10th Postgraduate Course for Training in Reproductive Medicine and Reproductive Biology

Fertilization

 F. Urner
Department of Obstetrics and Gynecology,  Geneva University Hospital

1.    Sperm penetration of cumulus cell complex

  • The ovulated oocyte is enclosed in a cumulus cell complex. Cumulus cells are somatic cells surrounding the oocyte and embedded in an extracellular matrix containing hyaluronic acid

  • Only capacitated and acrosome-intact sperm can penetrate the cumulus complex to reach the oocyte

  • Only capacitated and acrosome-intact sperm can penetrate the cumulus complex to reach the oocyte

  • Forward mobility is required for sperm to penetrate the cumulus complex and to reach the oocyte

  • Hyaluronidase activity of sperm surface molecules may participate in sperm progression through the cumulus complex

  • Hyaluronidase activity of sperm surface molecules may participate in sperm progression through the cumulus complexe

2.      Sperm interaction with the zona pellucida

    2.1.    The zona pellucida (ZP)

  • ZP is a glycoprotein coat surrounding the oocyte. It contains 3 glycoproteins : ZP1, ZP2, ZP3

  • ZP is the site for initial binding of sperm to the oocyte

  • ZP is the main barrier to interspecies fertilization   

  • ZP is the main barrier to interspecies fertilization

  • ZP is the barrier to polyspermy      

    2.2.    Binding of sperm to the zona pellucida

        2.2.1.    Primary binding

  • The primary binding is the binding of capacitated and acrosome-intact sperm to ZP3 (oligosaccharide moiety of ZP3)

  • Specific receptors for ZP3 are present in the plasma membrane of the sperm, but many different molecules have been proposed as candidate receptors.

    2.2.2.    Acrosome reaction (AR)

  • The acrosome reaction is triggered in sperm bound to ZP3

  • The acrosome reaction is triggered in sperm bound to ZP3

  • AR is essential for  sperm penetration of the zona pellucida and sperm fusion with the oocyte plasma membrane

  • AR leads to the loss of the acrosome vesicle which overlays the sperm nucleus

  • Acrosome reaction steps:

a) fusion between the plasma membrane and the outer acrosomal membrane of the sperm  

b) exposure of the inner acrosomal membrane

c) release of the acrosomal content (acrosin is an important acrosomal enzyme which is  released during AR)  

d) apparition of the equatorial segment which is involved in sperm-oocyte fusion

         2.2.3.    Secondary binding

  • The secondary binding is the binding of acrosome-reacted sperm to ZP2 following their binding to ZP3

  • This secondary binding is probably mediated by specifics receptors which are present on the inner acrosomal membrane of the acrosome-reacted sperm (different molecules have been proposed as receptors candidate receptors)

    2.3.    Penetration of the zona pellucida

  • Strong motility of sperm (hyperactivated motility) is required

  • Strong motility of sperm (hyperactivated motility) is required

  • Enzymatic digestion of the zona by acrosin facilitates penetration

3.    Sperm interaction with the oolemma

    3.1.    Sperm binding to the oolemma

  • Membrane contact occurs between the sperm equatorial segment and the microvilli of the oocyte. Microvilli are present on the whole surface of the oocyte, except the region overlaying the meiotic spindle

  • Membranes contact occurs between the sperm equatorial segment and the microvilli of the oocyte. Microvilli are present on the whole surface of the oocyte, except the region overlaying the meiotic spindle

  • Specific surface molecules have been proposed to be involved in binding of the gametes:

  • In the oocyte, integrins have been evidenced on the oolemma and have been proposed as binding sites for the fertilizing sperm. Generally speaking, integrins are transmembrane proteins which play a role in adhesion and signal transduction through the plasma membrane. Integrin isare composed of 2 subunits named a and b

  • In sperm, fertilin may be the surface molecule which binds to the oocyte integrin. Fertilin   is composed of 2 subunits: the :b subunit  is responsible for binding to integrins   and the a subunit iswould be responsible for fusion with the oolemma.

     3.2.       Sperm fusion with the oolemma

  • Only acrosome-reacted sperm are able to fuse with the oolemma

  • Only acrosome-reacted sperm are able to fuse with the oolemma

  • Sperm mobility is probably not required for fusion 

  • Following fusion of the membranes of the fertilizing sperm and the oocyte, the whole sperm is incorporated in the ooplasm:

    •        the nucleus which contains the paternal genome           

    •        the cytoplasm which contains important factors involved in oocyte activation    

    •        the mitochondria role ? ?

    •         the centriole which is involved in migration of the pronuclei and organization of the  ---mitotic spindle of the 1rst division

    •        the tail

4.    Sperm chromatin decondensation in the oocyte

Following sperm penetration into the oocyte, sperm chromatin remodelling is rapidly initiated.  Although this event takes place during oocyte activation , the initial sperm decondensationit does not depend on oocyte activation while, except for the pronucleus formation depends on activation.

    4.1.    Different steps of sSperm chromatin remodelling in ovo:

  • Removal of sperm nuclear envelope

  • Reduction of disulfide bonds of protamines by glutathione 

  • Protamines are nucleoproteins which have been associated with sperm DNA during spermiogenesis and which are required for highly compacted DNA. 

  • Glutathione is a oocyte factor which has accumulated during the meiotic maturation of the oocyte

  • Initiation of sperm chromatin decondensation

  • Removal of sperm nuclear envelope

  • Reduction of disulfide bonds of protamines by glutathioneProtamines are nucleoproteins which have been associated with sperm DNA during spermiogenesis and which are required for highly compacted DNA. 

  • Glutathione is a oocyte factor which has been accumulated during the meiotic maturation of the oocyte

  • Initiation of sperm chromatin decondensation 

  • Removal of protamines and replacement with  maternal histones

  • Chromatin recondensation

  • Chromatin decondensation male pronucleus formation

  • Chromatin recondensation

  • Chromatin decondensation and male pronucleus formation

5.    Oocyte activation

  • Oocyte activation is a sequence of events triggered by the fertilizing sperm and that are required to initiate embryonic development 

  • Early events of oocyte activation include : intracellular calcium increase and cortical granules exocytosis 

  • Late events of oocyte activation include :  resumption of meiosis, pronucleus formation, DNA synthesis and cleavage

Two different hypothesis have been proposed concerning the signalling system leading to activation:

  1. « Receptor hypothesis » : the sperm bound to the oolemma receptor activates a signal transduction pathway ; activation is triggered prior to gamete fusion

  2. « Fusion hypothesis » : activation is initiated  by a soluble sperm factor introduced into the ooplasm following gamete fusion

    5.1.    Increase in intracellular calcium

  • Calcium comes from intracellular stores and is release by the intracellular messenger IP3

  • Following the first increase in calcium , oscillatory calcium transients are observed for several  hours

  • These calcium transients regulate different events associated with activation      

   5.2.    Exocytosis of cortical granules

  • In mammals, exocytosis of cortical granules is the main mechanism preventing polyspermia

  • A few minutes after gamete fusion, the cortical granules, which are exocytotic vesicles present in the oocyte cortex, fuse with the oocyte plasma membrane and release their content into the perivitelline space

  • A modification of the zona pellucida is induced by the content of the cortical granules so as no additional sperm can bind to and penetrate the ZP

  • Few minutes after gamete fusion, the cortical granules, which are exocytotic vesicles present in the oocyte cortex, fuse with the oocyte plasma membrane and release their content into the perivitelline space

  • A modification of the zona pellucida is induced by the content of the cortical granules so as no additional sperm can bind to and penetrate the ZP

5.3.    Resumption of meiosis

  • During sperm chromatin decondensation, meiosis which is arrrested at the metaphase II stage is reinitiated to the telophase II stage

  • The metaphase II oocyte is characterized by the presence of the first polar body in the perivitelline space. At the end of meiosis, a second polar body is extruded in the perivitelline space and the oocyte becomes is haploid

  • and is characterized by the presence of 2 polar bodies in the perivitelline space. The fist polar body was extruded when the oocyte reached  metaphase II and contains 2n chromosomes while  the second polar body was extruded at telophase II and contains 1n chromosomes 

5.4.    Pronuclei 

The sperm chromatin (following its decondensation/recondensation in ovo) and the oocyte chromatin at the telophase II stage will transform into a paternal and maternal  pronuclerus respectively. Although asynchrony may be observed in the apparition of the 2 pronuclei, their formation and development are usually concomitant.

    Formation and development of the pronuclei:

  • Assembly of the pronuclear envelope (~4h post-fusion)

  • Enlargement of the pronuclei

  • Migration of the pronuclei 

  • DNA replication in both  pronuclei (~12h post-fusion)

  • Apposition of the 2 pronuclei (~20h post-fusion)

  • Disparition of the pronuclear membranes

  • Condensation of the paternal and maternal chromosomes on a metaphase plate

  • Assembly of the pronuclear envelope (4h post-fusion)

  • Enlargement of the pronuclei

  • Migration of the pronuclei 

  • DNA replication in both  pronuclei (12h post-fusion)

  • Apposition of the 2 pronuclei (20h post-fusion)

  • Disparition of the pronuclear membranes

  • Condensation of the paternal and maternal chromosomes on a metaphase plate

SUMMARY

  • Penetration of the cumulus cell complexe

  • Binding to the zona

  • Acrosome reaction

  • Zona penetration

  • Binding to the oolemma (Oocyte activation ?)

  • Fusion with the oolemma (Oocyte activation ?)

  • Sperm decondensation

  • Resumption of meiosis

  • Pronuclei formation

  • Association of paternal and maternal chromosomes

  • First cleavage

References

  1. Wassarman P. Mammalian fertilization : molecular aspects of gamete adhesion, exocytosis, and fusion. Cell 96 : 175-183, 1999

  2. Schultz RM and Kopf GS. Molecular basis of mammalian egg activation. Curr Topics Dev Biol 30 :21-62. 1995

  3. Wright SJ. Sperm nuclear activation  during fertilization. Curr Topics Dev Biol 46 : 133-178, 1999

 
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Edited by Aldo Campana,