Zigote, Embryo, IVF and ED

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


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


Nunc dishes


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


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

Effect of VOC


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

ED Sources

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


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


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)



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)


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


• 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:


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

In vitro exposure of bovine oocytes to PCBs

Pocar P et al. Mol. Reprod. Dev., (2001) 58:411-416

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.

Documents

Zigote, Embryo, IVF and ED