HEAT SHOCK DURING IN VITRO

MATURATION INDUCES CHROMATIN

MODIFICATIONS IN THE BOVINE EMBRYO
Luiz Sergio Almeida Camargo1, Tiphaine Aguirre-Lavin2, Pierre Adenot2,
Thamiris Dornelas Araujo3, Vivian Rachel Araujo Mendes4, Iuri Drumond Louro5,
Nathalie Beaujean and Eliza Diniz Souza

JOURNAL HUB 2020-2021

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 Animal Biotechnology
Group 02

Lê Nguyễn Lam Ngọc Nguyễn Thị Trang

BTBTIU18345 BTBTIU18248
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 CONTENTS

I. Introduction
II. Materials and methods
III. Results and discussion
IV. Conclusion

In vitro mature bovine oocytes. American Society of Animal

Science. Image of the Week (November 9th, 2017). 3
I. INTRODUCTION

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o Heat shock can disturb the physiology of the follicle-enclosed oocyte
during follicular development.

Low developmental competence in different stages of bovine embryos.

o One of the little-studied effects of heat stress on oocytes is

epigenetic modification.

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Epigenetic modifications in bovine
Epigenetic modifications are changes on chromatin structures, gene
expression, not change on DNA => No heritage

• Heterochromatin Gene expression

• Euchromatin Gene expression

The major embryonic Chromatin

Healthy embryos
genome activation (EGA) remodeling

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H3K9me3 and HP1
At 8-cell stage

Disturb the EGA, impair

embryonic development
H3K9me3 HP1

Transcriptional
repression  H3K9me3: : histone H3 lysine 9
trimethylation
HP1: heterochromatin protein 1
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RESEARCH HYPOTHESIS

o One the consequences of heat shock on chromatin remodeling of

early-stage bovine embryos which interferes with embryonic
genome activation (EGA) and further embryonic development
into blastocysts. 

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RESEARCH PURPOSE
Evaluate the effect of heat shock during oocyte in vitro maturation
on development, gene expression and chromatin organization of in
vitro fertilized embryos.

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II. MATERIALS AND
METHODS

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Materials
All materials are purchased from the Sigma-Aldrich

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Methods

01 02
Evaluation of developmetal Chromatin organization of
competence 4- and 8-cell embryos

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Gene expression of
8-cell embryos
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 Bos indicus In vitro maturation
Ovaries Follicles + COCs

Randomization

Control group Heat shock

group
IVM IVM
24h at 38.5°C in TCM-199 + 10% • 12h at 41.5°C under 7% CO2
estrus cow serum + 20μg/mL FSH in • 12h at 38.5°C under the same
5% CO2 + humidified air. conditions of control group

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1. Evaluation of developmental competence of heat-
shocked oocytes
IVC Record cleavage rate on
IVF Day 3, blastocyst rate on
Day 7 and 8
CR2aa medium +
20h in Fert-TALP, n=470
2.5% fetal calf serum
5% CO2 , 38.8°C
at 38.5°C , 5% CO2,
5% O2, 90% N2,
humified air Blastocysts
Keys
COCs – Cumulus-oocyte complexes
Record cell number, TUNEL
Sperms apoptotic index labeling

Zygotes
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2. Chromatin organization of 4- and 8-cell embryos

IVF, IVC Embryos Immunofluorescence

52h post fertilization at 4 analysis of H3K9me3
and 8 cell embryos (n=58) and HP1

Image analysis of Confocal

H3K9me3 & HP1 microscopy

(chromatin compaction
patterns)

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 3. Gene expression of 8-cell embryos
IVF, IVC Wash in PBS
8-cell Embryos Frozen, store
at -80°C

RNA
Real–time PCR extraction (3 pools of 10
Data analysis
embryos/group)

The volume of cDNA for every

primer was calculated
(YWHAZ, GAPDH, ACTB, HSP40,
YWHAZ, GAPDH, ACTB: OCT4, HSF1 and HSP90A1 genes)
endogenous reference 16
III. RESULTS AND
DISCUSSION

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 01 02
Total cell number and apoptosis Chromatin structure analysis of
in different embryonic stages early-staged embryos

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Transcriptional levels
in 8-cell embryos
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1. Total cell number and apoptosis in different
embryonic stages

Reduction in the percentage of blastocysts in Day 7, Day 8 and hatched blastocysts

 Embryo quality.

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1. Total cell number and apoptosis in different
embryonic stages

Heat exposure results in molecular

alterations, which consequently
affect embryo quality and cellular
apoptosis.
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2. Chromatin structure analysis of early-staged
embryos

Up-regulation of H3K9 trimethylation with aggregation of spots in 4-cell embryos.

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2. Chromatin structure analysis of early-staged
embryos
Figure A: representative merged images
of immunofluorescence in 4 and 8-cell
stages embryos.
Figure B: the patterns of
chromatin compaction at 4-cell stage
a, a’. Spread
b, b’. Fibrous
c, c’. Globular
Green: HP1
Red: H3K9m3

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In 4-cell embryos
H3K9me3: high in globular pattern
HP1: high in fibrous pattern

Heterochromatinization in
normal 4-cell embryos represses
some genes in the embryonic gene
activation (EGA)

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3. Transcriptional levels in 8-cell embryos

Down-regulation of HSP40 –
a co-chaperone protein
associated with heat stress
resistance.

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IV. CONCLUSION

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o Heat shock can affect H3K9me3 and HP1 accumulation in 4-cell and 8-
cell embryos which has a negative effect on EGA.

o Oocyte quality and embryonic development can be interfered with gene

expressions associated with chromatin alterations.

o Understanding cellular, molecular and epigenetic process will be helped

to investigate how heat stress affects bovine reproduction and find out
effective solutions.

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 New insights
o Conduct research about heat stress on bovine oocyte and determine
important proteins involving in EGA for further studies.

o Make in vitro heat-resistant bovine embryos to estimate their survival and

quality, thus provide heat-tolerant breeds.

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Thank you for listening!

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