mechanisms of mammalian fertilization jacob thundathil dept. of production animal health faculty of...
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Mechanisms of mammalian fertilization
Jacob Thundathil
Dept. of Production Animal Health
Faculty of Veterinary Medicine
University of Calgary, AB
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Learning objectives
• Describe the cellular and molecular mechanisms of mammalian fertilization
• Understand common causes of fertilization failure
• Future areas of research
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-8 -6 -4 -2 0 20
20
40
60
80
100
Ovulation
Fe
rtili
zati
on
rat
e (
%)
(Chang, 1951)
Insemination time (hours)
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Capacitation • Vagina
– 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– Sperm reservoir
• Oviduct– Isthmus (sperm reservoir and site of final stages of capacitation)– Ampullary isthmic junction (site of fertilization) – Review article (Theriogenology 2007; 68S:S138-S146)
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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
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Mol Hum Reprod 1997; 3:175-194
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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
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Cryopreservation procedures induce premature capacitation in spermatozoa
Collection Ejaculate Extender
Storage Insemination
Uncapacitated (%)
0
10
20
30
40
50
60
70
80
0 50 100
Fer
tilit
y (%
)
(Int. J. Androl., 22: 366-373, 1999)
Packaging
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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 ?
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Accessory sperm
Capacitated sperm binds to the zona pellucida
Cortical granules
Sperm-oocyte fusion
Acrosome reaction
Block topolyspermy
Pre
fert
iliza
tion
Gamete fusion
Fertilization
Zona penetration
+
Gamete interaction and fertilization
Zona pellucida
Polar bodies
Oolemma
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Sperm-oocyte binding
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 ZP3• Role of ZP3 in sperm zona binding has NOT been confirmed by gene knockout studies (Science 2002; 296:2183-2185)
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
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Interaction of sperm receptors with zona glycoproteins (ZP3?) activates signaling mechanisms leading to acrosome reaction
Nature Cell Biol 2002; Fertil Suppl: s57-s63
Acrosome reaction
Transient Ca 2+ Sustained Ca 2+
PM
ZP3 receptor
ZP3
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Acrosome reaction
Proteolytic and glycolytic enzymes released during acrosome reaction enable sperm to penetrate ZP
Acrosomal contents are proteolytic and glycolytic in nature
• Fusion between sperm plasma membrane and outer acrosomal membrane• Acrosomal exocytosis• Acrosomal contents modify equatorial region of spermatozoa
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( Primakoff and Myles, 2007)
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Gamete fusion and egg activation
(Nature Cell Biol 2002; Fertil Suppl: s57-s63)
AdhesionFusion
Ca 2+
Egg PM
InsP3
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Cortical granule exocytosis
DAGActivation of PKC
Cortical granule exocytosis
AdhesionFusion
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Resumption of meiosis
• 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
MII
Mitosis
Meiotic Prophase
1st meiotic division
Fertilization and 2nd meiotic
division
New born
Puberty
Female germ line
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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
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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
[Biol Reprod 2001; 65: 1359-1363Biol Reprod 2005; 72: 2-13]
Centrosome
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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: 582-590)
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Accessory sperm
Capacitated sperm binds to the zona pellucida
Cortical granules
Sperm-oocyte fusion
Acrosome reaction
Block topolyspermy
Pre
fert
iliza
tion
Gamete fusion
Fertilization
Zona penetration
+
Summary
Zona pellucida
Polar bodies
Oolemma
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(Biol Reprod 1991; 44:569-574)
DNA packaging in sperm head
Nucleosome Protamine
Chromatin fiberChromatin fiber
Chromatin fiber
Supercoiled Side by side linear array
Histone protein