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“Porcine sperm receptors in the acrosomal ridge mature in the
epididymis and bind to the zona pellucida of the egg during fertilization.”
Student project:
Jamie
Thomas
Email address:
jlynn1223@hotmail.com
Statement of project:
This is a summary of current research in the field of development, specifically
fertilization based on the research article:
“Zona Pellucida Protein Binding Ability of Porcine Sperm during Epididymal Maturation and the Acrosomal Reaction.” Burkin, H.; Miller, D. (2000) Developmental Biology 222: 99-109
My
project is for the course BIOL 480, Development Biology, Dr. Grant Mastick,
Biology Department, University of Nevada, Reno. Link to course.
Introduction:
Fertilization is the first step in an organism’s life
cycle, and without sperm and oocyte interaction fertilization does not
occur. Several steps are involved in
the process of fertilization such as:
(1) sperm penetration through the layers surrounding
the egg (cumulus and zona pellucida),
(2) sperm adhesion to the egg and
(3) egg and sperm fusion.
This study focused on information concerning the
sperm’s attachment to the zona pellucida, a thick layer surrounding the egg
consisting of three types of glycoproteins, and where the attachment
actually occurs on the sperm body, which leads to acrosomal activation
and penetration through the zona pellucida. The acrosome, a large
secretory granule with enzymatic contents, is activated within the sperm when
the sperm attaches to the zona pellucida, and then that activation leads to
digestion through the zona pellucida layer. Also, it is still debatable which regions of the sperm are
actually involved in attaching to zona pellucida proteins and which type of
proteins they bind.
This
study set out to determine where the receptors were located within the sperm
body in hope of settling the roles for zona pellucida proteins in future
studies. In addition, determining the location and timing of the first
appearance of sperm receptors specific for zona pellucida proteins was also a
goal.
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Layers around the egg cell include the cumulus cells
(red) and the compact zona pellucida layer (blue). The
sperm must make it way through both layers in order to reach the egg (yellow) for full gamete fusion. This study focused on sperm binding to the
zona pellucida and how that binding initiates acrosomal reaction, which
propels the sperm through the entire zona pellucida layer ( the gray represents the perivitelline space, which
is the space between the egg membrane and zona pellucida).
(Illustration modified after Primakoff, P.; Myles,
D. “ Penetration, Adhesion, and
Fusion in mammalian sperm-egg interaction.”
Reproductive Biology
296:2183-2185 2002) |
Experimental System:
This study focused on the sperm and eggs of pigs.
Advantages:
• Information gathered could be compared to additional
information from past studies on mammalian sperm.
• Many past studies focus on fertilization in mice, so
the pig seemed to be a beneficial choice because it also is a mammal
• There is easy access to gametes in pigs.
• Part of this study used Alexa 488 fluorophore
conjugated to zona pellucida proteins for fluorescence because Alexa bound zona
proteins showed the same activity as zona proteins unbound. This was beneficial
because the Alexa did not alter the
sperm and zona protein iteraction.
Disadvantages:
• There are some differences between porcine and mouse
fertilization such as the disparity in sperm head shape, which affects where
the sperm binds
• Because some aspects of sperm and zona pellucida
molecular interactions can be species specific, it is uncertain how closely
information on pig and mouse interactions relate to interactions within humans.
Experiment:
There were several different steps in the
experiment.
(1)
oocytes were freed from
the ovaries of the pigs, and the eggs were centrifuged and washed to isolate
the zona pellucida contents from the rest of the egg contents; the proteins
were also solubilized.
(2)
Some zona proteins were
biotinylated for fluorescence and run on SDS page gels. Others were conjugated with Alexa 488
fluoropore for fluorescence.
(3)
Semen samples collected
from boars were washed and pelleted, and specifically, sperm was collected from
the epididymis and preserved.
(4)
Fixed sperm (dead sperm
conjugated with Ionophore) that were allowed to dry on cover slips were
incubated for 2 hours in a moist chamber with biotinylated zona pellucida
proteins, so that the interaction between the egg and sperm were labeled and
obtained by color video. Ovalbumin was
used as a control for the zona pelluicda proteins.
(5)
Live sperm was incubated for 30 minutes with
Alexa 488 fluorophore zona pellucida proteins for comparison, and interactions
were labeled and obtained by color video.
(6)
To determine the acrosomal status of the
sperm as it goes through maturation, sperm form different developmental stages
in the testes were incubated for one hour with Alexa 488 fluorphore zona
proteins and the activation of the sperm’s acrosome was observed.
Results:
• The acrosomal region and the base of the sperm head
in fixed (dead) boar sperm bound zona pellucida proteins
• 90% of the live sperm only bound the zona
proteins on acrosomal ridge and 10% bound the zona proteins at the ridge and at
the base of the head
• In live sperm once the initial binding to the zona
pellucida occurred, the acrosome was activated to further increase binding to
the zona pellucida and in broader regions on the sperm.
• Sperm acquires receptors for the zona pellucida
during epididymal maturation.
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Areas in which fluorescent porcine zona pellucida proteins bound fixed boar sperm. (blue) bound
acrosomal ridge (green) bound base of sperm head (Illustration
modified after Burkin, H.; Miller, D. “Zona Pellucida Protein Binding
Ability of Porcine Sperm during Epididymal Maturation and the Acrosomal
Reaction.” Developmental Biology 222: 99-109. 2000) |
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Areas in which fluorescent porcine zona pellucida proteins bound live boar sperm. (red) bound acrosomal ridge (Illustration
modified after Burkin, H.; Miller, D. “Zona Pellucida Protein Binding
Ability of Porcine Sperm during Epididymal Maturation and the Acrosomal
Reaction.” Developmental Biology 222: 99-109. 2000. ) |
Conclusions:
Primarily,
importance was given to the fact that in live sperm primary and secondary sperm
binding to the zona pellucida incorporated different areas of the sperm
acrosome. Primary binding involved the
acrosomal ridge binding to zona proteins, and this lead to acrosomal activation
and a further and broader binding of the acrosomal region (secondary
binding). Fixed sperm bound zona
proteins in a broader region (the base and ridge) because the ionophore
conjugate that was used in the fixed sperm sample made the membrane more
soluble and more apt to bind proteins.
Lastly, this study offered information about the location and time that
zona protein receptors appeared within the sperm, and sperm that had not
reached epididymal maturation, specifically in the caudal and corpus regions,
were unable to bind zona pellucida protein and become activated.
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Simplistic example of primary and secondary
binding: Initially the sperm only binds one zona pellucida protein, which is
primary binding. This interaction
activates the acrosome of the sperm, which promotes further and broader
binding to the zona pellucida. Green = sperm Blue= receptor for zona proteins Red= zona proteins |
Significance:
Understanding
primary and secondary binding is critical because it can account for the
discrepancy between studies pertaining to what region of the sperm actually
binds to the zona proteins. Past
studies have conflicted on this issue because it may appear that in primary
binding the proteins are interacting with a larger region of the acrosome, but
really it is secondary binding. Also,
in contrast to the epididymal maturation of zona receptors in pig sperm, mice
sperm acquire protein receptors in an earlier stage and are able to bind
proteins before the pig sperm. This
suggests that the process of sperm maturation differs between species of
mammals.
These studies are critical to modern science because
they provide a better understanding of sperm-egg interaction, which allows for
advancements in the studies of contraceptives effects and infertility. Understanding the interactions between the
sperm and the zona pellucida layer are critical to improving our current
knowledge of the problems that occur with fertilization because that specific
interaction is an extremely crucial step.
Future directions:
Because more information is being gathered about how
the sperm binds to the zona pellucida layer its seems that the next step would
to be identify more specifically the type of zona pellucida proteins that are
attaching to the sperm regions.
Identifying these proteins would benefit fertility research. In addition, detecting methods of promoting
epididymal maturation of sperm that may lack zona protein receptors would
benefit males who are unable to have children.
Lastly, it seems that more specific research on human sperm would profit
current fertility and contraceptive research, but, of course, finding available
human eggs could be a difficult task.
References:
Primary research article:
(1) Burkin, H.; Miller, D. “Zona Pellucida
Protein Binding Ability of Porcine Sperm during Epididymal Maturation and the
Acrosomal Reaction.” Developmental
Biology 222: 99-109. 2000.
(2) Kenji, M. et. al. “Requirement of the CD9 on the egg plasma membrane for Fertilization.” Science 287: 321-24 (2000)
http://www.sciencemag.org/cgi/content/full/287/5451/321
Annotation: CD 9 is an integral protein (within the
membrane) and it is needed for sperm and egg fusion. CD9 knock out mice were unable to fertilize and became infertile.
Review articles:
(1) Barros, C.; Crosby, J.; Moreno, R. “Early steps of sperm-egg interaction during mammalian fertilization.” Cell Biology International 20:33-39 (1996)
Annotation: The mammalian egg has two
protective layers surrounding it: the cumulus and the zona pellucida. The zona peluucida can activate the acrosome
f sperm because it is a natural substrate for acrosin, which is the active
enzyme within the acrosome.
Fertilization can be inhibited by anti-acrosin monoclonal antibodies. There is conflicting research that shows
acrosin is not essential for the process of fertilization.
(2)
Primakoff, P.; Myles, D. “
Penetration, Adhesion, and Fusion in mammalian sperm-egg interaction.” Reproductive Biology 296:2183-2185 (2002)
http://www.sciencemag.org/cgi/content/abstract/296/5576/2183
Annotation: The sperm must make its way through two layers surrounding the egg
and it uses motility and enzymatic reactions to accomplish this. A temporary fusion of the sperm to the zona
pellucida activates the acrosome, which leads to gamete signaling and gamete
fusion.
(3)
Wassarman, P. “ Sperm receptors and
fertilization in mammals.” Mount Sinai
Journal of Medicine 69:148-152 (2002)
http://www.mssm.edu/msjournal/69/693148.shtml
Annotation: The mechanisms of fertilization are species specific
and the process includes activation of the sperm’s acrosome, penetration trough
the egg’s layers, binding of the sperm to the egg’s surface and then fusion of
the two gametes. In mammals a specific
zona protein ZP3, ad it is thought that this protein is what the sperm
binds. The evolution of these proteins
needs further research.
This
page was constructed by:
Jamie
Thomas
4/9/03