Effect of Fibroblast Co-culture on In Vitro Maturation and Fertilization of Mouse Preantral Follicles

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 (5).

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 (Fig 1D). Since the oocytes were not denuded, the exact oocyte nuclear maturity (MII) potential could not be scored.

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 (Fig 1E-F).

We prepared MEF according to Hatoya et al. with some modification (18). Briefly, fetuses were collected from female NMRI mice at days 12 to 13 of pregnancy and washed thoroughly in Dulbecco’s phosphate-buffer saline (PBS). The head and liver were removed, samples cut into small pieces and cultured in α-MEM supplemented with 10% FBS, 100 IU/mL penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO2 in air at 37ºC. Primary fibroblasts were cultured until confluent and proliferated through two subsequent passages. For preparation of a feeder layer, MEF was inactivated by 10 μg/ml mitomycin C (Kyowa, Tokyo, Japan) for 3 hours in a humidified atmosphere of 5% CO2 in air at 37ºC, and washed five times in PBS. These cells were subsequently frozen at -80ºC in a volume of 106 cells/ml in the freezing medium that contained α-MEM supplemented with 100 IU/ml penicillin and 100 mg/ml streptomycin, 60% FBS and 10% DMSO (dimethyl sulfoxide). Frozen cells were thawed in water at 37ºC, cultured and plated at a density of 1×105 cells/ml one day before use. On the day of culture, the fibroblasts had made a feeder layer in the culture droplets (Fig 1A).

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 (Fig 1B). Most follicles lost their follicular structure and developed a 'diffuse appearance' on day four of culture. The follicles reached a 'diffuse' pattern by day 10. Antrumlike cavity formation (indicated by the appearance of lucid patches within the granulosa cell mass) was recognized from day eight onwards. By day 12, clearly visible and well developed antral-like cavities were recognized in the fully grown antral follicles (Fig 1C). Follicles showing a spontaneous oocyte release were maintained for further evaluation.

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.

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.

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 (Table 1).

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 recovery (Table 1). Ovulation rates in the two groups were between 80.2%-84 %. There was no significant difference in ovulation rates between the two groups as shown in table 1.

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.

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.