UNSW Embryology
Week 1 - In Vivo Fertilization
© Dr Mark Hill (2009)
Introduction
Up until the last century we would not have needed a separate definition for biological fertilization : in vivo covers the normal fertilization process within the living animal, in vitro fertilization which is an aided fertilization process.
Both processes have the same biological outcome, fusion of male and female gametes to form a diploid zygote.
In vivo fertilization occurs at a less defined time and place than in the laboratory. If it occurs too late in the
menstral cycle after ovulation then the blastocyst may not have matured enough to “hatch” from the surrounding zona pellucida, which is required for adplantation, then implantation to occur. |
Animation of Fertilization process (More? Movies page) |
Page Links: Introduction | Some Recent Findings | Fertilization Overview | Reading | Fertilization Movies | Zygote - Sperm Contribution |Quick Links | Glossary
Some Recent Findings
Motosugi N, Dietrich JE, Polanski Z, Solter D, Hiiragi T. Space asymmetry directs preferential sperm entry in the absence of polarity in the mouse oocyte.
PLoS Biol. 2006 May;4(5):e135.
"Time-lapse recordings confirmed that sperm swim around the perivitelline space before fertilization. Experimental enlargement of the perivitelline space in the non-polar body half increased the regional probability of fertilization. Based on these experiments, we propose a model in which the space asymmetry exerted by the first polar body and the zona pellucida directs sperm entry preferentially to the polar body half, with no need for oocyte polarity."
Human fertilization- the egg zona pellucida glycoprotein ZP2 cleavage is required for sperm binding and acrosome reaction induction, but not zona pellucida penetration.
Fertilization Overview
The following text is extracted and modified from lecture slides and should be used as a "trigger" to remind you of key concepts in fertilization.
- Fertilization and Early Development
- Gamete formation
- egg formation-oogenesis
- sperm formation- spermatogenesis
- Fertilization
- sperm and egg migration
- sperm- activation, binding, acrosome
reaction
- egg- 2nd meiotic division, cortical
reaction
- Formation of the Zygote
- male and female pronuclei
- first mitotic division
- sex determination
- Formation of the Blastocyst
- Female Reproductive
- Early cell division
- Gamete formation- Oogenesis
- process of oogonia mature into oocytes (ova, ovum,
egg)
- all oogonia form primary oocytes before birth
- maturation of preexisting cells in the female
gonad- ovary
- humans usually only 1 ovum released (IVF-
superovulation)
- oocyte and its surrounding cells = follicle
- primary -> secondary -> ovulation
releases
- Ovary- Histology - whole transverse section (cortex, medulla)
- Menstrual Cycle
- Oogenesis- preantral follicle
- Primary Oocyte
- arrested at early Meiosis 1
- diploid: 22 chromosome pairs + 1 pair X
chromosomes (46, XX)
- autosomes and sex chromosome
- Oogenesis- antral follicle
- Secondary oocyte
- 1 Day before ovulation completes (stim by LH)
Meiosis 1
- haploid: 22 chromosomes + 1 X chromosome (23,
X)
- nondisjunction- abnormal chromosome
segregation
- begins Meiosis 2 and arrests at metaphase
- note no interphase replication of DNA
- only fertilization will complete Meiosis 2
- 2nd Meiotic Division
- Ovulation
- Hypothalmus (GRH) -> Pituitary FSH and LH ->
ovary
- GRH- gonadotropin releasing hormone
- FSH- follicle stimulating hormone
- LH- lutenizing hormone
- release of the secondary oocyte and formation of
corpus luteum
- secondary oocyte encased in zona pellucida and
corona radiata
- Ovulation- ampulla movement
- Ovulation- follicle
- Zona Pellucida
- glycoprotein shell ZP1, ZP2, ZP3
- mechanical protection of egg
- involved in the fertilization process
- sperm binding
- adhesion of sperm to egg
- acrosome reaction
- releases enzymes to locally breakdown
- block of polyspermy
- altered to prevent more than 1 sperm
penetrating
- may also have a role in development of the
blastocyst
- Corona Radiata
- granulosa cells and extracellular matrix
- protective cells for egg durin transport
- cells are also lost duringtransport along
oviduct
- nutrition
- Testis- anatomy and histology
- Gamete formation- Spermatogenesis
- process of spermatagonia mature into spermatazoa
(sperm)
- continuously throughout life occurs in the
seminiferous tubules in the male gonad- testis (plural
testes)
- at puberty spermatagonia activate and proliferate
(mitosis)
- primary spermatocyte -> secondary
spermatocyte-> spermatid->sperm
- Maturation involves meiosis and
spermeogenesis
- Seminiferous Tubule
- Spermatogenesis- Meiosis
- meiosis is reductive cell division
- 1 spermatagonia (diploid) = 4 sperm (haploid)
- 46, XY (also written 44+XY)
- Spermiogenesis - morphological (shape) change
- Spermatogenesis- spermeogenesis
- cell shape change round spermatids -> elongated sperm
- loose cytoplasm
- Transform golgi apparatus into acrosome (in head)
- Organize microtubules for motility (in tail, flagellum)
- Segregate mitochondria for energy (in tail
- Ejaculate
- 3.5 ml, 200-600 million sperm
- Infertility - Oligospermia, Azoospermia, Immotile Cilia Syndrome
- Oligospermia (Low Sperm Count)
- less than 20 million sperm after 72 hour abstinence from sex
- Azoospermia (Absent Sperm)
- Immotile Cilia Syndrome
- By volume <10 % sperm
- Accessory Glands
- 60 % seminal vesicle
- 10 % bulbourethral
- 30 % prostate
- Fertilization-sperm and egg migration
- Sperm migration
- deposited in vagina
- have approximately 24-48h to fertilize
- migrate through cervix into uterus
- from uterus into oviduct (= uterine horn, fallopian tube)
- Egg migration
- following ovulation from ovary into fimbria
- then into oviduct
- Fertilization Site
- Fertilization usually occurs in first 1/3 of
oviduct
- Fertilization can occur outside oviduct
- associated with ectopic pregnancy
- In virto Fertilization- IVF, GIFT, ZIFT....
- the majority of fertilized eggs do not go on to
form an embryo
- Fertilization- Sperm
- capacitation
- removal of glycoprotein coat and seminal
proteins
- alteration of sperm mitochondria
- binding
- ZP3 acts as receptor for sperm
- acrosome reaction
- exyocytosis of acrosome contents (Ca
mediated)
- enzymes to digest the zona pellucida
- exposes sperm surface proteins to bind ZP2
- membrane fusion
- between sperm and egg
- allows sperm nuclei passage into egg
cytoplasm
- Fertilization Figure
- Fertilization- Egg
- sperm membrane fusion
- causes depolarization of egg membrane
- primary block to polyspermy
- cortical reaction
- IP3 pathway elevates intracellular Calcium
- exocytosis of cortical granules
- enzyme alters ZP3 so it will no longer bind sperm
plasma membrane
- 2nd meiotic division
- completion of 2nd meiotic division
- forms second polar body
- Egg Cortical Reaction 1
- Egg Cortical Reaction 2
- Egg Cortical Reaction 3
- Formation of the Zygote
- Male and Female Pronuclei
- 2 haploid nuclei approach each other
- nuclear membranes break down
- chromosomal pairing
- DNA replicates
- first mitotic division
- Sperm contributes - centriole which organizes
mitotic spindle
- Oocyte contributes - mitochondria (maternally inherited)
- Sex Determination
- based upon whether an X or Y carrying sperm has
fertilized the egg
- should be 1.0 sex ratio
- is actually 1.05, 105 males for every 100
females.
- some studies show more males 2+ days after
ovulation
- cell totipotent (equivalent to a stem cell, can form any tissue of the body)
- Men- Y Chromosome
- carries sry gene
- gene activates pathway for male gonad (covered in genital development)
- Women- X Chromosome
- one X chromosome in each woman's cell has to be
inactivated
(because men only have 1 X chromosome, if abnormal, this leads to X-linked diseases more common in male that female where bothe X's need to be abnormal)
- process is apparently random
- therefore 50% of cells have father's X, 50% have
mother's
Sperm Contact
The act of fertilization changes the egg from a stage of slow structural and metabolic decline to one of renewed activation. Morphologically egg activation is a series of surface changes immediately following sperm contact.
- Marine invertebrates and some aquatic vertebrates elevate a fertilization membrane in order to prevent polyspermy. More than one reaction involved in the prevention of
polyspermy. The first reaction must be a very fast one. In oyster and echinoderm eggs the cortical granules swell and break down, lifting the vitelline membrane off the egg surface and
causing it to fuse with the plasma membrane of the ovum. Structures similar to this fertilization membrane occur in amphibians and teleosts. Cortical granules formed in Golgi
region and occur throughout the egg cytoplasm in immature eggs. They accumulate in the cortical regions as maturation proceeds.
- Mammals - No phenomenon comparable to the raising of the fertilization membrane is displayed. Mammalian eggs are surrounded by the zona pellucida which undergoes a structural
change known as the zonal reaction after sperm penetration. On sperm contact with the egg plasma membrane, cortical granules break down as in above forms, substances liberated into the
perivitelline space rapidly modify the zona pellucida resulting in a block to further sperm penetration.
The Acrosome Reaction
Penetration of egg by sperm is initiated by the acrosome reaction which takes different forms in different species.
The central part of the acrosome elongates into a tube which extends form the head of the spermatozoon. On contact with the egg the acrosomal
membrane fuses with the sperm plasma membrane thus opening the acrosomal vesicle and liberating the granules containing acrosomal lysins. The inner
portion of the acrosomal membrane everts and lengthens to form the acrosomal tubule through which the sperm nucleus enters the egg. The
mammalian sperm must remain for a time in the female genital tract before being capable of fertilization - Capacitation
- which is essentially a modification of the acrosomal reaction. Mammalian acrosomal lysins contain proteinases which lyse the glycoproteins of the zona pellucida.
Sperm Activation of Egg
During fertilization sperm activates the egg by induction of a calcium ion (Ca2+) oscillation within the egg's cytoplasm. Induction occurs by a sperm protein factor (unidentified) which can
stimulate only once Ca 2+ oscillations in metaphase eggs. Another sperm derived factor is then responsible for the inactivation of this oscillation. The activation of the egg by this Ca2+
oscillation is essential for entry of the egg into the first mitotic cycle.
- Ca 2+ oscillations induced by a cytosolic sperm protein factor are mediated by a maternal machinery that functions only once in mammalian eggs. Tie-Shan Tang, Jian-Bo Dong, Xiu-Ying
Huang and Fang-Zhen Sun Development 127, 1141-1150 (2000)
Formation and Fusion of pro-nuclei
Zona Pellucida
ZONA PELLUCIDA SPERM-BINDING PROTEINS
- ZONA PELLUCIDA SPERM-BINDING PROTEIN 2
- FUNCTION: ZP2 FORMS WITH
ZP1 AND ZP3 THE ZONA PELLUCIDA, IN WHICH ZP2
AND ZP3 COMPLEX INTO COPOLYMERS CROSS-LINKED
BY ZP1. ZP2 ACTS AS A SECONDARY SPERM
RECEPTOR.
- SUBCELLULAR LOCATION: TYPE
I MEMBRANE PROTEIN. EXTRACELLULAR
MATRIX.
- TISSUE SPECIFICITY:
OOCYTES.
- DEVELOPMENTAL STAGE:
EXPRESSED DURING THE 2-WEEK GROWTH PHASE OF
OOGENESIS, PRIOR TO OVULATION.
- PTM: IS PROTEOLYTICALLY
CLEAVED AFTER FERTILIZATION, AND THIS
MODIFICATION, ALONG WITH PRESUMED CHANGES IN
ZP3 MAY PLAY AN IMPORTANT ROLE IN THE
POSTFERTILIZATION BLOCK TO POLYSPERMY.
- PTM: SULFATED GLYCOPROTEIN
WITH O-LINKED OLIGOSACCHARIDES (BY
SIMILARITY).
- SIMILARITY: CONTAINS 1 ZP
DOMAIN.
- ZONA
PELLUCIDA SPERM-BINDING PROTEIN 3A
- FUNCTION: FUNCTIONS AS A
SPERM-RECEPTOR. IT IS RESPONSIBLE FOR
SPERM-ADHESION TO THE ZONA PELLUCIDA, AND MAY
CONTRIBUTE TO THE SPECIES-SPECIFICITY OF THE
INSEMINATION.
- SUBUNIT: ZP3 FORMS WITH ZP1
AND ZP2 THE ZONA PELLUCIDA, IN WHICH ZP2 AND
ZP3 COMPLEX INTO COPOLYMERS CROSS-LINKED BY
ZP1.
- SUBCELLULAR LOCATION: TYPE
I MEMBRANE PROTEIN. EXTRACELLULAR
MATRIX.
- PTM: SULFATED GLYCOPROTEIN
WITH O-LINKED OLIGOSACCHARIDES.
- SIMILARITY: CONTAINS 1 ZP
DOMAIN.
- ZONA
PELLUCIDA SPERM-BINDING PROTEIN 3B
- FUNCTION: FUNCTIONS AS A
SPERM-RECEPTOR. IT IS RESPONSIBLE FOR
SPERM-ADHESION TO THE ZONA PELLUCIDA, AND MAY
CONTRIBUTE TO THE SPECIES-SPECIFICITY OF THE
INSEMINATION.
- SUBUNIT: ZP3 FORMS WITH ZP1
AND ZP2 THE ZONA PELLUCIDA, IN WHICH ZP2 AND
ZP3 COMPLEX INTO COPOLYMERS CROSS-LINKED BY
ZP1.
- SUBCELLULAR LOCATION:
EXTRACELLULAR MATRIX.
- PTM: SULFATED GLYCOPROTEIN
WITH O-LINKED OLIGOSACCHARIDES.
- SIMILARITY: CONTAINS 1 ZP
DOMAIN.
Human Genbank Entries
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Database Online Mendelian Inheritence in Man Database.
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References
Reviews
Olszanska B, Stepinska U.
Molecular aspects of avian oogenesis and fertilisation.
Int J Dev Biol. 2008;52(2-3):187-94.
Nixon B, Aitken RJ, McLaughlin EA
New insights into the molecular mechanisms of sperm-egg interaction.
Cell Mol Life Sci. 2007 Jul;64(14):1805-23.
Articles
Search PubMed: Aug 2008 "fertilization" 55,398 reference articles of which 5,651 were reviews.
Search PubMed Now: fertilization |
fertilisation
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