fertilisation and blasocyst development

time of ovulation in cows sheep goats swine human

• 10-12 hours after end of estrous

• late of estrous

• mid estrous

• day 14 after initiation of mestrual cycle

oocyte fertile life for cows sheep pig human

• 8- 12 hours

• 16-24 hours

• 8-10 hours

• 6-24 hours

where is the site of semen deposition in cows sheep pig and human

all vagina except pig which is cervix+uterus

stage one- intermediate transport

• retrograde loss

• phagocytosis

• entrance into cervix/ uterus

• low motility sperm cannot pass into the cervix

• annular rings and interdigitations act as barriers

avoiding retrogade in ewes, pig and horse/dog

• how much percent lost in a cow

• 60

• sperm deposited in external cervical os in wew

• intra uterine semen deposition in pig

• semen squirted through cervix at copulation in horse and dog

variation

azoospermia

absence of sperm in the semen

oligospermia

low conc of sperm in the ejaculate

asthenospermia

reduced sperm motility

cryptorchism

failure of testes descending into the scrotum

examples of sperm head abnormalities

tail abnormalities examples

stage 2- cervix

• ‘priveliged pathways’

• removal of non motile sperm

• removal of some abnormalities

what are the 2 phases in sperm transport in the female

rapid

• reaches oviduct within minutes

• unable to fertilise the oocyte

sustained

sperm undergo

• capacitation

• hyperactivation

• acrosome reaction

what is capacitation

• ejaculated sperm cannot fertilise the occyte and sperm must undergo capacitation

• progressive destabilisation of the plasma membrane:

• glycoprotein coating sperm head removed

• exposure of zona pellucida bindng proteins

• this allows sperm to bind to oocyte at fertilisation

• surface change altered- may attract sperm to oocyte

• membrane fluidity increased to aid breakdown of acrosome

epididymis, ejaculation, capcitation

• epidymis- surface molecules added (protein and carb)

• ejaculation- surface moleules coated with seminal plasma proteins (decapacitation factors)

• capacitation- female tract strips some proteins leaving exposed areas for sperm egg binding. this happens in the uterus.

stage 3- uterus

• capacitation

• phagocytosis

hyperactivation

-what is it

what happens intracellularly

why does it happen

• Capacitated sperm exhibit hyperactivated motility

• Strong, wide amplitude, whiplashing tail beats

• Increased intracellular calcium leads to elevated cAMP

• Increased force required to swim through the viscous environment within the oviduct

stage 4- oviduct

• capacitation completed

• hyperactive motility

stage 5- fertilisation

• acrosome reaction

• spermatozzon penetrates oocyte

• male and female pronuclie form

sperm storage

• times in pig, human, horse and bat

• and what is cervical storage

• Extends ‘window’ for fertilisation • Storage time varies between species • Pig – 6 days • Human – 4 days • Horse – 6 days (up to 3 months?) • Bat – 1 year • Cervical storage • Sperm held in cervical crypts • Cervix will only release sperm in the absence of progesterone • Cervix also selects against morphological abnormalities

oviduct sperm storage

• where does sperm bind

• what does this coincide with

• what can maturation be delayed by

• Sperm bind to cells in the oviductal isthmus

• Release from the isthmus coincides with ovulation

• In vitro, sperm maturation can be delayed by

– Co-incubation with oviduct epithelial cells

– Co-incubation with proteins extracted from the plasma membrane of oviductal epithelia

events of fertilisation

what is ZP3

• structural component of the zona pellucida

• is a sperm receptor

• inducer of acroosme reaction

what is the acrosome

• what is it derived from

• where is it

• golgi derived lysosome like organelle

• overlies the nucleus in apical region of sperm head

acrosome reaction

• mulitple fusions between the outer acroosmal membrane and the plasma membrane

• extensive formation of hybrid membrane vesicles

• exposure of inner arosomal membrane and acrosomal content

fertilisation event

• what do acrosomal enzymes do

• where do sperm go

• what fuses

• what blocks polyspermy

• acrosomal enzymes digest small hole in zona pellucida

• penetration of zona pellucida is a rapid process

• sperm move into perivitelline space between zona and oocyte plasma membrane

• oocyte plasma membrane fuses with sperm equatorial segmanet

• sperm engulfed

• cortical grnaules from oocyte moe into perivitelline sace to cause zona block to polyspermy

fertilisation

sperm oocyte activaton

• how does the sperm activate the egg (2 ways)

• what is the mitochondrial DNA from

Sperm activates egg by:

• Causing calcium oscillations signaling event

• Providing centriole to contribute to initial cell division

• To form male and female pronuclei which arrange on the first mitotic spindle for the division of the zygote NB. Although sperm mitochondria enter the egg cytoplasm they are subsequently destroyed and do not contribute to mitochondrial DNA. Therefore mitochondrial DNA is entirely from the maternal side

acrosome reaction

• what are the receptors

• zona pellucida binds to at least 2 different receptors in the plasma membrane

• receptor R is a G coupled receptor that activates phospholipase C

• receptor TK is a tyrosine kinase receptor coupled to PLC gamma

• binding to r regulates adenylyl cyclase leading to elevation of cAMP and PKA activation

• PKA activates voltage dependent calcium channer which releases calcium from inside to cytosol

• PLC gamma is activated and more calcium increase through cascade

• TK can alson activate Na/H exvhanger leading to alkalinisaion of the cytosol

• increase in calcium and pH leads to membrane fusion and acrosomal exocytosis

fertilisation

• sperm penetrates cumulus oophorus

• ZP contact and acrosome reaction is triggered by ZP3

• sperm penetration of ZP and adhere to and fuse with the plasma membrane

• meiosis is complete and 2nd PB extrusion

Mechanism of PLCζ-induced Ca2+ release

• PLCζ from spermatozoa (phospholipase C) is considered to be the responsible activator

• it activates phospholipase C which catalyses the hydrolysis of PIP2 to DAG and IP3

• IP3 acts on its receptor in the ER whihc causes intracellular release of Ca

block to polyspermy

• cortical granules are vesicles and when sperm fuses with egg they release contents

• enzymes released by the cortical granules harden the zona pellucida- cross linking of zona pellucida proteins form

• this blocks additional sperm from entering

• BUT cannot overcome simultaneous penetration

pre implantation embryo development

• cleavage - zygote undergoes mitotic divisions in the zona pellucida

  • fallopian tube

  • asynchronous and occurs eery 10-12 hours from 2 cell

• compaction - cells increase cell to cell adhesion

  • forms a morula

• no net growth during pre implantation period

what is a pronucleus

stage of the fertilised egg where sperm and egg has not yet fused