Effect of preantral mouse follicle culture period on meiotic maturation and developmental competence of oocytes
ABSTRACT PurposeThe aim of this study is to determine the optimal culture period for meiotic maturation and developmental competence of invitro-grown
MethodsEarly preantral follicles with diameter of 100–130μm were collected mechanically from day14 mouse ovaries and cultured for
8, 10, and 12days. The diameters of follicles and oocytes were measured, and chromatin configuration in oocytes was observed.
We also examined meiotic maturation by human chorionic gonadotropin (hCG)/epidermal growth factor (EGF) stimulation, developmental
competence of fertilized oocytes to blastocysts, and apoptosis in blastocysts.
ResultsThe follicular diameter increased significantly from days4 to 10, and the diameter of day12 oocytes was significantly larger
than day8 or earlier oocytes. Chromatin configuration around the nucleolus was transformed from “nonsurrounded (immature)”
to “surrounded (mature)” after 10days. Furthermore, MII rate of day10 and 12 oocytes was significantly higher than that
of day8 oocytes. The blastocyst rate of day10 oocytes was higher than that of day8 or 12 oocytes. The blastocyst apoptotic
rate of day12 oocytes was higher than that of day10 oocytes.
ConclusionsLong culture periods of invitro-grown oocytes affect meiotic maturation, developmental competence to blastocysts, and apoptosis.
KeywordsApoptosis-Culture period-Invitro growth-Mouse-Preantral follicle
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ABSTRACT: To analyze the effects of combined FSH and variable doses of LH on the nuclear maturity and capacity to resume meiosis in oocytes from preantral follicles from prepubertal mice. Prospective, randomized, and controlled in vitro laboratory experiment. Academic research environment. Meiosis was studied after somatic cell removal or after stimulation with hCG plus epidermal growth factor in three culture conditions: maturation medium with FSH alone and with two different doses of LH. The nuclear maturation of the oocytes and the E2, progesterone, and alpha-specific inhibin content of the conditioned medium. Somatic cell removal and hormonal stimulation were equally effective in inducing germinal vesicle breakdown, but the hormonal stimulus was essential for the completion of meiosis, which was maximal (70%) on day 13 of culture. Continuous addition of LH to FSH during the oocytes' growth made them more prone to spontaneous resumption of meiosis I but resulted in a higher proportion of oocytes reaching the completion of meiosis. Estradiol and progesterone measurements demonstrated that the presence of LH influences luteinization. In contrast to oocytes grown in vivo, cumulus cell removal by itself is an insufficient stimulus for oocytes cultured in vitro to complete meiosis. Timed stimulation with hCG and epidermal growth factor increases nuclear maturation rates. A maximum number of metaphase II oocytes are obtained after a 13-day in vitro growth period when LH is added to the maturation medium.Fertility and Sterility 01/1999; 70(6):1114-25. · 4.17 Impact Factor
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ABSTRACT: Aim: Human serum albumin (HSA) is usually added to human in vitro fertilization culture medium to support embryo development, but carries some risk of contamination with various other compounds. Recombinant human albumin (rHA) has been shown to be a chemically defined protein. We evaluated the effect of rHA on mouse embryo development.Methods: B6D2F1 pronuclear oocytes were cultured in protein-free potassium simplex optimized medium with non-essential and essential amino acids supplemented with rHA, HSA (1 mg/mL) or polyvinyl alcohol (PVA; 0.1 mg/mL) for 96 h. The incidence of apoptosis and the generation of nitric oxide (NO) in blastocysts and fetal development after embryo transfer were examined.Results: Blastocyst development was equal in the three supplements. The incidence of apoptosis and the generation of NO in blastocysts developed in rHA was significantly (P < 0.05) lower than in HSA or PVA. After transfer of blastocysts developed in rHA, the percentage of fetal development was significantly (P < 0.05) higher than in HSA or PVA (75.8 ± 2.2%vs 46.8 ± 7.5% or 42.4 ± 3.9%, respectively).Conclusions: Supplementing culture medium with rHA resulted in decreased apoptosis and increased fetal development after embryo transfer. The results show that the presence of rHA in the culture medium is beneficial for producing high-quality blastocysts. (Reprod Med Biol 2007; 6: 195–201)Reproductive Medicine and Biology 11/2007; 6(4):195 - 201.
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ABSTRACT: This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes form preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk.Molecular Reproduction and Development 07/1996; 44(2):260-73. · 2.81 Impact Factor
Effect of preantral mouse follicle culture period on meiotic
maturation and developmental competence of oocytes
Shouko Nonowaki•Katsuhiko Takahashi•
Received: 13 October 2009/Accepted: 26 November 2009/Published online: 23 December 2009
? Japan Society for Reproductive Medicine 2009
culture period for meiotic maturation and developmental
competence of in vitro-grown mouse oocytes.
Early preantral follicles with diameter of 100–
130 lm were collected mechanically from day 14 mouse
ovaries and cultured for 8, 10, and 12 days. The diameters
of follicles and oocytes were measured, and chromatin
configuration in oocytes was observed. We also examined
meiotic maturation by human chorionic gonadotropin
(hCG)/epidermal growth factor (EGF) stimulation, devel-
opmental competence of fertilized oocytes to blastocysts,
and apoptosis in blastocysts.
The follicular diameter increased significantly
from days 4 to 10, and the diameter of day 12 oocytes was
significantly larger than day 8 or earlier oocytes. Chromatin
configuration around the nucleolus was transformed from
‘‘nonsurrounded (immature)’’ to ‘‘surrounded (mature)’’
after 10 days. Furthermore, MII rate of day 10 and 12
oocytes was significantly higher than that of day 8 oocytes.
The blastocyst rate of day 10 oocytes was higher than that
of day 8 or 12 oocytes. The blastocyst apoptotic rate of
day 12 oocytes was higher than that of day 10 oocytes.
Long culture periods of in vitro-grown
oocytes affect meiotic maturation, developmental compe-
tence to blastocysts, and apoptosis.
The aim of this study is to determine the optimal
Mouse ? Preantral follicle
Apoptosis ? Culture period ? In vitro growth ?
Mammalian ovaries contain many follicular oocytes; how-
ever, only very few go on to develop into mature oocytes
in vivo. Using in vitro culture techniques that encourage
follicular growth and oocyte maturation, it is possible to
To date, the two in vitro culture systems used most often to
grow preantral mouse follicles are mechanical dissection of
follicles that are then individually cultured in microdroplets
, and enzymatic isolation of oocyte–granulosa cell com-
. Mechanical dissection is useful for studying the mech-
anism of follicular development in the presence of thecal
cells, but it cannot be performed on a large scale . Fur-
thermore, there is the risk that follicles may be affected by
steroid hormone outflow into the oil covering. In contrast,
thecal cells and the original follicular form, with this pro-
cedure, large numbers of follicles can be cultured on a col-
lagen-coated membrane without the use of an oil covering
. Both procedures, however, result in very low levels of
blastocyst formation and low birth rates following implan-
tation of mature oocytes derived from in vitro growth,
compared with in vivo-matured oocytes [2–6]. In our study,
preantral follicles were mechanically dissected, and large
numbers of them, with thecal cells included, were cultured
on a collagen-treated dish with no oil overlay. We then
studied the effect of culture period on follicular develop-
ment, maturation capacity of oocytes, and oocyte develop-
S. Nonowaki ? T. Horiuchi (&)
Graduate School of Comprehensive Scientific Research,
Prefectural University of Hiroshima, Hiroshima 727-0023, Japan
S. Nonowaki ? K. Takahashi
Hiroshima HART Clinic, Hiroshima 730-0051, Japan
Reprod Med Biol (2010) 9:83–89
The size (diameter) of follicular oocytes increases as the
in vitro culture period progresses; it is a contributing factor
to oocyte size. Oocytes extracted from preantral follicles
are unable to induce in vitro germinal vesicle breakdown
(GVBD). However, as the diameters of growing oocytes
increase with increasingly longer culture periods, the
GVBD rate of follicular oocytes rises [7–9]. This indicates
that the size of the oocyte itself affects maturation capacity
in the later stages. Moreover, as the oocyte increases in
volume (size) during growth, it stores in the ooplasm the
elements necessary for future development. For this reason,
oocyte size is a vital component of developmentally
The resumption of meiosis and embryonic development
following fertilization are related to the chromatin config-
uration of the oocytes, as well as to their size [10, 11].
There are two chromatin configurations in oocytes: chro-
matin compacted as a ring around the nucleolus (sur-
rounded nucleolus, SN) and chromatin distributed diffusely
around the nucleolus (nonsurrounded nucleolus, NSN).
Compared with NSN chromatin, SN chromatin occurs in a
more advanced stage of oocyte development, closer to
ovulation [12, 13]. How the change in nucleolar chromatin
configuration and the increase in oocyte diameter correlate
with the in vitro culture period of the follicular oocyte is
not entirely clear, but the culture period is clearly vital to
the development of oocytes.
Lengthy periods of in vitro follicle culture may cause
damaging effects; the low embryonic developmental rate of
create large quantities of high-quality mature oocytes, the
optimal follicle culture period must be determined. In this
study, we investigated the progress of follicle growth and
culture, we analyzed follicle survival and antral-like cavity
formation, in addition to follicular mucification and oocyte
maturation after stimulation with hCG/EGF. In addition, to
confirm that the culture period affects the developmental
competence of in vitro-grown oocytes and embryo quality,
we studied blastocyst development and apoptosis.
Materials and methods
Follicles were harvested from F1 hybrid female mice
(C57BL/6 9 DBA/2; 14 days old) for in vitro growth.
Spermatozoa were collected from adult F1 males (3–
5 months old) with proven fertility. The mice were
obtained from C57BL/6 females mated with DBA/2
males. All of the mice were purchased from CLEA Japan
(Shizuoka, Japan). They were fed a standard diet ad libitum
and maintained in a temperature- and light-controlled room
at 22?C, 14 h light and 10 h darkness, with light starting at
06:00 h. The experiments were approved by the Committee
for Ethics on Animal Experiments of the Prefectural
University of Hiroshima, Japan.
Isolation of preantral follicles
Fourteen-day-old B6D2F1 mouse ovaries were placed in
Leibovitz L-15 medium (Invitrogen Life Technologies;
GIBCO, Grand Island, NY, USA) supplemented with
10% heat-inactivated fetal bovine serum (FBS; Hyclone,
Logan, UT, USA) at 37?C. Early preantral follicles were
mechanically dissected from the ovaries with fine 26-G
needles. We selected follicles with diameter of 100–
130 lm, that were enclosed by an intact basal membrane,
and that had at least a few thecal cells. In a preliminary
experiment, these cells were detected by cytochemical
staining of alkaline phosphatase, the activity of which is
specifically localized to thecal cells .
Culture medium and conditions
The culture medium for the follicle culture was a-minimum
essential medium (a-MEM) (Invitrogen Life Technologies;
GIBCO) with 5% FBS (Hyclone), 5 lg/ml insulin, 5 lg/ml
transferrin, 5 ng/ml selenium (ITS; Sigma, St. Louis, MO,
USA), 100 mIU/ml recombinant human FSH (rhFSH,
nant human LH (rhLH, Luveris; Serono), and 0.5% genta-
One hundred follicles were cultured in a 35-mm colla-
gen type I-coated dish (BD BioCoat; BD Falcon, Bedford,
MA, USA) containing 2 ml culture medium without a
mineral oil overlay. Three replicates were performed in
each experimental group. The culture dishes were main-
tained in an incubator at 37?C with 5% CO2in air for a
maximum of 12 days. Half of the medium was changed
every 2 days.
Measurement of follicle and oocyte diameters
Follicles were observed under an inverted microscope
(Nikon, Tokyo, Japan). After 0, 2, 4, 6, 8, 10, and 12 days
of culture, for each follicle, two perpendicular diameters
were measured using a calibrated ocular micrometer at
2009. On each day of culture, 30 follicles were chosen at
random and the oocytes were mechanically removed. The
oocyte diameter was measured using a calibrated ocular
micrometer at 4009.
84Reprod Med Biol (2010) 9:83–89
Fluorescence detection and classification
of chromatin configuration
After measuring the oocyte diameter, the chromatin con-
figuration of the germinal vesicle stage (GV) oocytes was
investigated by labeling the oocytes with 20 lg/ml Hoechst
33342 (Sigma) and mounting them on slides. The oocytes
were examined with an epifluorescence microscope
equipped with an ultraviolet (UV) filter (Nikon, Tokyo,
Japan). Images were recorded with a charge-coupled
device (CCD) camera (ORCA-cooled CCD; Hamamatsu
Photonics, Shizuoka, Japan) and analyzed with Image Pro
Plus software (Media Cybernetic, Silver Spring, MD,
The chromatin configuration of GV oocytes was clas-
sified as one of three types: chromatin distributed diffusely
around the nucleolus [nonsurrounded nucleolus (NSN)],
chromatin compacted in a ring around the nucleolus [sur-
rounded nucleolus (SN)], and an intermediate chromatin
configuration stage between NSN and SN [intermediated
nucleus (int.)] .
Evaluation of follicle survival and antral-like
The development of in vitro-cultured follicles was evalu-
ated morphologically on days 8, 10, and 12, and follicle
survival and antral-like cavity formation were recorded in
accordance with Cortvrindt et al. . In our culture system,
survival was defined as those follicles that retained their
oocyte completely embedded within the granulosa cell
mass; this was expressed as a percentage of the follicles
present at the beginning of culture. Antral-like cavity for-
mation was defined as thecal cells attached to the bottom of
the dish, a ring of large mural granulosa cells enclosing the
antral-like cavity, and a cumulus oophorus located cen-
trally with small cumulus cells in close contact with the
Ovulation induction (mucification)
and oocyte maturation
To induce meiotic maturation of follicular oocytes after 8,
10 or 12 days of culture, 1.5 IU/ml recombinant human
chorionic gonadotropin (rhCG, Ovidrel; Serono) and
5 ng/ml recombinant human epidermal growth factor (rhEGF;
Upstate, Lake Placid, NY, USA) were added to the culture
medium. After 15–17 h, the number of mucified follicles
was recorded in each experiment, and a portion of the
follicles was used to evaluate the nuclear maturation of
oocytes after the follicular cells were removed with 0.1%
hyaluronidase (Sigma) for 2 min followed by repeated
pipetting by narrow-bore micropipette. The oocytes were
fixed and stained with aceto-lacmoid solution. Nuclear
maturation was assessed by the presence of the first polar
body and metaphase chromosome. The majority of muci-
fied follicles were used for in vitro fertilization (IVF).
In vitro fertilization
Mucified follicles treated with hCG/EGF were subjected to
IVF. Spermatozoa collected from the caudal epididymis of
matured male mice were incubated for 90 min in a 0.4-ml
droplet of TYH medium  under paraffin oil in a
humidified atmosphere of 5% CO2 in air at 37?C for
capacitation. An aliquot of sperm suspension was added to
a 0.2-ml droplet of TYH medium containing the mucified
oocytes togivea final
1.5 9 105sperm/ml. The droplets were incubated for 5 h
under the same gas and temperature condition.
After IVF, presumptive zygotes were transferred to 50-ll
droplets of KSOMaa medium and cultured in vitro in a
humidified atmosphere of 5% CO2, 5% O2, and 90% N2at
37?C . Cleavage and development into blastocysts
were examined 24 and 120 h, respectively, after IVF.
Analysis of blastocyst apoptosis
The number of apoptotic cells in blastocysts was deter-
mined by the terminal deoxynucleotidyl transferase-
mediated dUTP nick-end labeling (TUNEL) method,
using the In Situ Cell Death detection kit (Roche Diag-
nostics, East Sussex, UK) in accordance with the manu-
facturer’s instructions. Blastocysts were fixed in 4%
paraformaldehyde in phosphate-buffered saline (PBS)
supplemented with 0.1% PVP-40 (PBS/PVP) for at least
1 h at room temperature, and permeabilized by immers-
ing them in 0.5% Triton X-100 in PBS for 1 h at room
temperature. Embryos were then washed twice in PBS/
PVP and treated with 0.02 mM fluorescein-conjugated
dUTP and 0.15 U/ll terminal deoxynucleotidyl transfer-
ase for 1 h at 37?C in the dark. Cell nuclei were coun-
terstained with 50 lg/ml propidium iodide. Embryos were
washed and then mounted between a slide and coverslip
using Vectashield mounting medium (Vector Laborato-
ries, Burlingame, CA, USA). The number, morphology,
and DNA fragmentation of cell nuclei were determined
using an epifluorescence microscope equipped with rho-
damine and fluorescein filters. The number of cells that
exhibited TUNEL was expressed as a percentage of the
total cell number of each blastocyst in accordance with
Takenaka et al. .
Reprod Med Biol (2010) 9:83–8985
The percentage data in each replicate were arcsine trans-
formed before being subjected to one-way analysis of
variance (ANOVA). All of the experiments were replicated
at least three times. Differences among the means in dif-
ferent groups were compared by Bonferroni’s multiple-
comparison test by using GraphPad PRISM (GraphPad
Software; La Jolla, CA, USA). When P\0.05, the dif-
ference was considered significant.
Follicular development, oocyte growth,
and chromatin configuration
The mean ± standard deviation (SD) follicular diameter
increased from 108 ± 5.5 to 615.9 ± 128 lm after
12 days of culture. The follicular diameter significantly
increased from days 4 to 10 of culture (P\0.05,
Fig. 1a). In addition, the oocyte diameter increased from
56.7 ± 2.9 to 76.4 ± 2.6 lm after 12 days of culture.
The oocyte diameter significantly increased from days 0
to 8 of culture (P\0.05), and the diameters of day 12
oocytes were significantly larger than day 8 or earlier
oocytes (P\0.05, Fig. 1b). All of the follicular oocytes
displayed NSN chromatin on day 0 of culture. In contrast,
after 10 days of culture, all of the oocytes displayed SN
chromatin (Fig. 2). Thus, it is likely that the chromatin
configuration was completed during the first 10 days of
Follicle survival, antral-like cavity formation,
mucification, and oocyte maturation
Follicle survival, antral-like cavity formation on days 8,
10, and 12 of culture, and mucification of follicles after
induction with hCG/EGF are presented in Table 1. The
percentages of follicle survival were not significantly dif-
ferent among the three culture periods (89–92%). In con-
trast, higher percentages of antral-like cavity formation
were observed when follicles were cultured for 10 days
(52%) or 12 days (62%) than when cultured for 8 days
(16%). In all three groups, more than 86% of follicles
demonstrated mucification after stimulation with hCG/
Table 2. The highest percentage of GVBD was observed
on day 12 of culture (100%). However, there was no
difference in the percentages of MII oocytes between
days 10 (77%) and 12 (76%) of culture. On day 8 of
culture, only 41% of mucified oocytes had reached the
Developmental competence of in vitro-grown
oocytes and apoptosis in blastocysts
The in vitro development of in vitro-grown oocytes fertil-
ized by IVF is presented in Table 3. A higher percentage of
the oocytes cultured for 10 and 12 days (63% and 51%,
respectively) developed to the two-cell stage than did those
cultured for 8 days (18%). The blastocyst rate of the
oocytes cultured for 10 days (25%) was significantly
higher than the rate for those cultured for 8 and 12 days
(9% and 14%, respectively).
Apoptosis in blastocysts derived from day 8, 10, and 12
oocytes is presented in Table 4. The percentage of apop-
totic cells in the day 12 oocytes (7%) was significantly
higher than in the day 10 oocytes (4%).
Fig. 1 Diameter of follicles (a) and oocytes (b) on days 0, 2, 4, 6, 8,
10, and 12 of culture. Data are presented by using box and whisker
plots as the percentile distribution of follicles or oocytes. Different
letters indicate significant differences (a–f, P\0.05)
86Reprod Med Biol (2010) 9:83–89
The results of our study indicate that the length of the
preantral follicle culture period affects the ability of the
oocyte to complete maturation, developmental competence
to the blastocyst stage, and apoptosis in the blastocysts.
Follicular granulosa cells were propagated after 4 days of
culture, and follicular diameter increased gradually from
days 4 to 10 of culture. Meanwhile, oocyte diameter
increased from 56.7 ± 2.9 lm at culture initiation to
73.6 ± 2.2, 75.5 ± 2.1, and 76.4 ± 2.6 lm on days 8, 10,
and 12, respectively. Mouse oocytes that reached more
than 70 lm in diameter are indeed capable of spontane-
ously completing the maturation sequence in vitro [17–19].
However, we found that, even when the oocyte diameter
was greater than 70 lm, the percentage of MII oocytes
mucified by stimulation with hCG/EGF after 8 days of
culture was considerably lower than after 10 or 12 days of
culture. The chromatin configuration of oocytes is corre-
lated with meiotic competence [10, 11]. SN chromatin
correspondstoa moreadvancedstage ofoocyte
development that is closer to ovulation than does NSN
chromatin [12, 13]. The percentage of oocytes with SN
chromatin on day 8 of culture was significantly lower than
on days 10 and 12 of culture. It was evident that the oocyte
had not yet matured after 8 days of culture even if oocyte
diameter was[70 lm.
Cortvrindt et al.  reported that, for preantral follicles
with diameter of 100–130 lm, 12 day culture yielded the
highest number of MII oocytes. We found that the MII rate
after 10 days of culture was the same as that after 12 days
of culture. One of the differences between our study and
theirs was the culture system. Cortvrindt et al.  cul-
tured one follicle at a time in 20-ll droplets under an oil
overlay. In contrast, we cultured 100 follicles as a group,
on a collagen-coated dish with 2 ml culture medium,
without an oil overlay. In this culture system, it was easy to
change the medium in a short time. Nayudu et al. 
reported that mouse preantral follicles cultured in vitro
without an oil covering have a faster growth rate than those
cultured with an oil covering. They suggested that the
optimal culture period for yielding mature oocytes is dif-
ferent for each culture system.
We did not detect a significant difference in follicle
survival after 8, 10, and 12 days of culture. However, the
antral-like cavity formation rate after 8 days of culture was
exceptionally lower than it was after the other two culture
periods. Antral-like cavity formation is caused by the dif-
ferentiation of cumulus cells surrounding the oocytes and
mural granulosa cells. We observed antral-like cavity for-
mation from day 8 onwards. Concurrently, we did not note
a significant difference in mucification induced by hCG/
EGF among the 8, 10, and 12 day culture periods. This
suggests that the reactivity to hCG/EGF of follicular
cumulus–oocyte complexes increases on day 8 of culture.
In this study, there was no significant difference in the
number of two-cell embryos resulting from day 10 and 12
oocytes. However, the blastocyst rate of day 10 oocytes
was significantly higher than that of day 12 oocytes. The
results of Liu et al.  were similar to ours: they cultured
preantral mouse follicles with diameter of 110–160 lm
in vitro, reporting that the total cell numbers of blastocysts
were significantly lower in the group of day 12 oocytes
than in the day 10 oocyte group. In our study, the total cell
numbers of blastocysts in the day 10 oocyte group were
higher, although not statistically significantly so, than in
the other groups. However, the total cell numbers of the
in vitro blastocysts were lower than in vivo-grown and
ovulated oocytes (data not shown).
Apoptosis is a natural biological process that eliminates
damaged cells and those with inappropriate phenotypes or
impaired developmental potential . In mammalian
preimplantation embryos, most apoptosis occurs in the
inner cell mass of the blastocysts, and increased apoptosis
Fig. 2 Chromatin configuration of in vitro-grown oocytes on days 0,
2, 4, 6, 8, 10, and 12 of culture. a Chromatin distributed diffusely
around the nucleolus (NSN), c chromatin compacted in a ring around
the nucleolus (SN), and b intermediate chromatin configuration stage
between NSN and SN (int.). d Distribution of chromatin configuration
patterns on each day of culture. Different letters indicate significant
differences (a–c, P\0.05). Scale bars represent 10 lm
Reprod Med Biol (2010) 9:83–8987
in blastocysts impairs embryonic and fetal development
[24–26]. Moreover, apoptosis in blastocysts is induced by
environmental stress due to culture conditions [16, 27, 28].
In our study, the apoptotic cell rate was significantly higher
in the group of day 12 oocytes (7.0 ± 0.6) than in the other
groups (day 8, 4.8 ± 0.9; day 10, 4.4 ± 0.5: P\0.05).
The apoptotic cells were observed mostly in inner cell
mass, as in previous papers [16, 24–26]. This suggests that
the long culture period had negative effects on the quality
of follicular oocytes.
In conclusion, we demonstrated that, by day 10 of cul-
ture, the oocytes in preantral mouse follicles had grown,
chromatin had assumed the mature SN configuration, and
oocytes subjected to IVF developed into blastocysts. After
12 days of culture, the apoptotic cell rate in the blastocysts
increased, indicating that long in vitro culture periods of
oocytes affect blastocyst development and apoptosis.
Compared with in vivo-grown and matured oocytes,
developmental competence to blastocyst stage and total
cell numbers of blastocysts of in vitro-grown preantral
mouse follicles were not yet sufficient. Further efforts are
required to develop a culture system that will lead to highly
competent in vitro-grown oocytes.
1. Cortvrindt R, Smitz J, Van Steirteghem AC. In-vitro maturation,
fertilization and embryo development of immature oocytes from
early preantral follicles from prepuberal mice in a simplified
culture system. Hum Reprod. 1996;11:2656–66.
2. Eppig JJ, Schroeder AC. Capacity of mouse oocytes from pre-
antral follicles to undergo embryogenesis and development to
Table 1 Effect of culture period on follicle survival, antral-like cavity formation, and mucification after induction with rhCG/rhEGF
Total no. of follicles
(no. of replicates)
No. (%, mean ± SD)
of survived follicles
No. (%, mean ± SD) of
follicles with antral-like cavities
No. (%, mean ± SD) of
mucified follicles after hCG/EGF
8 300 (3) 268 (89.3 ± 4.0) 47 (15.7 ± 2.5)a
157 (52.3 ± 2.1)b
187 (62.3 ± 6.7)b
259 (86.3 ± 3.1)
10 300 (3)274 (91.3 ± 4.0)
264 (88.0 ± 4.4)
12300 (3)276 (92.0 ± 5.0)
269 (89.7 ± 4.0)
Values with different superscripts within the same column are significantly different (P\0.05)
Table 2 Effect of culture period on oocyte maturation after induction with rhCG/rhEGF
No. of mucified follicles
(no. of replicates)
No. (%, mean ± SD) of oocytes after hCG/EGF
884 (3)66 (74.4 ± 15.5)a
82 (89.3 ± 10.8)a
86 (100 ± 0.0)b
35 (41.4 ± 3.3)a
71 (76.8 ± 4.8)b
65 (75.6 ± 2.0)b
Values with different superscripts within the same column are significantly different (P\0.05)
Table 3 Developmental competence of day 8, 10, and 12 oocytes fertilized in vitro
No. of oocytes inseminated
(no. of replicates)
No.(%, mean ± SD) of oocytes developed to
Two-cell (24 h)Morulae/blastocysts (96 h)Blastocysts (120 h)
8175 (3) 32 (18.3 ± 3.2)a
109 (63.4 ± 13.0)b
90 (50.5 ± 2.1)b
19 (11.0 ± 3.9)a
57 (33.3 ± 9.7)b
34 (19.1 ± 5.9)ab
15 (8.6 ± 3.5)a
43 (24.9 ± 2.5)b
24 (13.5 ± 2.9)a
Values with different superscripts within the same column are significantly different (P\0.05)
Table 4 Apoptosis in blastocysts derived from day 8, 10, and 12
oocytes grown in vitro
81365.3 ± 13.23.1 ± 2.04.8 ± 3.3ab
4.4 ± 2.4a
7.0 ± 2.9b
102073.3 ± 11.83.3 ± 1.8
122065.1 ± 14.94.4 ± 1.8
Values with different superscripts within the same column are sig-
nificantly different (P\0.05)
Values represent mean ± SD
88Reprod Med Biol (2010) 9:83–89
live young after growth, maturation, and fertilization in vitro.
Biol Reprod. 1989;41:268–76.
3. Spears N, Boland NI, Murray AA, Gosden RG. Mouse oocytes
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Hum Reprod. 1994;9:527–32.
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