Credit seminar

on

Developmental block during

embryonic development

Presented by:
Dharmendra Kumar
Ph D. (Animal Biotechnology)
N.D.R.I., Karnal
Haryana-132001 (India)
E-Mail:
biotechndri@gmail.com
 Developmental block

A stage which generally arises during the

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

Time of occurrence is species specific

 Early Embryonic
Development

Location Day Development

Ampullary-Isthmic 0-2 One cell

Junction
Ampullary-Isthmic 1-3 Two cell
Junction
Isthmus 2-3 Four cell

Isthmus 3-5 Eight cell

Uterus 4-5 Sixteen cell

Uterus 5-8 Morula

 Time of Embryonic block

Species Cell stage of Reference

developmental block
Bovine 8-16 Camous et al., 1984

Human 4-8 Braude., 1988

Murine 2 Telford.,1990

Porcine 4 Davis., 1985

Buffalo 8-16 Chauhan et al., 1998

Ovine 8-16 Gandolphi & Moor., 1987

 Main mechanisms causing
embryonic developmental
block

Inability to react to injuries caused by

environment

Inability to activate transcription of

developmentally important genes (Meirelles et al., 2004)
 Proposed model for embryo death by environment
Relative gene products

Major Activation
of genome
Oocyte Quality

Telomerase activity

+ - - - - + + +

Sensitive to
Environmental stress

Telomere damage
(Betts & King, 2001)
Embryo senescence
 Transcription factor expression
pattern
in bovine embryos

YY1
HMGA1
RY1
P300
CREB
YAP65
HMGN1 & HMGN2
NFAR
OCT-4
TEAD-2
ATF-1
MYS2
TBP

(Vigneault et al., 2004)

 Maternal to zygotic transition
(MZT)

Initiation of transcription in the embryo and the

replacement 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)
 Inhibition of RNA pol II dependent transcription during any of the first four
embryonic cell cycles has a detrimental effect on progression of
embryonic development beyond 16-cell stage

Hyperphosphorylated Hyperphosphorylated

II O RNA Polymerase II II B
Form Form

Unphosphorylated

II A
Form

Initiates transcription
(Memili & First, 1998)
 Transcription initiation by RNA Pol II

TBP TFIID
TATA BOX

TFIIA TFIIB

TFIIF
tail

RNA pol II

TFIIH
TFIIE

ON
P
P
P
P
 Effect of α -amanitin on embryo development at different cell stages

No. (%) of embryo

Group N 2-cells 4-cells 8-cells 9-16 Blastocys
cells t
1/α- 90 37 (41) 41 (45) 35 (38) 28 (31) 0 (0)
amanitin
1/control 88 49 (55) 41 (46) 34 (38) 17 (19) 20 (22)
2/α- 90 77 (85) 69 (76) 46 (51) 0 (0)
amanitin

2/control 90 75 (83) 61 (67) 39 (43) 43 (47)

3/α- 90 82 (91) 42 (50) 0 (0)
amanitin

3/control 88 64 (72) 54 (61) 35 (39)

4/α- 90 56 (62) 0 (0)
amanitin

4/control 90 75 (86) 47 (35)

(Memili & First, 1998)
 Biological significance of
transcriptionally repressive state

Genome activation is relatively promiscous

Reduce the expression of inappropriately

expressed genes

Newly generated gene expression profile to

make it compatible with further
development
(Schultz, 2002)
 Biological function of MZT

Reprogramming of gene
Destroy oocyte
Expression with generation
specific transcripts
of novel transcripts

Replace maternal transcripts

With zygotic transcripts
 FERTILIZATION
G1 S G2 G1 S G2

Oocyte M II 1-cell 2-cell 4-cell

Degradation of maternal mRNA

Translation of maternal mRNA
Recruitment of maternal mRNA
Demethylation
P-H Exchange
TF/H4Ac

Transcription
TATA+
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

(Schultz, 2002)
Embryonic cell cycle in bovine species

Duration
Cell Total G1 S G2
cycle hr hr hr hr
no.
1 26 10 8-10 4-6
2 10 0 8-10 0
3 14 0 8-10 4
4 24-28 Asynchronous cell divisions
(Barnes & Eyestone, 1990)
Bovine embryonic cell cycles and zygotic/embryonic gene expression in cattle

(Barnes & Eyestone, 1990)

 Techniques to study genome
activation

Subtractive hybridization
RT-PCR &
sequencing

DD-PCR
Array technology Methods to study & characterize
gene products

Quantitative or semi-
Si RNA knockdown
Quantitative RT-PCR
 How to relieve the
developmental block

Reduction of glucose in the culture

medium

Using co-culture systems

Addition of serum
(Gandolphi & Moor, 1987)
 Effect of glucose on
developmental block

By affecting salvage pathway

By generating ROS
Salvage pathway

(Dienhart et al.,1997)
 By generating
ROS

Glucose increases ROS

(Iwata et al., 1998)

High glucose induces embryo fragmentation &

apoptosis
(Moley et al., 1998)

High glucose decreases GSH hence induces

oxidative stress
(Krisher., 2004)
Interactions between glucose, purine
metabolism & ROS production
HK
Glucose Glucose 6-P

Pentose Phosphate Pathway

Purines
HPRT

Hypoxanthine
XO
O2-.
(Guerin et al., 2001)
Xanthine
 Co-culture

Granulosa cells
Bovine oviductal
VERO cells
cells
BRL cells
 What does co-culture do?

Embryotrophic factors are provided

(Gondolfi et al., 1989,1992)

Decreases glucose concentration

(Bavister, 1995)

Secretes GSH, hupotaurine & taurine

(Guerin & Menezo, 1995)

Reduces oxygen tension

(Fukui et al., 1991)
 Development of human embryos on vero
cells

Group Total Blocked or Cavitatin Expande Hatched

(%) degenerated g d

Control 31 30a (97%) 1 - -

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

(Menezo et al., 1990)

 Medium modification by somatic cells

Treatment Metabolite concentration (mM)

Glucose L-lactate Pyruvate

Controla 5.55±0.20 0.22±0.03 0.06±0.03

BOE cells 2.67±0.03c 2.92±0.35c 0.11±0.01c

BRL cells 3.73±0.19c 1.17±0.11c 0.17±0.08c

3T3 cells 3.31±0.15c 0.54±0.03b 0.08±0.01

(Edwards et al., 1997)

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)
 Pyruvate prevents peroxide-induced injury

H2O2 Pyruvate Zygote Cleaved 5-8-cell Blasto

(10-5) (0.3mM) (n) (%) (%) (%)
day 3 day 3 day 3
_ + 79 76±7a 35±5a 25±2a

_ _ 82 75±5a 32±4a 21±2a

+ + 73 68±4a 33±4a 21±2a

+ _ 78 58±1a 12±3b 8±1b

(Morales et al., 1999)

 OOCYTE Somatic ells secrete GSH,……..

SOD GCS GPx

EMBRYO
Stored transcripts

Enzymes

Hypotaurine OH., O2-.

Cysteamine
Stored transcripts
Follicular Fluid
Ascorbic Acid SOD, CAT, GPX

OH., O2-.

OH.,
Hypotaurine, Tubal fluids
Taurine
- O2-.

SOD, GPX,
- **
CSD GCS, Cat Metallic Ions
-
-
GSH
Transferrin,
Albumin
(Guerin & Menezo, 2001)
OVIDUCTAL EPITHELIAL
CELLS
 Effect of serum in kinetics of bovine
embryos
Presence of serum Cycle Cycle Cycle Morula to Compact
1 2 (hr) 4 (hr) compact morula to
IVM-IVF IVC (hr) morula (hr) early
blastocyst (hr)
In vivo - na 8 45 19 24
(+)

+ - 28 10 43 16 26

- - 32 10 49 16 22

+ + 27 9 39 11 13

P values <0.01

(Holm et al., 2002)

Serum addition significantly decreases the duration of the fourth cell cycle
during which maternal-embryonic genome transition occurs
 Conclusion

 The first hurdle in in vitro development of

embryos is developmental block

 It is species specific

 It occurs due to improper activation of

maternal to zygotic transcription

 It can be overcome by providing suitable

culture conditions
 Future Prospects

To explore the molecular mechanism of the

developmental block

To completely understand factors involved in

MZT

To completely explore the effect of serum

supplementation on embryonic development