The basic structural and functional unit of ovaries
are follicles which provide the necessary microenvironment
for oocyte growth and maturation (
To date, different systems have been developed that
culture preantral follicles in many species, including
Therefore, tissues from different sources such as
reproductive and non-reproductive female organs
have been used to support the development of different
embryos such as human (
Recent studies have used embryonic fibroblast
cells for development and maintenance of mouse
This project was approved by the Islamic Azad University Science and Research Branch.
All reagents were obtained from Sigma-Aldrich (Germany) unless otherwise specified.
Male and female NMRI mice were housed and bred in the central animal house of the Mazandaran University of Medical Science under a 12 hour light/12 hour dark regime at 22-24°C, with adequate food ad libitum.
Female mice, 12-14 days old, were killed by cervical dislocation and their ovaries dissected free of fat and mesentery. Ovaries were immediately transferred to dissection medium that consisted of α-minimal essential medium (α-MEM, Gibco, UK) supplemented with 10% fetal bovine serum (FBS, Gibco, UK), 100 μg/ml penicillin and 50 μg/ml streptomycin under mineral oil to prevent evaporation and severe pH and temperature fluctuation. Preantral follicles from ovaries were isolated by mechanical dissection under a stereomicroscope, using 27-gauge needles to ensure that the follicular structure remained intact. Isolated follicles were selected according to the following criteria: 1. intact follicle with one or two layers of granulosa cells and some adhering theca cells; 2. visible, round and central oocyte; and 3. follicle diameter between 120-150 μm. However, if the follicle had a non-spherical structure, two oocytes or diameter above or below the range of 120-150 μm, they were not selected for culture. All selected follicles were pooled and randomly divided between the study culture conditions. Then, isolated follicles were transferred to fresh culture medium.
Isolated preantral follicles were cultured individually
in 60 mm petri dishes (Falcon, Becton Dickinson,
Belgium) that contained 19×30 μl droplets of
α-MEM (Gibco, UK) supplemented with 5% FBS,
100 mIU/ml recombinant follicle stimulating hormone
(rFSH or Gonal-f, Serono, Switzerland), 1%
insulin, transferrin and selenium mix (ITS mix: 5
μg/ml, 5μg/ml and 5 ng/ml, respectively; Gibco,
UK), 100 μg/ml penicillin and 50 μg/ml streptomycin
as base medium under mineral oil and incubated
at 37°C in a humidified atmosphere of 5%
CO2 in air for 12 days (
In the dishes, half of the medium was sampled from each droplet every two days without damaging the follicle. It was replaced by 15 μl of fresh pre-equlibrated medium. All 15 μl droplets, except those from non-proliferating follicles in one culture dish were pooled and stored at -20°C until analysis.
Measurement of follicle diameter was assessed with a precalibrated ocular micrometer at ×100 magnification every 48 hours during the culture period.
From day four, we could not measure the exact diameter of the growing follicles because of the irregular follicular shape that resulted from granulosa cells piercing the basement membrane and the onset of creating a monolayer around the follicle; thus we measured approximate follicle diameters. The survival rate of the follicles was verified by evaluation of follicle morphology under an inverted microscope. Follicle survival in culture was considered positive as long as an oocyte remained surrounded by granulosa cells attached to the culture dish.
On day 12 of the culture, ovulation was induced by
collecting the total volume of droplets and the addition
of fresh medium supplemented with 1.5 IU/
ml human chorionic gonadotropin (hCG; Organon)
to the droplets. Mucification of the cumulus oocyte
complexes (COC) was observed 14-16 hours later
under inverted microscope (
A sperm suspension was prepared using spermatozoa
collected from the cauda epididymis
of mature F1 males and preincubated at a concentration
of 10-15 × 106 cells/ml for 90 minutes
in 500 μl of HTF medium that contained
NaCl (5.935 g/1), NaHCO3 (2.1 g/1), α-Dglucose
(0.5 g/1), KC1 (0.35 g/1), KH2PO4
(0.05 g/1), sodium pyruvate (0.036 g/1), penicillin-
G (0.06 g/1), streptomycin sulphate (0.05
g/1), CaCl2-2H2O (0.3 g/1), EDTA (0.021 g/l
MgSO47H2O (0.024 g/1), sodium lactate syrup
(3.2 ml of 60% syrup) and phenol red (0.01 g/1)
supplemented with 4 mg/ml bovine serum albumin
(BSA) fraction v (Sigma) to induce sperm
capacitation. The in vitro released mucified
COCs were placed in 100 μl (10 COCs/drop)
fertilization droplets of HTF medium with 4
mg/ml BSA and 106/ml spermatozoa. After 4-5
hours, oocytes were washed and cultured in 20
μl droplets (10 oocytes/drop) of T6 medium with
4 mg/ml BSA under mineral oil at 37ºC in an
atomosphere of 5% CO2 in air for five days until
the blastocyst stage. The developmental stages
of inseminated oocytes were determined by
morphological evaluation every 24 hours under
an inverted microscope. Fertilization rate was
scored as the percentage of 2-cell embryos observed
24 hours after insemination (
We prepared MEF according to Hatoya et al.
with some modification (
To evaluate the supporting effect of fibroblast coculture, preantral follicles were cultured in two culture conditions. Follicle diameter, survival rate, antrum formation and embryonic development were studied in a total number of 226 intact preantral follicles with diameters between 120-150 μm. Two follicle culture conditions were studied and the experiments were repeated three times per group: i. follicle culture in base medium as control group (n=113) and ii. follicle culture in the base medium co-cultured with fibroblasts (n=113).
In the ~15 μl medium droplets, which were sampled and subsequently replaced by fresh medium, the following secretory products were measured: estradiol and progesterone. By measuring estradiol and progesterone, we wished to obtain information on the steroidogenic pathways functioning throughout the culture period and on the differentiation of the granulosa cells in culture. Every other day, all ~15 μl samples from surviving follicles of each group were pooled.
Estradiol and progestrone were measured using commercially available radio-immunoassay kits that included the IBL (Germany) kit with a sensitivity of 9.7 pg/ml and a total precision of <10% (% coefficient of variation; CV) and the Demeditec (Germany) kit with a sensitivity of 0.04 ng/ ml and a total precision of <10 % CV, respectively.
Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Statistical Package for Social Scieneces (SPSS version 15). Follicular diameters were analyzed by student’s t-test. Survival rate, antrum formation, COC recovery or antrum formation and embryonic development of fertilized oocytes in the two groups were compared by the chi-square test. P<0.05 was considered statistically significant.
On day two of culture, spindle-shaped cells originating
from the surface of the follicle attached
themselves to the dish and proliferated, with the
formation of a monolayer that surrounded and
attached the follicle to the dish. By day four,
follicles attached to the dish. Granulosa cells
proliferated and broke through the basal membrane,
spreading over the basal membrane and
the monolayer already formed the initial follicular
monolayer that surrounded and attached the
follicle to the dish (
Follicle diameters were estimated every two days to evaluate the effects of fibroblast co-culture on the development of preantral follicles during the 12 day culture period. The respective diameters of the follicles (mean ± SD) in the control and co-culture groups were as follows: 138 ± 7.7 μm and 137 ± 10.1 μm (day 0); 173.3 ± 12.3 μm and 191.3 ± 15.9 μm (day 2); 233.3 ± 38.6 μm and 276 ± 52.4 μm (day 4); 323.3 ± 40.3 μm and 386.6 ± 58.1 μm (day 6); 394 ± 64.8 μm and 455.3±71.7 μm (day 8); 460 ± 89 μm and 483.3 ± 64.5 μm (day 10); 493.3 ± 41.7 μm and 526.6±69.3 μm (day 12), respectively.
In vitro maturation of preantral follicle from 14 day old mice. A. Fibroblast monolayer for co-culturing preantral follicles (scale bar: 50 μm). B. Preantral follicle after 4 days of co-culture with fibroblast cells. Germinal vesicle stage oocyte surrounded by several layers of granulosa cells (scale bar: 50 μm). C. Follicle co-cultured with fibroblast cells for 12 days and developed antrum cavity (arrow) (scale bar: 150 μm). D. Released cumulus oocyte complex 16-24 hours post hCG (arrow) (scale bar: 100 μm). E. Embryos at 2-cell stage (scale bar: 50 μm). F. Embryos at blastocyst stage (scale bar: 50 μm).
A comparison of the follicular growth rates in the control and co-cultured groups is shown in figure 2 (significant difference between groups at p<0.05). As shown in figure 2, follicular diameter increased after 12 days of culture in both groups; but coculturing had a positive, significant effect on follicular diameter on days 4, 6 and 8 of the culture period. Therefore, fibroblast co-culture stimulated the growth of preantral follicles by granulosa cell proliferation at the middle of the culture period, the time when follicles lose their initial structure and proceed to the next step as a diffuse stage.
Comparison of the growth rates of follicles (mean follicular diameter ± SD) in the control and co-culture groups. Follicular diameters in the co-culture group were significantly different (*p<0.05) at days 4, 6 and 8 of culture when compared to the control group.
A normal follicular structure contains a centrally located
oocyte surrounded by several layers of granulosa
cells within the basal layer. In the present study,
follicles were considered viable if they retained a
normal structure with close contact between the
oocytes and the surrounding granulosa cells. Follicles
that failed to survive were defined as those
that had lost their oocytes, failed to increase in diameter
and became necrotic in appearance.
Follicular survival rates in the two groups after
12 days of culture were between 80.5%-88.5%
as shown in Table 1. Follicles in the co-culture
group had a higher survival rate (88.5 ± 1.4) compared
to the control group (80.5 ± 2.3) at p<0.05
Antrum-like cavities were recognized from day eight onwards. Antral rates of both groups were between 85.7%-87%. Despite significant differences between the two groups in survival rates, however, there was no significant difference in antrum formation rates between the co-culture and control groups as shown in table 1.
At the end of the culture period, hCG (1.5 IU/ml)
was supplemented to induce ovulation. After 18-
24 hours, cumulus oocyte complexes (COC) were
counted for evaluation of ovulation rate or COCs
In vitro fertilization evaluated the fertilizing potential as well as embryonic development of in vitro matured oocytes. Since the oocytes obtained from COCs were not denuded, the exact fertilization potential could not be scored.
Effect of fibroblast co-culture on in vitro maturation and embryonic development of oocytes derived from preantral follicles
|No. (%) of follicles||Percent (%) of oocytes developed to|
|Groups||Cultured follicles||Survival rates||Antral rates||Ovulation rates||2-cell||Morula||Blastocyst|
|113||91 (80.5)||78 (85.7)||73 (80.2)||30 (41.1)||15 (21.7)||9 (12.3)|
|113||100 (88.5)*||87 (87)||84 (84)||38 (45.2)||19 (22.6)||10 (11.9)|
*Significant differences are compared with control group (p<0.05).
Production of estradiol (pg/ml) and progesterone (ng/ml) in pooled media during culture period of preantral follicles. There are significant differences (p<0.05) in both estradiol and progesterone secretion at days 4, 6, 8, 10, 12 and days 10 and 12, respectively between the co-culture and control groups
|Estradiol (pg/ml)||Progesterone (ng/ml)|
|Day 4||Day 6||Day 8||Day 10||Day 12||Day 6||Day 8||Day 10||Day 12|
*Significant differences are compared with control group (p<0.05).
Therefore, the percentage of 2-cell embryos observed 24 hours after fertilization was assumed to be the fertilization rate. As shown in Table 1, the fertilization rates of both groups were 41.1%-45.2 % and the percentages of morula and blastocyst embryos were between 21.7%-22.6% and 11.9%- 12.3%, respectively. There was no significant difference (p<0.05) between control and co-culture groups regarding embryonic development.
Production of estradiol (pg/ml) and progesterone (ng/ml) are shown in table 2. The production of estradiol increased progressively up to day 12. On this day, the production of estradiol reached 3708 pg/ml in the co-culture group. There were significant differences (p<0.05) in estradiol secretion on days 6, 8, 10 and 12 between the co-culture and control groups. Progesterone production remained below the sensitivity of the radioimmunoassay up to days 6 and 10 for the co-culture and control groups, respectively. Basal progesterone production increased moderately after this day to a level of 22 ng/ml in the co-cultured group. Progesterone concentrations were also significant (p<0.05) in the co-culture group on days 10 and 12 compared to the control group.
We established follicle cultures to study essential
factors for oocyte development during in vitro folliculogenesis
In the present study, early preantral follicles were cultured for 12 days in co-culture conditions to evaluate whether MEF co-culture could enhance follicular growth rates as well as in vitro fertilization and embryonic development of in vitro matured oocytes.
The results of this study showed that the co-culture
group had a significant (p<0.05) growth rate (on
days 4, 6 and 8 of the culture period) and survival
rate when compared to the control group. From
days 4 until 8, granulosa cells proliferated and their
protrusion through the basement membrane led to
the formation of large preantral follicles (
The results of this study also showed no significant
difference between the two groups in antrum
formation and ovulation rates of follicles that
survived. However, compared to the findings of
a study (
Our results showed no significant difference in
embryonic development between the co-culture
and control groups. This result suggests that the
co-culture system used in this study has no remarkable
effect on embryonic development of
oocytes from in vitro matured preantral follicles,
however it might be caused by insufficient
cytoplasmic maturation. A similar result was
reported by Haidari et al. who found no difference
in the rates of fertilization and subsequent
development to the blastocyst stage between the
oocytes derived from cumulus co-cultured preantral
follicles and those derived from control
preantral follicles (
Since steroid hormones might be important regulators
of crucial changes in oocyte cytoplasm for
normal fertilization, measurements of estradiol
and progesterone in conditioned media from cultured
follicles can provide precise information on
the functionality of the culture condition (
Our results suggest that fibroblast co-culture increases the growth and survival rate of cultured preantral follicles in a significant manner by enhancement of granulosa cell proliferation; however, the co-culture had no effect on embryonic development of the ovulated oocyte. This research investigated, for the first time, the effect of MEF co-culture on preantral follicles. However, more research is necessary for the improvement of in vitro maturation of preantral follicles.