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Zygote, Embryo, IVF and ED
Brian Dale
International Congress
Endocrine Distruptors: Endometriosis and Infertility
Thursday 8 April 2010
Napoli
The Zygote
• Gamete quality is critical to generate azygote with the highest potential fordevelopment.
• In vitro embryo production has allowedus to study the role played by eachgamete in embryo development
• Although gamete quality is crucial,culture conditions are also determinate.
Embryo development
• Oocyte competence is acquired within the ovary preceeding ovulation
through a process referred to as oocyte capacitation
• The oocyte role is critical during the interval between fertilization and the so-
called maternal-embryonic transition when the transcriptional activity of the
embryonic genome becomes functional
Any perturbation of these process may have an effect on gene expression
and, therefore, cause an arrest in embryo development
Culture and quality control of embryos
• Reduced light – no windows
• Limited and controlled access
• Easy to clean
• No alcohols, no perfumes, no smoking
• Non toxic painting
• Air conditioning
• Air filtration –HEPA
• Positive pressure
• Sterility precautions
The quality of the air and the environment have always
been a matter for concern in IVF labs
Temperature
• Transient cooling to room temperature can cause irreversible disruption of the
meiotic spindle in the human oocyte (-1°C = irreversible damage to meiotic
spindle)
• Repolarisation of the spindle, oocytes are more sensitive then embryos
- 0.5 °C/min
pH regulation in the human oocyte
• The intracellular pH (pHi) is not significantly different between germinal vesicle-stage oocytes, mature metaphase II stage oocytes, aged, failed fertilizedmetaphase II oocytes and fertilized, two pronuclear stage human zygote
• The response of individual oocytes is equivalent whether placed in pH 8.0medium or pH 7.0 medium
pH regulation in the human oocyte
• The response to alkaline or acidic shock does not change in human embryosup until the morula stage. Human blastocysts are characterized by a completerecovery to acid shock
• The optimal extracellular pH for oocyte insemination is in the 7.5 range asopposed to 7.9-8.0
VOC: sources
• Traffic – pollution
• Paintings – glue
• Packing materials – isolation
• Plastics
• Machines
• Laboratory equipment
• Compressed gasses
• Staff
• Alcohol - anesthetics
VOC in urban areas
20<1Acetilene
10<1Cis-2-Butene
10<11,3 Butadiene
20<1Isobutylene
1001Propene
2002Isopentane
4004N-Butane
1001Isobutane
3003Propane
5005Ethane
150001200Methane
Maximum parts per billionMinimum parts per billionComponent
Cohen et al. Human Reprod, (1997) 12:1742-1749
Range for low molecular hydrocarbons in typical urban areas
CO2
Air quality – standards, control and impact on
embryonic development
• Testing shows that most
laboratories conducting
human gamete and
embryo culture have an air
quality and source of
contamination that exceed
the levels measured in
homes, businesses and
schools
Select volatile compounds exposure
Cohen et al. Human Reprod, (1997) 12:1742-1749
0
5
10
15
20
25
Outside air Return air Hallw ays Procedure
room
Laminair f low
units
Incubators
μg/m
3
Benzene
Toluene
Xilenes
Styrene
VOC levels in compressed CO2
1.6Ethylbenzene
3.8m-& p- Xilenes
4.7Trichloroethene
7C12H26 alkane
9C7H16 alkane
10n-Undecane
10C9H12 alkyl benzene
10n-Heptane
12Toluene
20Ethanol
24Acetone
30Isohexane
30n-Butane
50Acetaldehyde
50n-Pentane
80Isopropanol
100Unknown freon
100Benzene
μg/m3Volatile organic
compound
Cohen et al. Human Reprod, (1997) 12:1742-1749
Nunc dishes
Cohen et al. Human Reprod, (1997) 12:1742-1749
5.80O- Xilene
7.5M- & p- Xilenes
10Octanal
10Propylbenzene
10Cumene
20Decanal
20N-Nonane58.002-Hexanone
20N-Octane64.00Ethylbenzene
23Benzene70Hexanal
30Butene isomer100Benzaldehyde
30n-Hexane100N-Butane
403-Pentanone100Acetaldehyde
40Butanal130.002-Butanone
50Nonanal150.00Acetone
503-Methylpentane180.00Toluene
50N-Pentane920.00Styrene
≤50 ng/sample>50 ng/sampleMaterial
Compounds released from cell tissue culture grade petri dishes
Effect of VOC
Cohen et al. Human Reprod, (1997) 12:1742-1749
1719One day after bench-top installation7 July 1995
344During installation of floor tiles3 July 1995
171712 days after use of water-based paint26 June 1995
Expanded
blastocystsa
Zygotes in
culture
Construction activity in neighbouring
space
Date
Effects of floor tile adhesive on mouse embryo development in vitro
Endocrine Disrupters
An endocrine disruptor is defined as an exogenous agent that interferes with
the synthesis, secretion, transport, metabolism, binding, action or elimination of
natural blood-borne hormones in the body that are responsable for
homeostasis, reproduction and developmental processes
DTT PBCs
2,3,7,8-TCDD BPA
Endocrine Disrupters Mode of Action
• Mimics naturally produced hormones
such as oestrogen and testosterone
• Hormone blockers “lock up” hormone
receptors, preventing naturally
produced hormone performing their
function
• Triggers act through hormone-like
pathways but initiate abnormal reaction
in the cellResponse/No response/Abnormal response
EDs interfere with the functioning of the endocrine system in at least 3
possible ways:
Selected examples of contaminants linked to
reproductive, fertiliy or developmental problems
Type of
contaminants
and examples
Sources and exposure curcumstances
Metal
Mercury Occurs from energy production emissions and naturally. Enters the aquatic food chain through
a complex sistem. Primary exposure by consumption of contaminated seafood
Lead Found in older homes where lead-based paints were used and in or on some toys and
children’s jewelry. Exposure by incidental ingestion
Organic compounds
Ethylene oxide Occupational exposure to workers sterilizing medical supplies or engaged in manufacturing
Pentaclhlorophenol Wood preservative for utility poles, railroad ties, wharf pilings; formerly a multi-use pesticide.
Found in soil, water, food, breast milk
Bisphenol A Chemical intermediate for polycarbonate plastic, resins. Found in consumer products and
packaging. Exposure through inalation, ingestion, dermal absorption
PCBs Used as industrial insulator and lubrificants. Banned in the 1970sbut persistent. Present in the
acquatic and terrestrial food chainsresultingin exposure by ingestion
Dioxins Byproducts of manufacture and combustion of chlorine-containing products. Persistent in the
environment; present in the acquatic and terrestrial food chainsresultingin exposure by
ingestion
Selected examples of contaminants linked to
reproductive, fertiliy or developmental problems
Type of
contaminants
and examples
Sources and exposure curcumstances
PFOS Perfluorinated compound used in consumer products as stain- and water-repellant. Persists in
the environment. Occupatinal exposureto workers and general population exposure by
inhalation, ingestion, dermal contact
DEHP Phthalates are plasticizers in consumer products and used as solvents for personal care
products. Exposure occur by inalation, ingestion, dermal absorption
Pesticides
Chlorpyrifos Organophodphate pesticide used in agricultural production and for home pest control (home
uses are now restricted). Exposure routes include inhalation, dietary and non-dietary ingestion,
and dermal contact
DDT Organochlorine insecticide banned in USA in the 1970s but still used abroad. Persistent in soil.
Enter the food chain resulting in ingestion exposures
Air contaminants
ETS common air
pollutants (e.g. PM,
ozone, Pb)
Burning of tobacco products. Exposure by inhalation from active or passive smoking. Sorces
include combustion of wood and fossil fuels, and industrial production. Exposure by inhalation
Glicol ethers Used in enamels, paints, varnishes, stains, elecronics, cosmetics.
Synthetic compounds known as or suspected to be
endocrine disruptors
Herbicides and fungicides• Dichlorophenoxyacetic acid (2,4-D)
• Trichlorophenoxyacetic acid (2,4,5-T)
• Alachlor
• Amitrole
• Atrazine
• Metribuzin
• Nitrofen
• Trifluralin
Insecticides• Benzenehexachlorcyclohexane (B-HCH)
• Methoxychlor
• Toxaphene
• DDT and metabolites (DDE)
• Carbaryl
• Endosulfan
• Mirex
• Transnonachlor
• Chlordane
Nematocides• Aldicarb
Industrial chemicals• Dioxins
• Polychlorinated biphenyls (PCBs)
• Polybrominate biphenyls (PBBs), pentachlorophenol (PCP)
• Benomyl
• Mancozeb
• Zineb
• Matriam complex
• Maneb
• Ziram
• Tributyltin
• Hexachlorobenzene
• Oxychlordane
• Dicofol
• Heptachlor and heptachlor epoxide
• Dieldrin
• Parathion
• Methomyl
• Lindane (Y-HCH)
• Synthetic pyrethroids
• Chlordecone (kepone)
• 1,2-Dibromo-3-chloropropane (DBCP)
• Penta- to nonyl phenols
• Phthalates
• Styrene
Mechanisms of developmental toxicity
• The best characterized mechanism for the action of dioxin (TCDD) and related
compounds, involves the arylhydrocarbon receptor (AhR)
• The AhR pathway is a widely expressed orphan receptor pathway activated by
meny enviromental toxicants and carcinogens
• Ahr ligands, including dioxins and PCBs, induce a spectrum of developmental and
toxic responses by modifying gene expression, altering hormonal profiles and
disrupting cell proliferation and differentation
• Animal studies show embryonic lethality, teratogenesis, cleft palate,
hydronephrosis and growth retardation among the many adverse effects observed
following gestational exposure to AhR ligands
The arylhydrocarbon receptor
• The AhR receptor is present in the cytoplasm
bound with at least three additional proteins
thought to keep the AhR in a state responsive
to ligand binding.
• When ligand binding occurs the AhR enters the
nucleus where it complexes with its nuclear
partner AhRnuclear translocator (Arnt)
• The newly formed heterodimer acquires the
ability to bind specific DNA enhancer
sequences know as xenobiotic responsive
elements acting as a transactivator of gene
expression
• Products of these gene belong to: drugs-
metabolizing enzymes and grow-regulatory
proteins.
• The most extensively studied AhR-target gene
is cytochrome P4501A1 (CYP1A1)
Pocar P et al. Reproduction, (2003) 125:313-325
Styrene
0
20
40
60
80
100
% b
las
toc
ys
ts
oil overlay oil-free
Styrene in mouse culture media• Styrene: an oil soluble pollutant
• A common air pollutant from plasticranges from 0-20 μg/m3
• In embryo culture with mineral oil thestyrene is selectively absorbed
• Log P = Log (butanol)/(water)
• Styrene Partition coefficient = 3.05
Cohen et al. Human Reprod, (1997) 12:1742-1749
Bisphenol A
• Bisphenol A (BPA) levels measured in follicular fluid of
women undergoing in vitro fertilization procedures
showed an average of 2.0 ng/ml
• Orally administered low-dose BPA in adult mice causes
congression failure and aneuploidy in oocyte
• BPA levels measured in human semen showed an
average concentration of 5.1 ng/ml
Effect of selected ED on meiotic maturation of
porcine oocytes
• At a concentration of 100 μM BPA andCMP significantly reduced the numberof OCC reaching complete expansion
Mlynarcikova A et al. Toxicology in Vitro, (2009) 23:371-377
Effect of selected ED on meiotic maturation of
porcine oocytes
• The mechanism of the inibitory action could involve alteration ofextracelluler matrix formation rather than reduction of synthesis ofits major component hyaluronic acid (HA)
Mlynarcikova A et al. Toxicology in Vitro, (2009) 23:371-377
Effect of selected ED on meiotic maturation of
porcine oocytes
• At low concentration, none of thetested agent disrupted nuclearmaturation of in vitro cultured oocyte
• After 100 μM BPA exposure, numberof oocytes that remained at the GVstage was significantly higher whencompared to the control group(21.3% vs. 10.4%)
• The number of oocytes thatunderwent GVBD (to 78.7% vs.89.6% in the control group) as well asthe percentage of oocytes that reachthe MII stage was significantlydecreased (50.0% vs. 81.7% incontrol)
Mlynarcikova A et al. Toxicology in Vitro, (2009) 23:371-377
TCDD disrupts morphogenesis of the rat pre-
implantation embryo
• Monopolar spindle formation
• F-actin capping and fragmentation
due to aberrant cytokinesis
• Size, shape and position of nuclei
Hutt KJ et al. BMC Developmental Biology, (2008) 8:I
Maternal TCDD exposure disrupts morphogenesis at the compaction stage
(8-16 cell), with defects including:
In vitro exposure of mouse embryos to TCDD
• The percentage of eight-cell embryos exposed to
TCDD at 1, 2, and 5 pMconcentration is significantly
lower than that of controls
• Blastocyst formation of the surviving eight-cell
embryos was accelerated, with the number of cells
in the blastocysts increased in a dose dependent
manner
• The effects of TCDD on embryos of the 8-cell or
later stage are probably not an expression of overt
toxicity, but rather indicate a stimulatory or mitogenic
role of TCDD on development
Tsutsumi O et al. Biochem Biophys Res Commun, (1998) 250:498-501
In vitro exposure of mouse embryos to TCDD
• Exposure of 1- and 2- cell
embryos for 12 or 24 h did not
change their relative CYP1A1,
AhR, and Arnt mRNA levels
• Exposure of 8-cell embryos
drammatically induced CYP1A1
mRNA expression in a TCDD
dose-dependent manner at the
blastocyst stage
Wu Q et al. Toxicology, (2002) 174:119-129
Sensitivity to TCDD differs with the embryonic stage:
In vitro exposure of mouse embryos to TCDD
Wu Q et al. Biol. Reprod, (2004) 70:1790-1797
• Real-time reverse transcription-
polymerase chain reaction analysis
revealed that exposure of
preimplantation embryos to TCDD
tended to decrease the expression
levels of the imprented genes H19 and
Igf2
• Use of bisulfite genomic sequencing
demonstrated that the methylation
level of the 430-base pair H19/Igf2
imprint control region was higher in
TCDDexposed embryos
• Methyltransferase activity was also
higher in the TCDD-exposed embryos
Alteration of mRNA stored in the ooplasm
Pocar P et al. Mol. Reprod. Dev., (2001) 60:535-541
• A-1254 induces changes to thepolyadenylation pattern of genesindicating a perturbing effect exerted bythis contaminant on the translationalregulation of this transcripts
• Exposure of oocytes to A-1254 duringIVM affects polyadenylation in a variedway:
• Pronounced deadenylation of some of thegene that would deadenylate in controlconditions (e.g. glucose transporter type1, connexin-43 and plakophilin)
• A longer poly(A) tail is observed at the 3’-end of connexin-32, a gene that normallyre-adenylates during maturation
• Heat shock protein 70 instead ofundergoing a deadenylation process as incontrol conditions, shows an extension ofthe tail at the end of in vitro maturation
Disruption of cytoplasmatic remodelling
Brevini TA et al. Eur. J. Histochem., (2004) 48:347-356
• Oocytes exposed to A-1254 exhibit delayedmigration and dispersal of cortical granules
• A significantly higher percentage of fertilizedoocytes fail to release cortical granules aftersperm penetrationand present multiplefertilization after IVF
• It is possible that PCBs block the molecularpathway that trigger exocytosis of corticalgranules
• Exposure to PBCs alters mitochondriarelocation during maturation this is associatedwith the lack of a cytoplasmatic microtubulenetwork
• A-1254 exposure also perturbsgap-junctionmediated comunication between oocyte andcumulus cells
A common attribute of cytoplasmatic maturation is the migration and
redistribution of organelles
IVF may contribute to imprinting disorders
• Differences in DNA methylation patterns in placental and umbilical
blood sample taken from children born after IVF and children
conceived naturally has been reported
• The IVF process may lead to imprinting changes from exposure of
the oocyte or developing embryo to exogenous factors
• IVF may indirectly contribute by allowing propagation using
abnormal sperm containing imprinting defects. These processes
could potentially lead to transgenerational effects
IVF may contribute to imprinting disorders
• Ovarian stimulation leads to elevated level of follicular homocysteine whichmay have an effect on methylation
• There has been a trend to colture early human embryos in culture mediumlacking essential amino acids. Consequently, methionine is not availableduring the first 3 days of in vitro culture,a time when methylation is of majorimportance
• Mammalian embryos exhibit remarkable plasticity and will struggle to formblastocysts under a wide range of culture condition, although presumably atsome adaptive cost to their postgestational development programme
• Although all animals are to some extent a model for others, great cautionshould be exercised in extrapolating data.
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