1. Fertilization and Cleavage
Zona pellucida, compaction, blastomers, infertility
Fertilization and Cleavage
Dr. Nandor Nagy
Semmelweis University

3. Process of fertilization
-maturation of sperms: capacitation
-The chemoattraction of sperm to the egg
-attachment to and penetration of the zona pellucida
The exocytosis of acrosomal vesicle to release enzymes
The binding of the sperm to the extracellular envelope
The passage of the sperm through the envelope
The membrane fusion between sperm and egg
-the process end with the intermingling of maternal and paternal chromosomes

4. Spermatozoa Undergo a Terminal Step of Functional Maturation
Called Capacitation
devbio8e-fig jpg
-freshly ejaculated sperm are unable to fertilize oocytes

5. Increased membran fluidity and Increased motility
capacitated sperm is metabolically more active and beats its flagellum rapidly
Thermotaxis: capacitated sperm can sense thermal gradient between isthmus of oviduct and ampulla (about 2oC).
devbio8e-fig jpg
Acrosome (Proacrosin-GFP)
Nucleus/mitochondria/mictotubule
Increased membran fluidity and Increased motility
albumin and lipid transfer protein (LTP-1; both protein present in female genital organ)  decrease the level of cholesterol more fluid spermatozoa cell membrane

6. Chemotaxis: secreted molecules from cumulus cells and oocyte.
obstacles…
First obstacle:
-granulosa cells embedded in a loose extracellular matrix
(hyaluronic acid)
Before the encounter

7. second obstacle: Zona pellucida (13 µm)

8. Events Leading to the Fusion of Egg and Sperm Plasma Membranes
Recognition of egg and sperm:
1. Chemoattraction of the sperm to the egg by soluble molecules
2. The binding of the sperm to the extracellular envelop of the egg
3. The exocytosis of the acrosomal vesicle to release its enzyme
4. The passage of the sperm through this envelop
5. Fusion of egg and sperm cell membrane
Figures\Chapter07\DevBio7e07081.jpg
Events Leading to the Fusion of Egg and Sperm Plasma Membranes

9. zona pellicida
3 protein of zona pellucida : ZP1 (200 kDa), ZP2 (120 kDa), ZP3 (83 kDa).
When a spermatozoon reaches the tough zona
pellucida surrounding the oocyte, it binds in a species-
(that is, human-) specific interaction with a glycoprotein
sperm receptor molecule in the zona (ZP3, one
of three glycoproteins composing the zona pellucida).

10. A zona pellucida fehérjéi:
ZP1 (200 kDa)
ZP2 (120 kDa)
ZP3 (83 kDa, ebből csak 44 kDa a polipeptidváz, a többi a piros „dobverőként” illusztrált oligoszaccharid oldallácok révén)
galactosyltransferase-I
-binds to ZP-3 –activate G protein—open Ca channel—acrsomal rxn-
Release proteases that lyse the ZP-make a hole in egg
Binding to ZP3 is mediated by a sperm surface protein
called SED1

11. The acrosome reaction
-The exocytosis of the acrosomal vesicle to release its enzyme
-Enzymes are released from the acrosome [esterases, collagenase, acrosin and neuraminidase]
-Enzymes cause lysis of the zona pellucida

12. As a result of this binding, the acrosome
is induced to release degradative enzymes that allow
the sperm to penetrate the zona pellucida.

13. The cell membrane of the two cells fuse.
Molecules required for this event:
-CD9
-Fertilin
-tetraspanin
-Izumo

14. Robert Edwards

16. ART – assisted reproductive technology
ZIFT - Zygote Intra-Fallopian Transfer
But: laparascopy
IUI - Intra-Uterine Insemination
IVF – In Vitro Fertilization
Nobel prize 2010 Edwards
Ovarian hyperstimulation
ICSI -
Intra-Cytoplasmatic Sperm Injection
GIFT – Gamete Intra-Fallopian Transfer
But: laparascopy

17. Number of months attempting to get pregnant
Infertility
Statistics on conception in normal fertile couples:
within a month: 20–25%.
within a year: 90%
Definition of infertility: the inability of a couple to conceive after a year of regular intercourse without contraception.
Number of months attempting to get pregnant
Figure from website
Data from HFEA (human fertilization embryology authority) 2

18. Binding and fusion of sperm cell and egg
-in normal monospermy: only one sperm enters
a haploid sperm nucleus + a haploid egg nuleus combine:
diploid nucleus of the fertilized egg (zygote)
Restoration of the chromosome number
Polyspermy leads to disastrous consequences in most organism
-two sperm entry results in triploid nucleus
-each sperm’centriole divides to form the two poles of mitotic apparatus
-triploid chromosomes
-such cells either die or develop abnormally (triploid embryos nearly
always abort)
-amorphous, nodular embryos
3 pronuclei are seen in the center of the cell Each pronucleus contains 23 chromosomes This embryo has 69 chromosomes instead of the normal 46 Zona pellucida (shell) is visible as a halo around periphery Sperm are visible at 1 and 8 o'clock - outside of the zona

19. polyspermy block
Membrane fusion immediately causes two events
to occur: formation of a calcium wave that
radiates over the surface of the egg from the point
of sperm contact; and release of the contents of
thousands of small cortical granules, located just
beneath the oocyte cell membrane, into the
perivitelline space between the oocyte and the
zona pellucida. These two events alter the sperm receptor
molecules, causing the zona to become impenetrable
by additional spermatozoa. –
- These changes prevent polyspermy or the fertilization
of the oocyte by more than one spermatozoon.

20. polyspermy block: step 1
Wave of Ca2+ release across a sea urchin egg during fertilization induces cortical granule reaction
polyspermy block: step 1
devbio8e-fig jpg

21. Cortical granule exocytosis
polyspermy block: step 2
devbio8e-fig jpg

23. Morula Early blastomeres lack well developed intercellular junctions.
Cells are in contact through microvilli and other cellular projections
Morula
microvilli

25. Compaction E-cadherin
                                                                                                                                                                        
E-cadherin
E-cadherin=uvomorulun: 54,000 D cell adhesion molecule

26. Compacting Human Morula
Biology of Reproduction
Nikas et al. 1996, Biol. Reprod. 55:32-37

27. compaction
Before and after

28. Blastocyst cavity forms within the morula (cavitation)
- Sodium/potassium ATPase pumps sodium interior of the morula and water follows through osmosis

30. Early Embryonic Development
Location Day Development
Ampullary One cell
Isthmic
Junction
All cell divisions
are mitotic Smaller
cell size with each
cleavage division
Ampullary-
Isthmic
Junction
Two cell
Isthmus Four cell
Isthmus Eight cell
Uterus Sixteen
cell
Uterus Morula

31. Day Development
Compact Morula form
tight cell contacts so that
Blastocyst formation is
possible
Tight Morula
Early Blastocyst
Fills with fluid in center -
Cells have established
polarity
Blastocyst
Blastocoel
Inner Cell Mass -
Forms Embryo
Trophoblast -
Forms Placenta