Mechanisms of mammalian fertilization Jacob Thundathil Dept. of Production Animal Health Faculty of...
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Transcript of Mechanisms of mammalian fertilization Jacob Thundathil Dept. of Production Animal Health Faculty of...
Mechanisms of mammalian fertilization
Jacob Thundathil
Dept. of Production Animal Health
Faculty of Veterinary Medicine
University of Calgary, AB
Learning objectives
• Describe the cellular and molecular mechanisms of mammalian fertilization
• Understand common causes of fertilization failure
• Future areas of research
-8 -6 -4 -2 0 20
20
40
60
80
100
Ovulation
Fe
rtili
zati
on
rat
e (
%)
(Chang, 1951)
Insemination time (hours)
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)
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
Mol Hum Reprod 1997; 3:175-194
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
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
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 ?
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
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
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
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
( Primakoff and Myles, 2007)
Gamete fusion and egg activation
(Nature Cell Biol 2002; Fertil Suppl: s57-s63)
AdhesionFusion
Ca 2+
Egg PM
InsP3
Cortical granule exocytosis
DAGActivation of PKC
Cortical granule exocytosis
AdhesionFusion
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
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
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
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)
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
(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