Capacitation

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Chapter 8

• Capacitation

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Capacitation

• Physiological change spermatozoa must undergo to
  be capable of fertilizing the ovum
• -occurs most frequently in the oviduct
• See diagram
• -Plasma membrane surrounds the entire cell
• -Acrosomal membrane is what gets changed with
  capacitation

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Capacitation

• A. In most species, spermatozoa are not capable
  of fertilization immediately upon entering the
  female reproductive tract
• -Sperm must undergo capacitation before they are
  fertilized
• Definition Morphologic, physiologic, and
  biochemical changes which occur to the sperm
  result in sperm capable of penetrating through
  the cumulus, corona radiata and zona pellucida of
  the ovum. (Removal of macro-molecular material
  from the sperm surface)

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• 1. The Ovum
• 2.The endometrium (uterus) and its fluids
  (endometrial secretions) play a major role.
• a. Estrogens (pro capacitation) stimulate
  capacitation (increase secretions)
• b. Progesterone is inhibitory
• c. Physiologically, mating occurs relatively
  early in estrus, ovulation occurs toward the end
  or even after estrus
• -sperm usually reach the site of fertilization
  several hours before the ova

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• 3. Spermatozoa
• a. Movement from the vagina (poor sperm
  survival) to the uterus (improved survival) is
  rapid
• b. Sperm have the opportunity for capacitation
  in the most favorable environment

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• 4. Changes in sperm occur near the acrosome.
  Increasing permeability of the sperm
  membrane-increase in the number of channels
  crossing the membrane
• a. These changes permit the sequential time
  release of a series of hydrolytic enzymes
  (permits passage through the egg layers).
• b. Hyaluronidase ( corona penetrating enzyme)
  and trypsin-like enzyme (acrosin) are associated
  with the sperm acrosome

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Acrosome contains 2 enzymes

• 1. Hyaluronidase (Corona Penetrating
  Enzyme)
• -Protein that breaks down mucopolysaccharides
• -Mucopolysaccharides (hyaluronic acid) is the
  material that holds the cumulus oopherus cells
  together
• 2. Acrosin
• -This enzyme plus sperm flagellum aid in
  penetrating to the perivitelline space

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Acrosomal reaction or capacitation

• -Removal of plasma membrane
• -Exposure of acrosome
• -Breakdown of acrosomal membrane
• -Release of enzymes
• -Exposure of nuclear membrane covering of head of
  sperm
• Theory Male contains a decapacitating factor
  which prevents capacitation within the male
  tract. Females tract contains a factor that is
  able to block or inhibit the decapacitating
  factor which then allows capacitation to occur

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• C. Decapacitation factor- macro-molecular
  material which normally coats the sperm surface
  (DF)
• 1. DF is present in the seminal plasma of bull,
  boar, stallion, monkey, and rabbit. Sperm are
  exposed in the epididymis and during ejaculation
• 2. Inhibits CPE, inhibits trypsin-like enzyme
• a. Removal of DF and proteinase inhibitor from
  the sperm results in the activation of acrosomal
  enzymes so that sperm can penetrate various
  layers of the sperm

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• b. Sperm leave the vicinity of a high
  concentration of excess DF by migrating into the
  uterus
• c. DF is destroyed or removed. Calcium ions,
  fertilizins, viruses, amylase, glucuronidase,
  serum and steriods are involved

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• 2. Penetration of the zona pellucida by
  spermatozoa
• -Occurs within 15 minutes after sperm attachment
• -Sperm comes in contact with vitelline membrane
  (the true egg membrane)
• -with regard to egg oogenesis is now taking
  place

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Fertilization
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• The entire process of sexual reproduction is
  centered around the act of fertilization
• Fertilization consists essentially of the fusion
  of two cells, the male and female gametes- forms
  a single cell- The Zygote
• a. The ovum is activated by the sperm. The ovum
  begins to cleave, embryologic development occurs.

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• b. Hereditary material from the sire is
  introduced into the ovum. Beneficial characters
  arising far apart in time and space, eventually
  to become combined in a single individual.

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• B. The sperm encounters the ovum
• 1. The number of sperm in an ejaculate is
  measured in hundreds or thousands or millions
  the number traveling as far as the ampulla is
  relatively small, not much more than 1,000-10,000
  in any mammal
• 2. In many species, the sperm have to undergo
  capacitation before they can activate the ova

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• 3. The fertile life of ova is short, usually
  less than 24 hours. Cattle 20-24 hours horse 6-8
  hours sheep 16-24 hours sow 8-10 hours.
• 4. The fertile life of sperm is also fairly
  short. Cattle 30-48 hours horse 73-120 hours
  sheep 30-48 hours swine 34-72 hours
• 5. Timing is a matter of the utmost importance
  in mating and artificial insemination.

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Fertilization

• Exceeds 90 in all domestic species
• Fertilization requires 3 critical events
• sperm migration between cumulus cells
• sperm attachment and migration through the zona
  pellucida
• fusion of sperm and ovum plasma membranes

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Cumulus Cells(cumulus oopherus cells)

• Lost in the cow after 3 to 4 hours post
  ovulation.
• Hyaluronidase in bull acrosome would allow
  penetration of C.O.
• Arylsulfatase (boar acrosome) causes c.o. cells
  to disperse.

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• C. Sperm entry into the ovum
• 1. Spermatozoa must penetrate the cumulus mass
  and corona radiata, the zona pellucida and the
  vitelline membrane
• a. Sperm moves through the cumulus by its own
  motility dissolving a tunnel through the
  hyaluronic acid matrix as it goes. Sperm contain
  the enzyme hyaluronidase which help to dissolve
  the cumulus. Aided by CPE and TLE.

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• b. The sperm next meet the zona pellucida.
  Glycoprotein receptors on the zona pellucida
  receive sperm or otherwise no attachment. The
  ovum produces fertilizin which reacts with
  sperm and permits it to remain in contact with
  the zona pellucida. Sperm continues to swim
  through the zona pellucida leaving a narrow
  tunnel behind it.

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Sperm Attachment

• Regulated by receptor sites on Z.P.
• Treatment with trypsin blocks sperm attachment
  (breaks down protein receptors)
• Sperm receptors (3 glycoproteins).
• ZP1
• ZP2 present in all
• ZP3 mammalian species
• ZP3 only bind sperm cells with intact acrosomes.

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Sperm Penetration

• Penetration of the zona by sperm occurs within 5
  to 15 minutes after attachment.
• Attachment of sperm head to ZP3 allows for
  release of acrosomal acrosin.
• Digest pathway through the ZP.
• Also requires sperm motility for penetration.
• Sperm moves to Perivitelline Space.

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• c. The acrosome (loosened capacitation) is lost,
  exposing the perforatorium. Proteolytic enzyme
  facilitates passage through the zona.
• d. Last stage before fertilization involves the
  attachment of the sperm head to the surface of
  the vitellus. Activation of the ovum occurs at
  this time.
• e. Sperm head (and the tail in some species)
  enters the vitellus by phagocytosis
  (endocytosis).

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• The zona pellucida undergoes some change after
  the passage of the first sperm which renders it
  less easy to penetrate subsequently. This is
  called zona reaction.
• a. The reaction consists in a propagated change
  in the zona set off when the first sperm makes
  contact with the surface of the vitellus.

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Gamete Fusion

• True oocyte membrane vitelline membrane.
• Vitelline membrane and sperm cell fusion occurs.
• Fertilization requires fusion of the equatorial
  segment and the oocyte plasma membrane.
• After fusion, the fertilizing spermatozoan is
  engulfed (endocytosis).

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After Fusion Cortical Reaction

• Cortical Reaction prevents penetration by
  additional spermatozoa.
• Results in Zona Reaction.
• ZP changes biochemically.
• No further sperm penetration.
• Also reduces the ability of oocyte membrane to
  fuse with additional spermatozoa.
• Vitelline Block
• Prevents Polyspermy

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• 3. Male and Female pronuclei form
• -One sperm cell is engulfed by the cytoplasm
  (endocytosis) in the oocyte and the vitelline
  block is evoked which prevents other sperm cells
  from fusing to the surface (block to polyspermy)
• -The cytoplasm shrinks and a second polar body
  is pushed into the perivitelline space

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Pronuclei Formation

• Sperm loses flagellum ?male pronuclei.
• Ovum extrudes second polar body ?female
  pronuclei.
• Male and female pronuclei unite.
• Syngamy
• True Fertilization

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• In the ovum second polar body is extruded from
  the ovum soon after sperm entry. Female pro
  nucleus begins to form. Male and female pronuclei
  develop synchronously, increasing in volume.
• Syngamy During their maximum development, the
  male and female pronuclei come into contact. They
  begin to shrink and at the same time to coalesce.
  Nucleoli and nuclear membranes disappear. Syngamy
  is true fertilization

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• 5. As the time for first cleavage draws near two
  chromosome groups become visible. They unit to
  form a single group which represents prophase of
  the first cleavage mitosis.
• Fertilization is now complete

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• b. Extra sperm which succeed in entering the
  vitellus in spite of the zona reaction and the
  vitelline block are called supernumerary sperm-
  creates polyspermy.
• c. Polyspermy is a disadvantageous state. One or
  2 in mammals. Occurs in aged ovum or with
  increased animal body heat. Delayed copulation
  increases this condition in pigs. Polyspermy
  leads to polyploidy which is lethal in animals.

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• 8. Polyspermy occurs in 1-2 of all mammals. It
  results in death of the embryo some time during
  pregnancy or at birth. It is associated with
• a. Aged ova or head stressed female
• b. Females bred too late (post-ovulation)
• c. Elevated body temperature