Establishment IVG for Oocyte isolated from Early Antral Follicle &

Oocyte-Like Cell differentiated from Female Germline Stem Cells

Fully grown oocyte (GV)

GTH (FSH, LH)
Growing Oocyte
oocytes
Maturation
Antral follicle

Secondary follicles

Primary follicles

Primordial
follicles
Mature oocyte
Oocyte Growth (M II)
Non-growing
Pair of human ovaries: oocytes
302.000 primordial follicles 1
 Schema of fertility preservation approaches
in cancer patients

A
I.

II.

2
II. Surgical isolation Study of fertility preservation

Topic 1 Topic 2

❖ How to bring small oocyte

become fully grown and undergo
maturation, then fertilization to
make full term development?

❖ How to establish Female Germ

line Stem Cell and make them
become functional oocyte after
undergo growing, maturation
and fertilization
3
 Experimental design:
Antrum formation
A. Isolation preantral follicles from B. Establishment of pFGSCs from
ovarian tissue biopsies fresh and vitrified ovarian tissues
Tissue
Cumulu Ovarian tissue cryopreservatio
Cryopreservatio Ovarian tissue s cells Antral follicle biopsies
n biopsies n
Granulosa cells
Preantral follicle
Secondary follicle
Warming
Isolate Primary follicle Ovulatory Enzymatic
Warming preantral Primordial follicle follicle isolation
follicle

C. Isolation oocyte from antral follicle Establish

pFGSCs
Culture follicle
In vitro
Growing oocytes differentiat
Fully ion
Isolate oocytes and grown OLCs?
OCGs from follicle oocyte
In Vitro
Maturatio Mature
In vitro oocyte In vitro
Growth n Growth
In vitro Maturation In vitro Maturation
Check mature Check mature
Sperm
oocytes? OLCs? Fully grown
Fully grown oocytes? OLCs?
Zygote formation and Zygote
embryonic development?
4
 In vitro growth of oocyte from small follicle

2-3 mm follicle
??? ???

Growing
oocyte Fully grown
Mature
oocyte
oocyte

Ascorbic
acid BMP15 Developmental
Suppress competence

Pre-IVM Oocyte
ROS Oxidative stress
&IVM quality
5
 Theriogenology 2020 (SCI/Q1; IF: 2.33)

IVM: In vitro maturation Oocyte

Pre-IVM: Pre-maturation Ascorbic Acid (AsA)
2-3 mm follicles
Non pre IVM pre IVM
hours
15

4-6 mm follicles 2-3 mm follicles

AsA0 AsA25 AsA50 AsA100
(0 µg/ml) (25 µg/ml) (50 µg/ml) (100 µg/ml)
hours
22

IVM IVM hCG added

IVM IVM IVM IVM

hours
57

Positive control Negative control

Parthenogenetic activation
days
7

Embryo Development

Quantity & DAPI staining Immuno-staining

Quality analysis analysis analysis
6
 L-ascorbic acid improved developmental competence
and acetylation of H3-K9 of porcine embryos derived
from small follicle
DAPI Ac-H3-K9
SMALL FOLLICLE (S) LARGE FOLLICLE (L)
AsA 0

a b
AsA 50

90
d S0

INTENSITY OF H3K9-Ac
80 d
c d 70 c S50
60 bc
b
50 S100
AsA 100

40
a
30 L0
20
10 L50
0
e f Large L100
Small 7
follicles follicles
Development 2014 (SCI/Q1; IF: 6.1)

Bui HT* (First and Corresponding author)

8
 Materials and Methods

Ovarian tissue
Collagenase 1mg/ml
Trypsin 0.25%
Single cells

Gelatin coated dish

Overnight

Floating cells were recovered

and cultured in 4-well dish

MEM-α StemPro-34 DMEM-F12

Zou et al 2009 Pacchiarotti et al 2010 Bui et al unpublish
10% FBS 1% FBS 10%FBS
STO feeder STO feeder MEF feeder

9
 Comparison of basic culture media MEM-α,
StemPro-34, and DMEM-F12 for PSCs
Basal Growth Supplement
components factors (s)
Results
1. MEM-α -Cells proliferated without forming cluster.
Similar for
EGF -After 1st culture for 1 week, most of small cells (5–7 m)
mouse FGSCs 10% FBS dispersed separately, arrested and degenerated.
bFGF
By Zou et al.
GDNF
STO cell -After 1 month culture, somatic cell proliferation is faster
2009.
LIF
feeders than PSCs. Many somatic cell types appear: neuron-
like cell, myoblast-like cells, and epithelial cells.
2. StemPro-34 -Cell proliferated with forming small clusters (<10 cells).
Similar for -After 1st culture for 1week, most of small cell (5–7 m)
mouse FGSCs EGF
1% FBS
dispersed separately, arrested and degenerated, and few
By: FGF2
MEF cell
of them grew to 10-12 m.
Pacchiarotti GDNF -After 1 month culture, proliferation of PSCs was
feeders
et al. 2010 LIF slowly. Some of PSCs differentiated to oocyte-like
structure and some somatic cell types appear.

-Cell proliferated with forming clusters (<20 cells).

3. DMEM-F12
EGF -After 1st culture for 1week, a number of small cells (5–7
(concentration of
bFGF
10% FBS m) formed groups and grew to 10-12 m.
components as in
GDNF
MEF cell -After 1 month culture, PSCs proliferated and formed
Methods of Bui et feeders groups of cell. Many PSCs differentiated to oocyte-
al. unpublish) LIF
like structure and some somatic cell types appear.
10
Appearance of PSCs on 3 kinds of media MEM-α,
StemPro-34, and DMEM-F12
MEM-α StemPro-34 DMEM-F12
1week
1 month

DMEM-F12

2 days after subculture 1 week after subculture 11

 Development 2014 (SCI/Q1; IF: 6.1)

Bui HT* (First and Corresponding author)

Ov RT- 1 2 3 Differentiation to oocyte like cells
actin
ZP Oocyte
SCP3 markers
Oct4
Fragilis Germ
Blimp1 cell
Vasa markers
C-kit
DAZL
Nanog FGSC 1 2 3 4 GV
Sox2 Stem actin
REX1 cell ZP
C-MYC markers Oocyte ZPC
KLF4 markers SCP3
GDF9b
Vasa
Germ Blimp 1
cell Fragilis
markers C-KIT 12
Bright Merge Stella Fragillis
How to bring Female Germline Stem Cells (FGSCs)
become Oocyte-Like Cells (OLCs: egg)?

Isolated Co-culture with

FGSCs Granulosa cells
Gelatin coated dish

Low attachment plate

Gelatin-coated dish
Granulosa
cells

Co-culture with MEF

MEF feeder layer

Mitomycin C-treated MEF

Low attachment plate
feeder layer
MEF cells 13
13
 J. Reproduction and Development 2019 (SCIE/Q1)

2 weeks 4 weeks
a b

Condition 1
*
c d

Condition 2

e f

Condition 3

g h

Condition 4

14
 Expression of oocyte-specific markers in oocyte-like
cells (OLC) after differentiation.
Merge with DAPI DAPI Vasa ZP2
Merge DAPI VASA ZP2

OLC
DAPI SCP3
a b

OLC
Oocyte
c d

Merge Ac-H3-K9 GDF9 Merge Oocyte

OLC
DAPI DAZL
a b
Oocyte
OLC

c d
a
Merge Me-H3-K4 GDF9 Merge
Oocyte
OLC

Oocyte
15
 CONCLUSION
FGSCs isolation
Somatic & fibroblast
Ovarian tissue cells
Enzyme
digestion Different adhesion
overnight selection
FGS 1-day isolated FGSCs
Collagenase
Cs
Trypsin N21 free-serum
supplemented culture
Using FGSCs 1-week after isolation,
Key: where ovarian somatic cells still
remained in the culture, was 1 week 4 weeks
: Female germline stem cells
necessary to study in vitro Vasa
: Ovarian somatic cells differentiation into oocyte-like cells. Oct4
: Fibroblast cells Stella
C-kit

FGSCs differentiate into OLCs

OLCs OLCs
70 µm
40 µm
1-week isolated
FGSCs
2 weeks 4 weeks

MEF Co-culture in 3D low GDF9

attachment well ZP2
SCP3

Differentiation medium
DMEM/F-12 16
+FSH, +LH, +Estradiol, +dbcAMP