Fertilization and Ovulation

Ovulation

• The oocyte is released from the follicle
• Oocyte and cumulus mass are moved by fimbria into the infundibulum of the uterine tube
• Meiosis continues to second meiotic metaphase, but is blocked unless fertilisation occurs
• Fertilization happens in the ampulla of the uterine tube
• Short window for fertilization, lasting 6-12 hours

Ovulation in dogs

• Oocytes ovulate at Prophase 1
• This is Before completion of meiosis 1
• Maturation finishes in the oviduct

Fertilisation - Sperm Transit

• Sperm gain the ability to fertilize during the epididymal transit
• Sperm move from the site of deposition (insemination x natural mating) to the uterine tube within minutes or hours
• Contractions of the female tract occur
• Millions or billions of sperm are ejaculated
• About a thousand reach the uterine tube
• Only hundreds are present for fertilization

Sperm Reservoir

• The isthmus of the uterine tube is the sperm reservoir
• It is near the uterotubal junction
• Sperm adhere to the tubal epithelium
• There is intimate junction with cell membrane interaction
• Adherent sperm have a prolonged lifespan
• Sperm are released at the time of ovulation
• The release mechanism is unknown

Sperm Capacitation

• Sperm become capable of undergoing acrosome reaction
• There are biochemical (non-morphologic) changes required in the female tract before sperm are capable of fertilization
• Seminal plasma components are removed and interaction with female secretions occur
• Hypermotility of the sperm occurs and there are changes in membrane cholesterol, gylcosaminoglycans which affect membrane fluidity
• Oocyte receptor expression occurs

Acrosome Reaction

• It's essential for fertilization, and allows sperm penetration into the oocyte
• It's calcium dependent
• It usually occurs when sperm contact the zona pellucida of the oocyte
• In some species, it occurs during cumulus penetration
• Allows fusion of the sperm plasma membrane with the outer acrosomal membrane
• Extensive vesiculation occurs over the surface of the sperm and acrosome
• Allows the orderly release of acrosomal enzymes

Fertilisation, Penetration of Zona Pellucida

• Sperm migrate between cumulus cells
• They attach to and migrate through the zona pellucida
• Fusion of the sperm and oocyte membrane occurs
• The zona pellucida has 3 major proteins
• ZP3 is the specific binding site for sperm
• Only acrosome-intact sperm bind to the zona
• Sperm motility and enzymes are required for zona penetration (e.g. acrosine)

Fertilisation, Gamete Fusion

• The oocyte membrane (vitelline membrane) is less species-specific in binding than the ZP
• Completion of the acrosome reaction is required before sperm can fuse with oocytes
• Fusion involves the post-acrosomal sperm membrane (equatorial region)
• It becomes fused ("intermingled") with the oolemma

Blocks to Polyspermy

• The zona pellucida becomes impermeable upon fertilization
• Extensive reorganisation of the ZP structure
• This is driven by oocyte cortical granules released into the perivitelline space, after the release of soluble factors and DNA

Fertilisation, After Sperm Penetration

• The Oocyte completes meiosis
  • Expels the second polar body
• Remaining maternal (haploid) chromosomes are enclosed in the pronucleus
• The sperm nuclear envelope disintegrates
  • DNA decondensation occurs
  • A new envelope of male pronucleus is formed
• Male and female pronuclei migrate to the egg cell center
• The nuclear envelopes disperse
• Intermixing (fusion) of chromosomes occurs
• Chromosomes aggregate in prophase of the first cleavage division
• The proximal centriole of sperm forms one of the zygote’s centrioles

Early embryo development

• Early cleavage occurs in the uterine tubes
• The zygote reaches the uterus:
  • In about 4 days in cows and most other species
  • Longer in horses, 6 days
  • Longer in dogs, 8 days
    • 16-cell/compact morula stage (cows, sheep)
    • Morula/early blastocyst (mares and dogs)

Mares

• Only fertilized zygotes are transported through the uterine tubes (oviductal papilla)
• The zygote is responsible for transport by secretion of PGE2

Early Embryo Development

• Several mitotic divisions occur
• Early divisions occur without increase in cell mass
  • "Cleavage" (or “Reduction Division”)
• Metabolic support is provided by maternal secretions
• Zygotic protein synthesis begins at 2 to 16-cell stage depending on species, totipotent cells

Hatched Blastocyst

• The blastocyst hatches out from the zona pellucida, the beginning of implantation
  • There is an increase in fluid in the blastocele
  • Proteolytic enzymes come from the trophoblast
  • Blastocysts experience Contraction

Timings for Species

• Most species hatching occurs in 7-10 days
• Mares hatching occurs in 9-10 days, however, equine embryos have a capsule surrounding the ZP, therefore, the rounded shape is maintained

Important Early Events

• Development inside the zona pellucida occurs
• Hatching from the ZP occurs
• Maternal recognition of pregnancy
• Formation of extraembryonic membranes:
  • Yolk sac is for nutrition and sex differentiation
  • Amnion amniotic fluid provides mechanic protection, hydration, temperature changes and fetus movement
  • Allantois collects secretions from the embryo and helps with gases exchanges
  • Chorion is the outermost fetal membrane which attaches with the uterus and provides nutrition to the embryo.

Maternal Recognition of Pregnancy

• It is critical in that the mother "recognizes" that she is pregnant

• The major consequence is to prolong the period of function of the corpus luteum

• It is a transition from a cyclic to pregnant state

• Different processes are used in each species

  • Two major strategies
    • Anti-luteolytic which consists of Diversion or Inhibition of PGF2α secretion
    • Luteotropic which consists of maintaining the CL (e.g. hCG in humans)

• The Pregnant and non-pregnant cycle are similar for the first 14-16 days

• The mechanism isn't completely understood in mares - Embryo mobility is seen in horses - Interferon tau (IFNT) is seen in cows and sheep - Estrogens are seen in pigs - Chorionic gonadotropin is seen in primates

Maternal Recognition of Pregnancy in Ruminants

• After hatching from the zona pellucida, the blastocyst elongates
• The blastocyst contacts most of the uterine luminal epithelium
• Trophoectoderm (future "placenta”) produces Interferon tau (IFNτ)
  • IFNτ Inhibits oxytocin receptor synthesis and Inhibits prostaglandin F2α
  • IFNτ Stimulates the endometrial glands

Maternal Recognition of Pregnancy in Swine

• The blastocyst produces Estradiol
• Estradiol changes direction of PGF2α secretion by the endometrium:
  • Endometrial-derived PGF2α is directed away from the uterine vasculature (endocrine secretion) and sequestration into the uterine lumen (exocrine secretion)
  • PGF2α is inactivated in the lumen or PGF2α is metabolized to its inactive 13, 14-dihydro-15-keto prostaglandin F2α metabolite

Maternal Recognition of Pregnancy in Horses

• Embryo mobility is present
• Fertilization happens in the uterine tube
• The viable embryo enters the uterus around day 6 after fertilization: this marks first maternal recognition of pregnancy and transport depends on PGE2
• The Equine embryo migrates through both uterine horns and body several times a day, up to 18 horn changes per day
• Movement is mediated by the uterus: although, if there are simulated fake vesicles then movement is less. Movement is maximal around days 10 – 12 and some signal from the embryo occurs (PGE)?
• The prevention of movement results in pregnancy loss