Part II (Assoc. Prof.

Thuy) – 25 pts
Answer for question: How to bring small oocyte to become fully grown oocyte, undergo
meiosis to fertilize with sperm, and develop to embryo?
1) Design experiment using one factor: antioxidant (Chemical: Vitamin C or
Astaxanthin…) or different timing to culture small oocyte (2-3 mm follicle) to
become fully grown oocyte (4-6 mm follicle). Then these oocytes undergo meiotic
division to become functional gametes through investigation of embryos
development? (15 points)
Collect bovine oocytes and divide them into two groups, according to the data in the
+ Group 1: 4-6 mm large antral follicle
+ Group 2: 2-3 mm small antral follicles (S(-); SC; and S)

 S(-): Oocytes derived from small antral follicles and cultured for 42 hours in IVM
medium.
 SC: Oocytes derived from small antral follicles were pre-matured for 22 hours with
dbcAMP and L-ascorbic acid concentrations of 0, 50, and 100mg/mL, and then
continuously transferred to IVM medium for 42 hours.
 S: Oocytes derived from small antral follicles were pre-matured for 22 hours with
dbcAMP and L-ascorbic acid (0, 50, and 100mg/mL), and hCG was added to the
culture medium during the last 7 hours of pre-IVM (15 h-22 h), and then
continuously transferred to IVM medium until a total of 42 hours of in vitro
maturation was reached.
 L: For 42 hours, oocytes from large antral follicles were cultured in IVM medium.
During the first 22 hours of culture, L-ascorbic acid was either supplemented or not.

Pre-IVM medium: alpha-MEM medium, 5% Fetal Bovine Serum (FBS), 5% porcine

follicular fluid (pFF), 0.91 mM Sodium Pyruvate, 0.01 IU/mL FSH, 1mM dibutyryl-cyclic
adenosine monophosphate dbcAMP, 0.28 ugM Androstenedione, 1.8uM Estradiol-
17beta.
IVM medium: TCM-199 medium, 10% FBS, 10% pFF, 0.1 mM Sodium Pyruvate, 10 IU/mL
hCG.
Activation medium: 0.3 mM mannitol, 0.01% (w/v) PVA, 0.05 mM CaCl 2, 0.1 mM MgSO4,
Embryo culture medium consists of NCSU23 medium, Cytochalasin B (first 6 hours), 10x
essential amino acids, 50x nonessential amino acids.
Immunostained dyes’ components include rabbit polyclonal anti-acetyl-histone H3-K9
(primary AB), rabbit polyclonal anti-dimethyl-histone H3-K4 (upstate), Alexa-Fluor-488-
labelled goat anti-rabbit IgC (secondary aB).
Independent variable: L-ascorbic concentration
Dependent variable: meiotic competence and developmental competence

After 57h, test the quality of oocytes: 

 o Cell number of blastocyst 
o Histone acetylation 
o Histone methylation

Small antral follicle oocytes had fewer cumulus cell layers and were smaller in size than
large antral follicle oocytes. The maturation rate of oocytes in the negative control group (S-
group) after IVM was significantly lower than the maturation rates of oocytes derived from
large antral follicles (L-group) and small antral follicles (S-group).
L-Ascorbic acid supplementation at 50 and 100 ug/mL significantly increased the proportion
in all stages of embryos derived from small antral follicle-derived oocytes when compared to
the non-treated group. L-Ascorbic acid supplementation thus promotes the developmental
competence of embryos derived from small antral follicles.
Furthermore, L-Ascorbic acid supplementation had a significant positive effect on the quality
of blastocysts formed from small antral follicles as well as the global level of acetylation of
H3K9 and methylation of H3eK4 in blastocysts formed from small follicles.
 2) What is the difference between a control group and an experimental group? How to
compare the difference between these experimental groups? (5 points)
- The control group is the group in which the independent variable is not altered; the control
group is only used to compare to the experimental group. The control group is divided into
two categories: positive control (conditions that are guaranteed to produce a positive result)
and negative control (conditions that produce negative outcomes to identify outside
influences).
- The experimental group is the group of test units that are exposed to a change in the
independent variable. In other words, the treatment group receives treatment, whereas the
control group does not.
To compare the differences, we look at the effect of a treatment in control versus
experimental groups. Except for the variables on which we want to see effects, we must
hold all other variables constant. We can compare different levels of a treatment to the
condition of no treatment at all and draw conclusions about the efficacy of each treatment
level, as well as whether the effects are greater or lesser than the no-treatment control, and
whether the effects are truly the result of those variables and no other noise factors.

3) Make conclusion to describe the best condition to obtain perfect oocyte. (5

points)
Despite numerous modifications to IVM culture, only a small proportion of growing oocytes
can reach metaphase II and subsequently improve developmental competence because
failure in cytoplasmic maturation at the latter stage of oogenesis causes oocytes to fail to
complete meiosis. As a result, oocytes from large antral follicles have a greater
developmental potential to reach the blastocyst stage than those from small antral follicles.
In IVM medium, L-Ascorbic acid serves as an antioxidant, lowering Reactive Oxygen Species
(light, oxygen concentration, medium ingredients, etc.) while increasing glutathione (GSH)
levels in mature oocytes. Also, L-Ascorbic acid is an epigenetic factor that causes global
epigenetic reprogramming by increasing H3K9-Ac and H3K4-Me2. As a result, L-Ascorbic acid
boosts DNA replication and transcription by altering chromosome structure and facilitating
transcription factor access.