

Figs. 11.20 and 11.23 (Gilbert, 2000)

 

 

 

Cell fate dictated by positional cues:

Blastocyst:

Cavitation:

Implantation:

Figures 11.26 and 11.27 (Gilbert, 2000)

 

 

 

Birds

Yolk – Telolecithal

Cleavage pattern – Meroblastic

Cleavage symmetry – Discoidal

Blastodisc:

Blastoderm:

Figure 5.30 (Gilbert, 1997)

Figure 11.9 (Gilbert, 2000)

 

 

 

Insects

Yolk – Centrolecithal

Cleavage pattern – Meroblastic

Cleavage symmetry – Superficial

Figure 5.35 (Gilbert, 1997)

Figure 9.1 (Gilbert, 2000)

 

 

 

 

 

Summary of major cleavage patterns:

Figure 8.5 (Gilbert, 2000)

 

Tracy O'Connor 2001