In spite of great scientific breakthrough for
Positive effects of RA on IVM of cumulus oocyte complexes
have previously been described (
In this experimental study, the animals were kept under controlled conditions (12 hour light: 12 hour dark), fed with water ad libitum. All procedures were performed in accordance with the approval of the Institutional Animal Care and Use Committee at the Kurdistan University of Medical Sciences (MUK, Iran). All reagents were purchased from Sigma-Aldrich Co, USA.
Outcome of oocytes IVM in different groups
|Group||GV numbersn||Arrested GV Mean ± SD||Degenerated GV Mean ± SD||GVBD Mean ± SD||MII Mean ± SD|
|Control||110||39.33 ± 2.08||10 ± 4||30.33 ± 1.52||31.66 ± 1.52|
|sham (ethanol)||120||41.33 ± 1.15||13.66 ± 2.30||34 ± 1.73||32.66 ± 0.57|
|bFGF||115||18.33 ± 1.52||12.33 ± 1.15||44.66 ± 3.21||40.66 ± 2.30|
|RA||125||16.33 ± 0.57||4.33 ± 0.57||47 ± 2||58 ± 1$,@|
|bFGF+RA||120||17.33 ± 2.30||6.33 ± 0.57||38.66 ± 1.15||57 ± 3.46*, #|
*; P<0.03 vs. bFGF, #; P<0.02 vs. control, $; P<0.02 vs. bFGF, @; P<0.001 vs. control and sham, IVM; In vitro maturation, GV; Germinal vesicle, GVBD; GV break down, MII; Miosis phase II, bFGF; Basic fibroblast growth factor, and RA; Retinoic acid.
Animals were superovulated by an intraperitoneal injection
of 10 IU pregnant mare’s serum gonadotropin (PMSG).
Mice were sacrificed 44 hours later by cervical dislocation
and their ovaries were placed in a-MEM culture medium
supplemented with 10% fetal bovine serum (FBS). Immature
oocytes in the germinal vesicle (GV) stage were mechanically
dissected using 26-G needles attached to a 1 ml
syringe under a stereo microscope (Olympus, Japan). The
collected GV-stage oocytes obtained from 2-months-old
NMRI mice were randomly divided into control, sham and
three experimental groups (
The collected GV-stage oocytes of each group were placed in 25 µl drops of maturation medium consisting of a-MEM supplemented with 10% FBS, 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ml epidermal growth factors (EGF), and then they were incubated in a humidified atmosphere of 5% CO2 at 37°C for 24 hours.
In the first experimental group, maturation medium was
incubated with 2 µM RA dissolved in pure ethanol (
Sperms of 12-weeks-old male NMRI mice were collected
from the tail of epididymis. Sperm suspension (1×106
motile spermatozoa/ml) was capacitated for 1 hour in 500
µl human tubular fluid (HTF) culture medium.
Data were analyzed using One-way ANOVA with a post- hoc Tukey and presented as mean ± SD. The differences in the values of maturation, fertilization and developmental rates were considered significant at P<0.05. All computations were carried out using SPSS 16 for Windows.
Development of oocytes from GV break down
(GVBD) to two-cells stage has been shown in in the
Figure 1. The maturation rate of cultured GV-stage oocytes
was low in both control and sham groups with
31.66 ± 1.52 and 32.66 ± 0.57, respectively. As compared
with the control group, the rate of maturation
was significantly increased in the RA (P<0.001) and
bFGF+RA (P<0.002) groups with 58 ± 1 and 57 ± 3.46,
respectively. The rate of maturation was significant in
the RA (P<0.02) and bFGF+RA (P<0.03) groups compared
to the bFGF group (
Data from Table 2 showed that the bFGF+RA group had
a higher rate 83 ± 1.52 (47.7%) of two-cells development,
compared to the control 33 ± 1 (34%) (P<0.001). The number
was significant in the bFGF+RA group in comparison
with the bFGF (P<0.001,
The number and percentage of oocytes attaining the two-cells stage after 24 hours of culture
|Group||Number of MIIn||Number of two-cells stage Mean ± SD (%)|
|Control||95||33 ± 1 (34)|
|sham (ethanol)||65||20 ± 0.57 (30)|
|bFGF||122||51 ± 1 (41)#|
|RA||174||58 ± 0.57 (50)*|
|bFGF+RA||116||83 ± 1.52 (47.7)*|
*; P<0.001 vs. bFGF, sham and control, #; P<0.001 vs. all groups, MII; Miosis phase II, bFGF; Basic fibroblast growth factor, and RA; Retinoic acid.
Oocytes in various stages of development. A. Germinal vesicle break down (GVBD), B. GV, C. Mature oocytes with polar bodies, and D. Two-cells stage.
In the present survey, we compared the effect of RA and
bFGF on maturation of mouse oocytes and their further
development into two-cells stage. We found that separate
usage of either RA or bFGF in basic culture medium could
improve outcomes of IVM. Achieving an efficient culture
system for IVM is an important criterion in reproductive
research. The advantageous roles of retinol metabolites in
bFGF has been known as an oocyte competency factor
due to its formation from theca, granulosa and cumulus
cells throughout folliculogenesis (
Our findings showed beneficial effects of 2 µM RA and 20 ng/ml bFGF on mouse oocyte IVM.