Embryo Development

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Timing of Insemination

Errors in fertilization

• Polyspermy - Polyandry
  • multiple sperm penetration into an oocyte and formation of multiple paternal pronuclei and one maternal pronuclei
  • in invertebrates, the excess sperm are eliminated because the sperm centriole contributes to the first embryonic cleavage spindle
  • in mammals the sperm centriole is not essential and development can continue but if two sperm penetrate, the zygote is triploid and development fails before midpregnancy at the latest
  • this may occur in aged oocytes due to failure or slow blocks to polyspermy
• Polygyny
  • multiple maternal pronuclei and one paternal pronuclei
  • does not ususally occur in nature
  • can be created by suppressing extrusion of polar body II
• Androgenote
  • from union of two paternal pronuclei to form the embryo
  • does not occur in nature but the result of pronuclear exchange (embryo manipulation in vitro)
• Gynegenote
  • union of two maternal pronuclei to form the embryo
  • does not occur in nature but can result from artificial activation of the oocyte (parthenogenesis) and suppressing extrusion of polar body II
• Parthenogenesis
  • activation of the oocyte and development without a sperm
  • embryo is either haploid or gynogenesis occurs to form diploid

Stages of development (timing relative to start of estrus, estrus = 0 hr or day 0)

• Fertilization (30 hr)
• Zygote (34 hr)
  • defined when pronuclei are present
• Cleavage
  • 2 cell (62 hr, day 2)
  • 4 cell (75 hr, day 3)
  • 8 cell (90 hr, day 3)
  • 16 cell (120 hr, day 4)
• Morula (day 5-7)
  • where compaction occurs
• Blastocyst (day 7-10)
  • has fluid filled cavity
  • has the inner cell mass and outer trophectoderm
• Hatching (day 9-11)

Fertilization to cleavage

• Imprinting
  • identified by experiments involving transplantation of pronuclei
  • normal - transplant a paternal and maternal pronuclei into an enucleated zygote
    • development
  • gynogenote - two maternal pronuclei
    • fails mid pregnancy
    • small placenta
  • androgenote - two paternal pronuclei
    • fails to form embryo proper (no inner cell mass forms)
    • has some placntal tissue
  • important because identifies that maternal and paternal genomes are not equivalent and need both to form viable embryo
    • there is differential gene expression from the maternal and paternal genomes
    • during gametogenesis, somehow the genomes are encoded for if or when they will be expressed in the developing embryo or in the adult
  • imprinting can also be tissue specific
    • in the embryo 50:50 if a cell has maternal or paternal X chromosome inactivated
    • in the extraembryonic tissue (placenta) it is the paternal X that is inactivated
• Maternal gene control
  • proteins are made from mRNA made during oocyte development and maturation
  • during these stages of embryo development, no new mRNA is made (no transcription)
  • many cell regulatory functions are accomplished by phosphorylation of proteins (post translational modification) that were made during oocyte growth or after fertilization (translation of pre-existing mRNA)
• Mitotic cell cycle control
  • first cell cycle in the bovine is 32 hours

Precompaction cleavage

• Change in cell size
  • as embryo divides, blastomeres get smaller but zona pellucida remains the same size
• Length of cell cycles
  • 1st - 32 hours, G1=16 hr, S=8 hr, G2+M=8 hr
  • 2nd - 13 hours, G1 <1 hr, S=8 hr, G2+M=2 hr
    • short with little G1 or G2
    • this continues until the switch to embryonic gene control of development
• Inner cell mass and trophectoderm development
  • asynchrony of cell divisions
    • some blastomeres divide faster and smaller blastomeres go to inside of embryo
    • inside-outside hypothesis of development
      • inner cells give rise to inner cell mass and embryo
      • outer cells give rise to trophectoderm and placenta
      • if put an 8 cell blastomere in an 8 cell embryo
        • if placed in the center, donor blastomere contributes to the inner cell mass
        • if placed on the outside, donor blastomere contributes to the placenta or trophectoderm
      • can use this principle to create embryos from different species
        • make placenta from one species (host mother species) make the embryo from the other species (donor mother species)
• Embryonic gene control
  • extensive transcription and translational activity resumes.
    • critical step in development
  • this is where blocks to embryo development occur in vitro
    • modifications to culture media have been needed to get embryos through this block
    • sometimes can co-culture with oviduct epithelial cells
  • transition is initiated during a pause in G1
    • appears embryo runs out of key factors needed to progress in the cell cycle
    • triggers new transcription
• Movement into uterus
  • after 8 cell stage
  • changes in estrogen and progesterone responsible in most species
• Early pregnancy factor (mice, hamster, sheep, swine, human and cattle)
  • secreted within 24 - 72 hours of conception
  • seen in viable pregnancy
  • may be responsible for movement into uterus
  • also thought to sensitize the uterus for implantation
  • basis for recent pregnancy detection kit in cattle

Morula to blastocyst

• Polarization
  • initially microvilli surround most of blastomere
  • as compaction procedes, microvilli move to one one side of blastomere
  • allows gap and tight junctions to form between blastomeres where microvilli are not present
    • tight junctions which form among blastomeres on the outside (polarized cells) of the embryo create a seal between blastomeres
      • this is important for blastocoel formation
      • the cells involved give rise to the trophectoderm
    • gap junctions form among the inner (non-polar) blastomeres and between inner and outer (polar) blastomeres, these cells communicate and give rise to the inner cell mass
  • division of a polarized blastomere can give rise to
    • 2 polarized blastomere if division is vertically
    • 1 polarized and 1 non-polarized blastomere if division is oblique or horizontal
  • division of a non-polar blastomere gives rise to 2 non-polar blastomere.
• Compaction
  • occurs at fixed time after fertilization not cell number
  • loss of observable boundries in between cells
  • a differentiational event controlled by the embryonic genome
  • microfilaments and tubules interact with plasma membrane to cause this to occur

Blastocyst formation

• Tight junctions form among outside cells that will give rise to trophectoderm
  • tight junctions prevent ion and water movement between cells
  • Na/K pumps move Na into center of embryo
    • leads to an increase in osmotic pressure between cells in center of embryo
    • water diffuses through cells, is pump and secreted into center of embryo to dilute the increasing ion concentration and return to iso-osmotic conditions
    • leads to the formation of a cavity called the blastocoel
• blastocoel not dependent on
  • cell number, number of cell divisions, but again is a differentiational event controlled by the embryonic genome

Blastocyst hatching

• enzymatic digestion of zona pellucida from embryo and/or uterus
  • plasminogen and plasminogen activator produced by embryo
  • softening of zona by uterine enzymes
• increase in size of blastocyst
  • most important - due to pumping of Na and then water into the blastocoel cavity
• occurs on day 9-10 in cattle, day 6 in swine and day 7 - 8 in horses or sheep

Twins

• dizygotic (not identical)
  • fertilization of two oocytes
  • most common
• monozygotic (identical)
  • 2 cell split
  • 2 inner cell masses form
  • a split of the inner cell masses
    • this may give rise to fetuses being attached to one another (Siamese twins)

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