Mechanisms of mammalian fertilization

Jacob Thundathil

Dept. of Production Animal Health

Faculty of Veterinary Medicine

University of Calgary, AB

jthundat@ucalgary.ca

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

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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

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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