Experiments

in vitro growth and

in vitro maturation
Bui Hong Thuy, Ph.D.
Associated Professor
School of Biotechnology,
International University
Email: bhthuy@hcmiu.edu.vn
1
Why need to study in vitro growth
of oocyte?
Food-consuming

Long-term feeding

Excellent quality

Consume less food

Grow fast
2
In vivo growth of Large number of growing
oocyte? oocytes from early antral
follicles (~ 84,000 growing
follicles /pair of ovaries in pig)

In vivo growth

Very few number of oocytes

undergo fully grown phase
In vivo
Maturation
Offsprings

3
 In vitro growth Large number of growing
(IVG) of oocyte? oocytes from early antral
follicles (~ 84,000 growing
follicles /pair of ovaries in pig)

In vitro growth

Increase large number

of fully grown oocytes

In vitro maturation
In vitro fertilization
Increase number of
embryos

Embryo transfer
Increase number of
offsprings 4
Numbers of ovarian follicles in mammals

No. of primordial No. of growing

Species follicles follicles
Mouse 4,270 676
Sheep 105,450 475
Cow 120,000 300
Pig 420,000 84,000
Human 302,000 12,090
Mean number per pair of ovaries (Gosden and Telfer, 1987; Erickson, 1966)

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 The size of oocytes
Primordial Fully grown
follicles follicles

Mouse Oocyte size

15 ｰ 20 µm
Oocyte size
75 – 80 µm

30 µm 120 ｰ 125 µm

Cow
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Schematic of the key stages of oocyte

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Oocyte growth and maturation in the ovary
Fully grown
oocyte (GV)
GTH (FSH, LH)
Growing Oocyte
oocytes
Maturation
Antral follicle

Secondary follicles

Primary follicles

Primordial
follicles
Mature oocyte
(M II)
Oocyte Growth
Non-growing
oocytes
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➔ Collect the growing oocytes for IVG and IVM culture

In vitro
growth

In vivo growth

9
What factors control oocyte during
growing and maturation phases ?

??? ???

Growing
oocyte Fully grown
Mature
oocyte
oocyte

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 Components of the growth phase

Follicle Stimulating
Hormone (FSH)

Estradiol 17β

Androstenedione

Follicular fluid

Cyclic adenosine
monophosphate Pre-Antral follicles
(cAMP) Growing oocytes
Antral follicles
Antral Follicle
……….
(4-6mm in diameter)
=> Fully-grown oocyte
(120µm)
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 Components of the growth phase

Follicle Stimulating
Hormone (FSH)

Estradiol 17β

Androstenedione

Follicular fluid

Cyclic adenosine
monophosphate Prevent nuclear breakdown and increase
(cAMP) intracellular levels of cAMP
=> Maintain the oocyte growing phase
and inhibit oocyte maturation

12
◎Androstenedione is an androgenic steroid produced by the
testes, adrenal cortex, and ovaries. They are also the parent
structure of estrone.

FSH

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 ❖ Increase oocyte size.
Follicle stimulating ❖ Stimulates proliferation and
hormone differentiation of granulosa cell.
(FSH) ❖ Maintained the cumulus cells attached to
oocytes through transzonal projections.

Androstenedione
(A)
❖ Contribute to granulosa cell differentiation
and follicle development.
❖ (A) added in the medium might have
compensated for the lack of theca cell.

❖ Secreted from granulosa cells – support the

Estradiol 17β proliferation and of cells surrounding oocyte.
❖ Promotes oocyte growth.
❖ Improve meiotic competence.
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 ❖ Exogenous nucleotide derivative.
Dibutyryl cyclic ❖ Mimics functions and increases
adenosine monophosphate concentration of endogenous cAMP.
(dbcAMP) ❖ Inhibit nuclear breakdown to prevent
oocytes mature and keep in growing.

Follicular fluid

❖ Provides the microenvironment for

oocyte development.
❖ Supplies macromolecules (ex: growth
factors, hormones, vitamins,…).

❖ Effect antrum formation of the oocyte

Polyvinylpyrrolidone granulosa cell complexes (OGCs).
(PVP) ❖ Effect the adhesion properties of granulosa
cells.
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 Antrum formation
A. Isolation preantral follicles
from ovarian tissue biopsies
Cumulus
Cryopreservation Ovarian tissue cells Antral follicle
biopsies
Granulosa cells
Preantral follicle
Secondary follicle
Isolate Primary follicle Rupturing
Warming preantral Primordial follicle follicle
follicle

B. Isolation oocytes from

Culture follicle Antral Follicle

Growing oocytes
Isolate oocytes and
OCGs from follicle Fully grown
oocyte
In vitro
Growth IVM Mature
In vitro Maturation oocyte
Check mature Zygote
oocytes?
Fully grown oocytes? Sperm Zygote formation and
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OCGs: Oocyte-Cumulus-Granulosa cells complexs embryonic development?
 IVM

B Dissect ovary Large Antral Fully grown oocyte

to isolate (Oocyte-Cumulus- Mature oocyte
follicle with
follicle 4-6 mm diameter of Granulosa cell
4-6 mm Complexes; OCGCs)
??? IVM
A Preantral follicle 0.5-1 mm

IVG ???

Culture preantral Isolate Oocyte-

follicle for 2 weeks Granulosa cell Floating OCGCs from
(Embedding in complexes (OGCs) drop preantral follicle
Collagen Gel) from preantral follicle after IVG
and culture for 2 weeks 17
Cortical slices were cut
 In vitro growth of oocyte, in vitro maturation
and in vitro fertilization

Growth Maturation Fertilization

Non-growing Growing Full-grown

Oocytes Oocytes Oocytes
Mature Zygotes
Immature Oocytes
Oocytes

IVG IVM IVF

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Produce offspring from growing oocytes

Ovarian small oocytes

Vitrification

IVG Xenotransplantation

Artificial control of
follicular selection
and development IVM ｰ IVF ｰ ET 19
 Process of performing floating drop
technique and culturing growing oocytes

Making drops of culture medium Covering drops with mineral oil

Growing oocytes

Culturing growing oocytes Adding more culture medium

on the top of the drops on the top of the existing drops

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Nuclear morphology of oocytes isolated from follicles of
various sizes

0.2- <1

1 - <2

2 - <3 Denuded
oocytes
Fixed
Pig ovary
3 - <4 and
stained
with 1%
orcein
4 - 6 mm
Cumulus-oocyte-
Antral follicles granulosa cell
complexes
(OCGs) 21
Chromatin morphology of in vivo
growth oocytes
FC SC GVI

Filamentous chromatin Stringy chromatin Germinal vesicle

22
Chromatin morphology after in vitro
growth of oocytes

chromosomal
10 µm
FC SC GV I

Filamentous Chromatin: FC
Stringy Chromatin: SC
Germinal Vesicle: GV
Early Diakinesis: ED GV II-IV ED
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Chromatin morphology after in vitro
maturation of oocytes

GV I GV II-IV Diakinesis
1st polar body

10 µm
MI AI-TI MII
Metaphase I : MI Anaphase I : AI Metaphase II : MII
Telophase I : TI 24
 Preparation and collection of pig
oocytes from early antral follicles

Pig ovaries Cortical slices were Early antral follicles were

were washed 3 cut seperated (1-1.5 mm )
times with PBS

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 In vitro growth culture of oocytes
HEPES
1 medium

3 2

DMEM
medium
Culture in 96-wells culture plate in basic medium
Wash twice in HEPES (BM) or BM+A under 5% CO2 and in 38.5oC
and once in DMEM

Staining with aceto-orcein After 3 days, oocytes

were denuded for fixing
*Basic medium was supplied by Bicarbonate - buffered DMEM (Dulbecco’s Modified Eagle Medium, Sigma)
supplemented with 0.1 mg/ml sodium pyruvate, 0.1µg/ml penicillin, 0.05 µg/ml streptomycin, 1 mM dbcAMP,
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0.01 IU/ml FSH and 5% fetal bovine serum (FBS), 0.01 µg/ ml estradiol-17β (E).
Le Kim Thoai Sem. I, 2015
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Pig oocytes isolated from 1-1.5 mm follicle
after in vitro growth for 3 days
a1 a2 a3

b1 b2 b3

0 day 1 day 3 days

Morphology of pig COCs collected from early antral follicle during growth culture. (a2,
b2) OGCs grown in the growth medium have promoted a thin layer and some of them
have 2-cell layer after 1 day culture. (a3, b3) After 3 days, these complex shows
development throughout the expansion of cumulus which reach to more than 3-cell
layer. Scale bar = 90 µm
Le Kim Thoai Sem. I, 2015 27
 In vitro growth of oocytes isolated
from 2-3 mm follicle (IVG)

Cumulus cells were

removed for fixing and
staining to observe
chromosome morphology

Oocytes were cultured

for 24 hours at 38.5°C
and 5% CO2

Culture medium: DMEM, 0.1mg/ml Sodium pyruvate, 0.1mg/ml Penicillin, 0.05

mg/ml Streptomycin, 10% fetal bovine serum (FBS), 5% follcilular fluid, 1mM
dbcAMP, 0.01IU/ml FSH and 0.001mg/ml 17β-Estradiol

Le Thu Hoai Sem. I, 2015 28

Pig oocytes isolated from 2-3 mm follicle
after in vitro growth for 1 days

The proliferation of cumulus cells after IVG

Figure 3.1.1: Porcine OCGCs (a) were collected from preantral follicles (2-3
mm) and cultured for 24 hours of in vitro growth (b). The degree of cumulus
cell layers were observed as (a) 1-cell and 2-cell layer, (b) 3-cell and more than
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3-cell layers. Scale bar is 110µm

Le Thu Hoai Sem. I, 2015 29

 Pig oocytes isolated from 1-1.5 mm
follicle after IVG for 4 days
Floating drop 96 well plate a a2 a3
1

b1 b2 b3

0 day 2 days 4 days

 Oocytes cultured in floating drop had better proliferation of
cumulus-granulosa cell complexes than 96 well culture plate in
both treat and untreated-Androstenedione group
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Pham Khanh Linh Sem. II, 2015
Morphology of in vivo an in vitro growth pig oocytes

Oocyte can reach to fully-grown stage after in vitro culture (a, b, c) showing the
similar chromatin morphology at FC, SC and GV stage as in vivo growth (d, e,f)
(B) Cumulus-oocyte complex after isolate from ovary (a) and in vitro (b) culture.
The number of oocyte surrounding cells increased significantly (c)
Bao Tran et al. BME. 2016 31
 Meiotic competence of oocytes after
in vitro growth and in vitro maturation

The morphologiesinclude stringy chromatin (SC, a), germinal vesicle I (GVI,

b), germinal vesicle II-IV (GVII-IV, c), diakinesis (D, d), metaphase I (MI,
e), anaphase I-telophase I (AI-TI, f) and metaphase II (MII, g).
Thu Hoai et al. BME. 2016 32
 How to improve meiotic
competence of porcine oocyte?
Problems: Heterogeneity of cytoplasmic and nuclear stage

Granulosa cell

Cumulus cell

Low
Aspiration
cAMP Developmental
competence of
Embryo
4-6mm antral follicle Fully grown oocyte
cAMP ↑ cAMP ↓
Meiotic arrest Meiotic resumption
Spontaneously
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Solution: Synchronization of nuclear and cytoplasmic
progression by inhibition of meiotic resumption using
dbcAMP
dbcAMP
20 hrs 21 hrs Parthenogetic
Embryo
development
activation
Pre-IVM Extend-IVM ??????

↑
dbcAMP 34
 Effect of dbcAMP on meiotic competence

1st pb

GV MI AI TI MII

Examined No. (%) of oocytes at

IVM treatment
(n) GV MI AI-TI MII
Basic medium (Control) 87 41 (45.0) 10 (11.8) 3 (4.0) 33 (39.2)

Basic + dbcAMP 0.5mM 129 36 (28.0) 23 (17.7) 6 (4.9) 64 (49.4)

Basic + dbcAMP 1mM 95 32 (33.2) 5 (6.2) 10 (7.4) 48 (50.3)

Pham Truong Duy Sem. I, 2015

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Experimental design:

Pre-IVM Extend-IVM
Experiment : Evaluate the effect of dbcAMP and FSH on cumulus
expansion and meiotic competence. 36
Truong Binh An Sem. I, 2015
 Basic Basic +dbcAMP Basic +dbcAMP
+FSH 0.01IU/mL +FSH 0.02IU/mL

Cumulus expansion of fully grown oocyte after IVM

IVM treatment Examined No. (%) of oocytes

(n) Expand Not Expand
Basic 241 114 (47.5)a 127 (52.5)a
Basic + dbcAMP 1mM + FSH 337 248 (74.2)b 89 (25.8)b
0.01IU/ml
Basic + dbcAMP 1mM + FSH 333 219 (65.9)b 114 (34.1)b
0.02IU/ml
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Truong Binh An Sem. II, 2015
 Effects of dbcAMP and FSH on meiotic competence

IVM treatment Examined No. (%) of oocytes

(n) GV MI AI/TI MII
Basic 256 76 (29.7)a 19 (7.0)a 13 (4.4)a 148 (58.9)a
Basic + dbcAMP 1mM 347 57 (16.6)b 20 (5.7)a 4 (1.2)a 266 (76.3)b
FSH 0.01IU/ml
Basic + dbcAMP 1mM 158 25 (16.0)b 9 (6.0)a 5 (3.6)a 119 (74.4)b
FSH 0.02IU/ml

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Truong Binh An Sem. II, 2015
 Examine combination effects of
dbcAMP and FSH on in vitro maturation
through pronuclear formation

The combination of dbcAMP and FSH

significantly promoted oocyte maturation
in vitro and normal fertilization in pig

Thuy Van et al., BME 2016

39
Effects of dbcAMP and FSH on the
developmental competence of embryos
Partheno-
activation

Germinal vesical (GV) Germinal vesical break down (GVBD)

20 hours 21 hours
1. Basic medium (control)
2. Basic 2. Basic medium +
medium + 1mM 0.01 IU/ml FSH
dbcAMP + 0.01
IU/ml FSH
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 Aging porcine oocyte
Maturation Mitogen Activated
Promoting P
Protein Kinase
Factor p34
cdc2
P (MAP kinase)
(MPF) MAPK
cyclin B P

Oocytes Maturation
GV
Polyspermy

Aging

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❑ Caffeine (1,3,7 –trimethylxanthine)
✓ A phosphordiesterase inhibitor
✓ Inhibit Myt1/Wee1 kinase activity 42
Caffeine reduce polyspermy in
aging porcine oocyte
Polar body Pronuclei
Pronuclei Arrested sperm

Pronuclei
Monospermic Polyspermic Monospermic
Metaphase II
pronucleus pronucleus pronucleus
Arrested
sperm Female
❖Monospermy: only one sperm Pronucleus
penetrated the oocyte
❖Polyspermy: more than one sperms
entered the oocytes
Polar body

43
 Caffeine promote developmetal
competence of aging porcine oocyte

The morphology of porcine aging oocytes. Typical morphology of 2-cell and 4-cell
embryos developed from A-B) Aging oocytes and C-D) Fresh oocytes. Scale bar= 35 μm.

Preimplantation development
of parthenogenetic porcine
embryos derived from aging
oocytes treated with caffeine.
A) Two-cell embryo. B) Four-
cell embryo. C) Eight-cell
embryo. D) Morula. E) Early
blastocyst. F) Blastocyst. Scale
bar= 35 μm.
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In vitro maturation
of Pig Oocytes
Bui Hong Thuy, Ph.D.
Associated Professor
School of Biotechnology,
International University
Email: bhthuy@hcmiu.edu.vn

45
Different types of porcine ovaries
from prepubertal gilts

A) Two ovaries (white arrows) from each prepubertal gilt. B) Ovary with
follicles during proestrus/ estrus (Superovulation). C) Ovary in luteal
phase (The black arrows pointed at the corpus luteum). D) Ovary with
small follicles during early proestrus (2-3 mm and 3-4 mm). E) Ovary
with hemorrhagic ovarian cyst. F) Ovary in follicular phase with large
follicles (4-6 mm). Scale bar= 15 mm.
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Effects of oocyte collection methods on the meiotic
competence of porcine oocytes

ASPIRATION DISSECTION
47
Effects of oocyte collection methods on the meiotic
competence of porcine oocytes

100
90
84.2
b
80 Oocytes collected by
70
PERCENTAGE

57.39
a dissection method
60
50 a
matured at a
40 significantly higher rate
30 compared to those
20 b
collected by aspiration
10
0
method.
Arrested MII

Aspiration Dissection

Dissection method was used to collect oocytes for the

Cellular Reprogramming Lab. 48
 MATERIALS AND METHODS
OOCYTE
COLLECTION

8mm 8mm 8mm

IN VITRO
MATURATION

DENUDED
OOCYTES
OPTIMIZE THE COLLECTION METHOD 49
Collection and in vitro culture for mature of
pig oocytes

Ovaries were collected Follicles 4-6 mm in

from abattoir Dissection diameter were
dissected from
ovaries

Collection of oocytes : *Aspiration Oocyte-cumulus- Oocyte-cumulus-

*Dissection
granulosa complexes complexes (OCGCs)
(OCGCs)
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51
In vitro culture for maturation of pig oocytes

38-42 h
OCGCs Culture medium: Mature oocyte
(M II)
TCM 199 + 10% FCS + 0.1 mg/ml
Na pyruvate + 0.08 mg/ml kanamycin
+ 0.1 IU/ml hMG
First polar body First polar body

Chromosome
52
 MATERIALS AND METHODS
OPTIMIZE THE ACTIVATION SYSTEM OPTIMIZE THE CULTURE MEDIA

ACTIVATION MACHINEACTIVATION
CHAMBER

OPTIMIZE HDACI (SCRIPTAID) TREATMENT

5x

ASSESSMENT

MEIOTIC DEVELOPMENTAL QUALITY 53

HISTONE
OOCYTE ACTIVATION COMPETENCE COMPETENCE OF EMBRYO MODIFICATION
Effects of different concentrations of FBS on the
preimplantation development of porcine embryos

A) Development of
blastocyst at day 8
from embryos treated
with different
concentrations of FBS :
0, 5, 10 and 15 %.
B) The development
rates were recorded at
two-cell, four-cell,
eight-cell, morula,
blastocyst, late-to-
hatched blastocyst with
different concentrations
of FBS

54
Effects of different timings of FBS supplementation on
the quality of porcine parthenogenetic diploid embryos

A) Development of blastocyst at day 6 and day 8 from embryos treated

with different timings of FBS supplementation: 0, Day 3, Day 4 and Day
5. DNA was stained with DAPI (blue) B) Blastocysts at Day 6 (a) and
hatched blastocysts at Day 8 (b). Scale bar = 200 µm.
55
 Effects of different timings of FBS supplementation on
the preimplantation development of porcine cloned embryos

56