1. Mechanisms of mammalian fertilization
Jacob Thundathil
Dept. of Production Animal Health
Faculty of Veterinary Medicine
University of Calgary, AB

3. Learning objectives
Describe the cellular and molecular mechanisms of mammalian fertilization
Understand common causes of fertilization failure
Future areas of research

4. Insemination time (hours)
100
Ovulation 80 60
Fertilization rate (%) 40 20
Insemination time (hours)
(Chang, 1951)

6. Capacitation Vagina Cervix Uterus Uterotubal junction Oviduct
Removal of seminal plasma cholesterol, Prostasome fusion and binding of FPP
Cervix
Sperm reservoir
Removal of adsorbed molecules and sterols
ROS generation
Removal of alpha-tocopherol
Uterus
Removal of cholesterol from sperm membrane
SABP mediated calcium influx ?
Uterotubal junction
Oviduct
Isthmus (sperm reservoir and site of final stages of capacitation)
Ampullary isthmic junction (site of fertilization)
Review article (Theriogenology 2007; 68S:S138-S146)

7. Biochemical mechanisms of capacitation
Removal of decapacitating
factors (rubbing off)
Cholesterol efflux (role of BSA)
Redistribution of phospholipids
and lipid protein interaction
Increased glycolytic activity and
oxygen consumption
Increased intracellular Ca, Na
and pH levels
Activation of PKA
and protein phosphorylations

8. Mol Hum Reprod 1997; 3:

9. Characteristics of capacitated spermatozoa
Hyperactivated motility
Tyrosine phosphorylation in a cohort of sperm proteins
Ability to undergo acrosome reaction
Capacitation
- +
Capacitation- associated tyrosine phosphorylation
in sperm proteins

10. Cryopreservation procedures induce premature capacitation in spermatozoa
Collection
Packaging
Ejaculate
Extender
Uncapacitated (%) 10 20 30 40 50 60 70 80
100
Fertility (%)
Storage
Insemination
(Int. J. Androl., 22: , 1999)

11. Sperm entry into the cumulus mass
Capacitated sperm with intact acrosome can penetrate cumulus mass
Sperm surface-adsorbed enzymes such as hyaluronidase, acrosin, beta galactosidase and arylsulfatase aids sperm penetration through the cumulus mass
Is cumulus mass necessary for fertilization ?

12. Gamete interaction and fertilization
Acrosome
reaction
Block to
polyspermy
Prefertilization
Gamete fusion
Fertilization
Zona
penetration +
Capacitated sperm binds to the zona pellucida
Zona pellucida
Oolemma
Cortical
granules
Accessory
sperm
Polar bodies
Sperm-oocyte fusion

13. Sperm-oocyte binding Acrosome Equatorial region Post acrosomal Sperm
ZP1
ZP3
ZP2
Zona pellucida consists of ZP1, ZP2
and ZP3
Preincubation of sperm with ZP3
inhibited sperm egg binding
Sperm binding can be blocked
by O-linked oligosacchrides of
Role of ZP3 in sperm zona binding
has NOT been confirmed by gene
knockout studies (Science 2002;
296: )
Acrosome
Equatorial region
Post acrosomal
Sperm
oocyte
~15 candidate proteins have
been identified as receptors for ZP
Galactosyl transferase
p47
sp56
zonadhesin
Fertilin β
cyritestin
Sperm-oocyte binding

14. Interaction of sperm receptors with zona glycoproteins (ZP3
Interaction of sperm receptors with zona glycoproteins (ZP3?) activates signaling mechanisms leading to acrosome reaction
Acrosome reaction
Transient Ca 2+
Sustained Ca 2+ PM
ZP3 receptor
ZP3
Nature Cell Biol 2002; Fertil Suppl: s57-s63

15. Acrosome reaction membrane and outer acrosomal membrane
Fusion between sperm plasma
membrane and outer acrosomal
membrane
Acrosomal exocytosis
Acrosomal contents modify
equatorial region of spermatozoa
Acrosomal contents are proteolytic and glycolytic in nature
Proteolytic and glycolytic enzymes released during acrosome reaction enable sperm to penetrate ZP

16. ( Primakoff and Myles, 2007)

17. Gamete fusion and egg activation
Egg PM
Fusion
Adhesion
Ca 2+
InsP3
(Nature Cell Biol 2002; Fertil Suppl: s57-s63)

18. Cortical granule exocytosis
DAG
Activation of PKC
Cortical granule exocytosis
Cortical granule exocytosis
Fusion
Adhesion

19. Resumption of meiosis
Female germ line
Ovulated egg is at the MII of the meiotic division
Interaction of sperm with oolemma leads to Increase in Ca 2+
Inhibition of MPF
Completion of meiosis and expulsion of 2nd polar body
Mitosis
Meiotic
Prophase
New born
1st meiotic
division
Puberty
Fertilization
and 2nd meiotic
division
MII

21. Sperm DNA decondensation and male pronucleus formation
DNA – protamine complex
DNA- histone complex
Glutathione reduces inter and intramolecular disulfide bonds
present in protamine
Histones associate with sperm DNA
Glutathione and histone proteins are present in the oocyte
Zona Intact oocyte Zona free oocyte

22. Fate of sperm components within the egg during fertilization
Sperm centrosome
Paternal inheritance in most animals
Sperm aster (microtubule) formation and aggregation of pronuclei
mitotic spindle
Defective centrosome function as a cause of infertility
Centrosome
[Biol Reprod 2001; 65:
Biol Reprod 2005; 72: 2-13]

23. Fate of sperm components within the egg during fertilization
Mitochondria
Maternal inheritance
paternal mitochondria are degraded in the oocyte by ubiquitination
Ubiquitin is a 8.5 kDa proteolytic polypeptide
Ubiquitin tagged mitochondria undergo lysosome-mediated degradation
Green: mitochondria; Red: Ubiquitin
(Biol Reprod 2000; 63: )

24. Summary + Zona pellucida Oolemma Fertilization Prefertilization
Acrosome
reaction
Block to
polyspermy
Prefertilization
Gamete fusion
Fertilization
Zona
penetration +
Capacitated sperm binds to the zona pellucida
Zona pellucida
Oolemma
Cortical
granules
Accessory
sperm
Polar bodies
Sperm-oocyte fusion

26. Side by side linear array
DNA packaging in sperm head
Nucleosome
Protamine
Histone
protein
Chromatin fiber
Chromatin fiber
Chromatin fiber
Supercoiled
Side by side linear array
(Biol Reprod 1991; 44: )