developmental block in embryos

8/14/2019 Developmental Block in Embryos

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Developmental block during

embryonic development

Presented by:Dharmendra Kumar

Ph D. (Animal Biotechnology)N.D.R.I., KarnalHaryana-132001 (India)E-Mail:[email protected]

Credit seminar

on


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Developmental block

A stage which generally arises during the

course of embryonic development in vitro,due to improper genome activation & resultsin death of the embryo

Time of occurrence is species specific


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Morula5-8Uterus

Sixteen cell4-5Uterus

Eight cell3-5Isthmus

Four cell2-3Isthmus

Two cell1-3Ampullary-Isthmic

Junction

One cell0-2Ampullary-Isthmic

Junction

DevelopmentDayLocation

Early Embryonic

Development


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Time of Embryonic block

8-16

8-16

4

2

4-8

8-16

Cell stage of

developmental block

Davis., 1985Porcine

Telford.,1990Murine

Gandolphi & Moor., 1987Ovine

Chauhan et al., 1998Buffalo

Braude., 1988Human

Camous et al., 1984Bovine

ReferenceSpecies


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Main mechanisms causingembryonic developmental

block

Inability to react to injuries caused byenvironment

Inability to activate transcription ofdevelopmentally important genes (Meirelles et al., 2004)


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Major Activation

of genomeOocyte Quality

Relativegeneproducts

Telomerase activity

+ - - - - + + +

Sensitive to

Environmental stress

Telomere damage

Embryo senescence

Proposed model for embryo death by environment

(Betts & King, 2001)


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Transcription factor expressionpattern

in bovine embryos

YY1HMGA1RY1

P300CREB

YAP65HMGN1 & HMGN2NFAROCT-4

TEAD-2ATF-1MYS2TBP

(Vigneaultet al

., 2004)


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Maternal to zygotic transition

(MZT)

Initiation of transcription in the embryo and thereplacement of maternal mRNA with embryonic mRNA

by RNA pol-II

Transcriptionally repressive state appears

Relieving this transcriptionally repressive state by

inducing histone hyperacetylation

(Schultz, 2002)


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TATA BOX

P PP

P

ON

TBP TFIID

TFIIA TFIIB

TFIIF

tail

RNA pol II

TFIIETFIIH

Transcription initiation by RNA Pol II


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Biological significance oftranscriptionally repressive state

Genome activation is relatively promiscous

Reduce the expression of inappropriatelyexpressed genes

Newly generated gene expression profile to

make it compatible with furtherdevelopment

(Schultz, 2002)


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Biological function of MZTBiological function of MZT

Destroy oocyte

specific transcripts

Replace maternal transcripts

With zygotic transcripts

Reprogramming of gene

Expression with generation

of novel transcripts


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Oocyte M II 2-cell 4-cell

FERTILIZATION

G1 S G2 G1 S G2

1-cell

Degradation of maternal mRNA

Translation of maternal mRNARecruitment of maternal mRNA

Demethylation

P-H Exchange

TF/H4Ac

Transcription

TATA-less preferred

M-TEAD2 Activity

Enhancer stimulation of promoters

Translation of zygotic mRNAs

Development of repressive state

Schematic diagram representing transcriptional activity during initial cell stages

TATA+

(Schultz, 2002)


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Asynchronous cell divisions24-28448-100143

08-100102

4-68-1010261

G2

hr

S

hr

G1

hr

Total

hr

Cell

cycleno.

Duration

Embryonic cell cycle in bovine species

(Barnes & Eyestone, 1990)


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Bovine embryonic cell cycles and zygotic/embryonic gene expression in cattle

(Barnes & Eyestone, 1990)


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Methods to study & characterize

gene products

RT-PCR

DD-PCRArray technology

Si RNA knockdownQuantitative or semi-

Quantitative RT-PCR

Subtractive hybridization

&

sequencing

Techniques to study genomeactivation


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How to relieve the

developmental block

Reduction of glucose in the culturemedium

Using co-culture systems

Addition of serum (Gandolphi & Moor, 1987)


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Effect of glucose on

developmental block

By affecting salvage pathway

By generating ROS


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Salvage pathway

(Dienhart et al.,1997)


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Interactions between glucose, purinemetabolism & ROS production

(Guerin et al., 2001)

GlucoseHK

Glucose 6-P

Pentose Phosphate Pathway

Purines

HPRT

Hypoxanthine

Xanthine

XOO2

-.


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Co-culture

Bovine oviductal

cells

Granulosa cells

VERO cells

BRL cells


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What does co-culture do?

Embryotrophic factors are provided

Decreases glucose concentration

Secretes GSH, hupotaurine & taurine

Reduces oxygen tension

(Guerin & Menezo, 1995)

(Bavister, 1995)

(Fukui et al., 1991)

(Gondolfi et al., 1989,1992)


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Development of human embryos on verocells

5 (12%)8 (20%)12 (29%)16b (39%)41Co-culture

(%)

--130a (97%)31Control

(%)

HatchedExpanded

Cavitating

Blocked ordegenerated

TotalGroup

(%)

(Menezo et al., 1990)


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Medium modification by somatic cells

0.080.010.540.03b3.310.15c3T3 cells

0.170.08c

1.170.11c

3.730.19c

BRL cells

0.110.01c2.920.35c2.670.03cBOE cells

0.060.030.220.035.550.20ControlaPyruvateL-lactateGlucose

Treatment Metabolite concentration (mM)

(Edwards et al., 1997)


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Pyruvate prevents peroxide-induced injury

Removing ammonia from embryos by converting

into alanine

Decarboxylated in presence of H2O2 to produce

acetate, CO2, & water

Acetate can be used as energy substrate

(Morales et al., 1999)


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Pyruvate prevents peroxide-induced injury

81b123b581a78_+

212a

334a

684a

73++

212a324a755a82__

252a355a767a79+_

Blasto(%)

day 3

5-8-cell(%)

day 3

Cleaved(%)

day 3

Zygote

(n)

Pyruvate(0.3mM)

H2O2

(10-5)

(Morales et al., 1999)


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SOD GCS GPx

Stored transcripts

Enzymes

OH., O2-.

Follicular Fluid

Ascorbic Acid

Hypotaurine

CysteamineStored transcripts

SOD, CAT, GPX

OH., O2-.

OOCYTE

EMBRYO

OH.,

O2-.Hypotaurine,

Taurine

CSD

OVIDUCTAL EPITHELIALCELLS

-

SOD, GPX,

GCS, Cat

-

GSH

-Metallic Ions

-

Transferrin,

Albumin

**

Tubal fluids

Somatic ells secrete GSH,..

(Guerin & Menezo, 2001)


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Effect of serum in kinetics of bovine

embryos


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Conclusion

The first hurdle in in vitro development ofembryos is developmental block

It is species specific

It occurs due to improper activation ofmaternal to zygotic transcription

It can be overcome by providing suitableculture conditions


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Future Prospects

To explore the molecular mechanism of thedevelopmental block

To completely understand factors involved inMZT

To completely explore the effect of serumsupplementation on embryonic development


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developmental block in embryos

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