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Biology of Reproduction 59, 255-262 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Maintenance of Bovine Oocytes in Meiotic Arrest and Subsequent Development In Vitro: A Comparative Evaluation of Antral Follicle Culture with Other Methods1

A.A. Fouladi Nashta2,c,d, D. Waddingtonc, , and K.H.S. Campbelle

c Roslin Institute, Roslin, Midlothian EH25 9PS, Scotland, United Kingdom d Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush, Midlothian EH25 9QR, Scotland, United Kingdom e PPL Therapeutics, Roslin, Midlothian, EH25 9PP, Scotland, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The frequency of development of bovine embryos produced by maturation, fertilization, and culture in vitro is lower than that observed in vivo. One factor that may affect both the frequency of development and the quality of the embryos produced is the developmental competence of the oocyte. In current in vitro production systems, oocyte maturation, characterized by the resumption of meiosis, occurs after oocyte aspiration from the follicle. The developmental competence of individual oocytes may be improved by inducing maturation after culturing under conditions that inhibit the resumption of meiosis. In order to test this hypothesis, a system has been established in which intact antral follicles (3–8 mm in diameter) are cultured in vitro. During this period the oocytes are maintained at the germinal vesicle (GV) stage under the inhibitory effects of the follicle. Culture of intact antral follicles was compared with two other "physiological" methods for the maintenance of GV arrest: oocytes were cultured attached to a small part of the follicle wall or within hemisections of follicles. It was found that 96.8% of oocytes recovered from intact antral follicles—as compared to 24.6% attached to a small part of the follicle wall and 62.7% within hemisections of follicles—were maintained at the GV stage after 24-h culture. The effects on GV arrest and subsequent maturation of the oocytes were evaluated after longer periods of antral follicle culture (2, 4, and 7 days). As the culture period increased, the number of GV-arrested oocytes decreased; the maximum percentage of GV arrest was observed after 24-h culture. The majority of these oocytes matured to metaphase II. A comparison of blastocyst production was made after fertilization and subsequent development of oocytes obtained following follicle culture and of control oocytes aspirated directly from antral follicles. The cleavage rate and percentage of blastocyst production in these two groups were 54.6 ± 13.9%, 48.4 ± 8.4% and 68.6 ± 8.6%, 32.8 ± 10.8%, respectively. Statistical analysis showed significant differences in both cleavage rate and blastocyst production between these two groups. Total cell numbers in the control group were 144.6 ± 7.28 and 152.0 ± 25.8 after follicle culture. It is concluded that culture of intact antral follicles for 24 h is an alternative method for the maintenance of bovine oocytes in meiotic arrest and that these oocytes acquire a greater developmental competence in vitro.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The in vitro maturation of bovine oocytes coupled to fertilization and culture is a useful tool for both agricultural and research purposes (for review see [1]). Modifications to the culture conditions used for oocyte maturation, fertilization, and embryo development have increased the frequency of bovine oocytes matured and fertilized in vitro, but embryo production is still hampered by a high loss during early development. The frequency of development, in terms of the number of blastocyst-stage embryos obtained per 100 cumulus-oocyte complexes (COCs) cultured, is inferior to that for in vivo production systems [2]. Furthermore, pregnancy rates and cryopreservation of bovine morulae and blastocysts produced in vivo still exceed those of embryos produced in vitro [3]. Several reports have demonstrated that in vitro, using coculture systems, 40–80% of COCs develop to the 2-cell stage [4]; however, only a low percentage of these will develop to the blastocyst stage. The ineffectiveness of in vitro embryo production systems may be due to suboptimal conditions during the maturation, fertilization, or culture procedures. Alternatively, it may reflect the developmental competence or status of the oocyte prior to the onset of maturation.

In vivo, meiotic maturation of the oocytes occurs within the follicle. However, in vitro, when oocytes are removed from the follicle, meiotic maturation resumes spontaneously [5]. The relationship between developmental competence and oocyte development is unclear. However, it is hypothesized that if oocytes can be cultured in vitro under conditions that maintain meiotic arrest at the germinal vesicle (GV) stage, then they may have the opportunity to acquire greater developmental competence. To date, treatment of oocytes with chemicals that elevate intracellular levels of cAMP, including dibutyryl cAMP [6], inhibitors of the cAMP-degrading enzyme phosphodiesterase, 3-isobutyl-1-methylxanthine [7–11] activators of adenylate cyclase, forskolin, cholera toxin, sodium fluoride, and prostaglandin E2 [12], have been used in various species. According to several studies, treatments that maintain high levels of cAMP exert only a transient suppression of germinal vesicle breakdown (GVBD) in bovine oocytes [10, 13, 14]. Most of these products are not compatible with long-term survival of the oocyte and therefore can not be used to enhance its developmental competence in culture [15].

Other studies have attempted to establish physiological methods for the maintenance of meiotic arrest, including addition of follicular fluid to the culture medium [16–18] and culture of oocytes on monolayers of granulosa or theca cells [19–21], within follicle hemisections [22, 23], or attached to a small part of the follicular wall [24]. An alternative to these methods is to isolate and culture intact antral follicles. This would supply a combination of the effects of follicular components to the enclosed oocyte. To date there are no published reports relating to the culture of intact bovine antral follicles. Several studies on ovine follicular steroidogenesis utilized an antral follicle culture system [25–28]. Here we report a system for the culture of intact, large bovine antral follicles in vitro by modification of the system used by Moor et al. [25]. We have evaluated the ability of this system to maintain bovine oocytes in meiotic arrest and the ability of oocytes subsequently released from cultured follicles to resume meiosis, and we have assessed the developmental competence of the resultant embryos up to the blastocyst stage. In addition, the maintenance of meiotic arrest by antral follicle culture was compared with that for oocytes cultured either in follicle hemisections or attached to a small part of the follicular wall.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture of Antral Follicles

Ovaries were obtained from local abattoirs and maintained at 28–35°C during transport to the laboratory (1–2 h). Ovaries were dissected from the rest of the reproductive tract and transferred into a beaker containing 200–300 ml of Dulbecco's PBS (pH = 7.4) at 39°C (Unipath Ltd., Basingstoke, England) containing 0.5 ml of gentamycin solution (Sigma Chemical Co., St. Louis, MO) in a clean glass beaker. They were then washed once in industrial methylated spirits and transferred to PBS at 39°C. Using a pair of scissors and forceps, follicles of 3- to 8-mm diameter (Fig. 1a) were dissected from the surrounding connective tissues. Individual follicles were transferred to PBS at 39°C and completely trimmed from remaining connective tissues. Nonatretic follicles were selected on the basis of morphological criteria including translucency, lack of free particles, and the presence of blood vessels [29] (Fig. 1b). Selected follicles were cultured in Waymouth medium MB752/1 (Gibco BRL, Oakville, ON, Canada) supplemented with 2240 mg/L sodium bicarbonate (Sigma), 0.23 mM pyruvic acid, 50 mg/L streptomycin sulfate, 75 mg/L penicillin G, 3 mg/ml BSA (A-6003; Sigma), 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium (Sigma; pH 7.4, osmolarity 280 mOsmol). Fetal calf serum (FCS; 5%) was added prior to gassing. A gaseous atmosphere of 45% O2:50% N2:5% CO2 was used according to the method of Moor et al. [25]. Selected follicles were transferred into 6-well culture dishes with or without netwell inserts of 500-µm pore diameter and containing the culture medium described above. The culture medium was gassed using an anaerobic jar and warmed to 39°C in an incubator prior to culture. After the follicles were transferred into the culture dishes, they were gassed again by the same system for 5 min. Culture was continued for up to 2 days without changing the medium; however, during longer periods of culture the medium was changed every 2 days.



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  		FIG. 1. Bovine antral follicles after dissection from the ovary. a) Range of follicle sizes. b) Good-quality follicle showing translucency and presence of blood vessels used as selection criteria (x20; reproduced at 47%).

Culture of Oocytes in Hemisections of Follicular Wall

Follicles were dissected from ovaries as described above, and selected follicles (3- to 8-mm diameter) were completely trimmed from the remaining stromal tissues. The position of the oocyte was located within each follicle using a stereomicroscope. The follicle wall was then pierced at the side opposite to the oocyte. After the follicular fluid was gently flushed out, the collapsed follicle was cut in half using fine scissors; the oocyte remained in one half of the follicle. Oocytes that detached during the process and others isolated by aspiration were placed inside the follicle hemisections. Two oocytes were placed into each hemisection. Three follicle hemisections were transferred to 500-µl drops of Waymouth medium supplemented with 5% FCS under mineral oil (Sigma) in a 35-mm tissue culture dish and were maintained in an atmosphere of 5% CO2 in air at 39°C for 24 h. In preliminary experiments, various volumes of culture medium (200 µl to 1 ml) were used to find the most effective volume.

Culture of Oocytes Attached to Part of the Follicle Wall

Follicles were dissected from the ovaries as explained above. Selected follicles were cut according to De Loos et al. [24]. Briefly, the COC was located using a stereomicroscope. Directly opposite the point of attachment of the COC to the follicle wall, the follicle was pierced with a needle, and the follicular fluid was gently flushed out, leaving a collapsed follicle. Three areas of the collapsed follicle were cut away using fine scissors to leave a sandwich of follicular wall with its COC positioned centrally. To obtain a preparation of follicular wall (2–3 mm2) with a COC attached, the upper part of this sandwich was pulled back and the follicular wall around the COC was trimmed (Fig. 2).



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  		FIG. 2. Schematic diagram of antral follicle dissection for the preparation of follicle hemisections or isolation of oocytes attached to part of the follicle wall. Isolated follicles were pierced, and collapsed follicle walls were cut in three places. Oocytes attached to hemisections of follicle wall were cultured for 24 h.

An alternative method employed for the isolation of oocytes attached to part of the follicle wall compartment was a vacuum aspiration system. An 18-gauge needle (1.2-mm internal diameter) connected to a vacuum line was inserted into individual follicles; the needle was used to gently scratch the follicular wall during aspiration. Using this technique, the majority of the oocytes isolated were attached to part of the follicular wall compartments. Five to ten isolated COC follicular wall complexes were cultured in 500 µl Waymouth maturation medium supplemented with 5% FCS in an atmosphere of 5% CO2 in air at 39°C for 24 h.

Aspiration and Selection of Oocytes

After dissecting and washing of the ovaries, COCs were aspirated from follicles of 3–8 mm in diameter using a 10-ml syringe fitted with an 18-gauge hypodermic needle. The aspirated follicular fluid was placed into sterile plastic universal containers in a warmed chamber (35°C) and allowed to settle for 10–15 min. The majority of the fluid was then removed by surface aspiration, and the remaining follicular material was diluted with an equal volume of dissection medium (Tissue Culture Medium 199 [TCM199] with Earle's salts [Gibco, Grand Island, NY], 75.0 mg/L kanamycin monosulfate [Sigma], 7.08 g/L Hepes [pH 7.8, osmolarity 279 mOsmol/kg H2O]) supplemented with 10% FCS. The diluted follicular fluid was transferred into an 85-mm petri dish and examined for COCs under a dissecting microscope (x40 magnification). Oocytes were selected on morphological criteria; good-quality oocytes with homogenous evenly distributed cytoplasm and 3–4 layers of compact cumulus investment were selected for maturation.

Test of Oocyte Viability and Cell Cycle Stage

After 24 h, oocytes from all culture procedures were recovered. A proportion of the oocytes were tested for viability by dye exclusion, using 0.5% trypan blue (Sigma; cell culture tested). Viable oocytes exclude trypan blue whereas nonviable oocytes take up this dye and stain blue.

Nuclear morphology was determined microscopically. Oocytes were denuded by incubating in dissection medium containing 500 IU/ml hyaluronidase enzyme (Sigma) for 10 min followed by repeated pipetting. Completely denuded oocytes were recovered and transferred to cleaned glass slides in small drops of 3 µl dissection medium. A mixture of Vaseline and paraffin wax (10:2) was used to attach a coverslip to the glass slide holding the oocytes in position without excessive pressure. Mounted oocytes were then fixed in methanol:acetic acid (3:1) for 24 h, stained with 1% aceto-orcein, and examined under phase contrast at x100 and x400 magnification. Oocytes were classified as GV stage if the nuclear membrane was present and the chromatin was uncondensed.

Test of Oocyte Reversibility

After 24-h culture, isolated COCs were washed twice in dissection medium and transferred into 500 µl maturation medium (TCM199 with Earle's salts [Gibco], 75 mg/L kanamycin, 4.75 g/L Hepes, 2.29 g/L NaHCO3 [pH 7.8, osmolarity 280 mOsmol/kg H2O]) supplemented with ovine FSH/LH (0.006 IU/ml), 5% FCS, in 35-mm tissue culture dishes. COCs were then incubated in humidified atmosphere of 5% CO2 in air at 39°C for 24 h. After culture, the oocytes were denuded, fixed, stained with aceto-orcein, and examined under phase contrast.

Use of Okadaic Acid

Antral follicles were immersed in culture medium using 6-well tissue culture dishes without netwell insert membranes for 24 h. Oocytes were recovered and transferred into maturation medium containing gonadotrophic hormone and 0.5 µM okadaic acid (Sigma). Okadaic acid has been shown to induce a rapid appearance of maturation-promoting factor activity in Xenopus and starfish oocytes [30] and accelerates GVBD in cattle and pig oocytes [31].

In Vitro Fertilization

In vitro-matured oocytes were fertilized according to the method described by Vergos et al. [32]. Briefly, COCs were gently pipetted in order to remove adhering granulosa cells and break up aggregated COCs. Disaggregated COCs were then washed once (in oocyte wash medium containing NaCl, 6.8 g/L; KCl, 230 mg/L; NaHCO3, 168 mg/L; Na2HPO4, 47 mg/L; Hepes, 4.8 g/L; kanamycin monosulfate, 75 mg/L; pyruvic acid, 11 mg/L; BSA, 6 g/L; 60% syrup lactic acid, 1.86 ml/L; MgCl2·6H2O, 100 mg/L; CaCl2·2H2O, 840 mg/L; pH 7.4, osmolarity 282 mOsmol/kg H2O) and transferred into 45-µl microdrops of fertilization medium (5–10 oocytes per drop) containing sperm (1.5 x 106/ml) and cultured for 48 h at 39°C in a humidified incubator of 5% CO2 in air.

In Vitro Embryo Culture

At 46–48 h after coincubation of the spermatozoa and oocytes, cleaved embryos with at least 4 cells were selected, washed twice in a Hepes-buffered synthetic oviductal fluid (Hepes-SOF) medium (pH 7.4), and transferred to 20-µl droplets of synthetic oviductal fluid (SOF) [33] medium supplemented with 4 mg/ml Pentex crystalline BSA (Bayer, Elkhart, IN). Embryo culture was carried out in 35-mm cell culture dishes at 39°C, in a humidified incubator with a gaseous atmosphere of 5% CO2:5% O2:90% N2.

Total Cell Counting

Blastocyst-stage embryos were incubated for 15 min in dissection medium containing 5 µg/ml bisbenzimide (Hoechst 33258; Sigma). The embryos were then placed onto clean glass slides in 5-µl drops of DABCO (Sigma) under coverslips. Counting took place using an inverted, differential interference contrast microscope fitted with epifluorescence (Nikon, Garden City, NY).

Design of Experiments and Statistical Analysis

Experiment 1. The aim of this preliminary experiment was to establish a system for culture of bovine antral follicles in vitro. A total of 90 antral follicles were submerged in culture medium, without the use of netwell insert membrane. After 24-h culture, the oocytes were recovered; a proportion of them were fixed and stained for examination of nuclear morphology, and the others were transferred to the maturation medium. Matured oocytes were fixed and stained for evaluation of nuclear maturation to metaphase II (MII) stage.

Experiment 2. This experiment was designed to evaluate the viability of oocytes derived from antral follicle culture in culture medium without the use of insert membrane netwell. A total of 52 oocytes were recovered after 24-h follicle culture inside culture medium. A proportion of these oocytes (n = 15) were stained by trypan blue, and the remainder (n = 37) were transferred to maturation medium containing gonadotrophic hormones and okadaic acid. After 24-h maturation, the oocytes were fixed and stained to assess GVBD and development to MII.

Experiment 3. The aim of this experiment was to compare the ability of three different methods of oocyte culture to maintain meiotic arrest. A total of 196 oocytes were cultured for 24 h as attached to the follicle wall (n = 73), within follicle hemisections (n = 59), or as intact antral follicles (n = 64). They were then fixed, stained with aceto-orcein, and examined microscopically to assess the organization of chromatin in the GV of arrested oocytes.

Experiment 4. In this experiment the effect of longer periods of antral follicle culture on the maintenance of meiotic arrest was evaluated. In total, 367 antral follicles were cultured for 24 h (n = 105), 48 h (n = 82), 72 h (n = 84), and 168 h (n = 96). At the end of each culture period, a proportion of the oocytes were fixed and examined to determine the cell cycle stage of the chromatin. The remainder were cultured in maturation medium for 24 h and then fixed, stained with aceto-orcein, and examined for cell cycle stage.

Experiment 5. This experiment was designed to evaluate the developmental competence of oocytes derived from antral follicles cultured for 24 h prior to oocyte maturation. A total of 94 oocytes derived from cultured follicles (7 replicates), and 186 oocytes (7 replicates) directly aspirated from follicles of the same sizes, were matured for 24 h. Mature oocytes were fertilized, and after 48 h, embryo cleavage was assessed. Embryos with 4 or more cells were transferred to pre-gassed 20-µl droplets of SOF medium (5 µl/embryo). Embryo development from the 4-cell to the blastocyst stage was evaluated at Day 8. In addition, the total cell numbers of blastocyst-stage embryos were determined.

Statistical Analysis

For experiment 3, which compared the effects of follicle culture, the proportion of oocytes at the GV stage were analyzed as binomial data using the marginal model of Breslow and Clayton [34], allowing for differences between days on the proportion of oocytes cleaving. The same model was used to analyze the proportion of cleaved oocytes becoming blastocysts, and an equivalent ANOVA was used to analyze cell counts in experiment 5. For experiment 4, the effects of different periods of culture on the proportion of oocytes at the GV stage or reaching MII were assessed by fitting polynomials of periods in a generalized linear model with binomial errors [35]. Tests of treatment effects in all models were made by comparisons with chi-squared distributions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Oocyte Maturation/Viability after Culture of Antral Follicles Immersed in Culture Medium

All of the oocytes derived after culture of the antral follicles immersed in culture medium were maintained at GV stage, although in some of these oocytes, abnormal chromatin condensation was observed. However, on subsequent culture, none of them were able to break down GV and develop to MII (Fig. 3a). Treatment with okadaic acid did not induce GVBD in these oocytes. Trypan blue exclusion as a test of oocyte viability revealed that the majority of these oocytes had lost viability during the culture period.



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  		FIG. 3. GV-stage oocytes isolated after 24 h culture. a) Group of GV oocytes showing abnormal chromatin condensation isolated after 24-h culture inside culture medium. b) GV-stage oocyte after 24-h antral follicle culture showing uncondensed chromatin. c) GV-stage oocyte after 24-h culture inside hemisection of the follicle wall showing abnormal chromatin condensation in the presence of nucleus membrane. a,b x200; c x400.

Maintenance of Meiotic Arrest

The number of oocytes cultured and the percentage of GV-stage oocytes derived from each of the three culture methods are shown in Table 1. Oocytes recovered after 24-h culture of intact antral follicles maintained the highest frequency of GV arrest (96.8%). This contrasted with 62.7% of oocytes cultured within follicle hemisections ({chi}21 = 37.1, p < 0.001), which in turn was greater than the 24.6% for oocytes attached to a portion of the follicle wall ({chi}21 = 48.5, p < 0.001). Visual assessment of oocyte quality suggested that the oocytes derived from whole follicle culture had a normal appearance (Fig. 3b); however, the oocytes cultured within hemisections of follicle wall showed an abnormal, slightly condensed chromatin morphology (Fig. 3c).


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  		TABLE 1. Number of oocytes and percentage remaining at the GV stage after 24 h culture in three different culture systems.

On prolonged culture of intact antral follicles, the percentages of the oocytes that remained at the GV stage after 24, 48, 96, and 168 hours were 96.8%, 89.1%, 28.5%, and 30.9%, respectively (Fig. 4). This gave a significant cubic decline with time ({chi}21 = 8.53, p < 0.01). Oocytes that resumed meiosis within the antral follicle during culture were found at different stages of maturation up to and including second metaphase and also showed clear signs of degeneration (Fig. 5; Table 2).



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  		FIG. 4. Maintenance of meiotic arrest in oocytes cultured within antral follicles for increasing time periods, and subsequent maturation to metaphase of the second meiotic division.



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  		FIG. 5. Graph showing the distribution of cell cycle phases in oocytes cultured within antral follicle. GV, intact germinal vesicle; Int, Intermediate, i.e., between GV and MII; M, arrested at MII.


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  		TABLE 2. Cell-cycle stage of oocytes derived from different periods of follicle culture before and after maturation.

Oocyte Maturation after Antral Follicle Culture

The percentages of oocytes recovered from each follicle culture period after maturation that developed to the MII stage were 80.0% (24 h), 68.8% (48 h), 22.8% (96 h), and 24.0% (168 h), respectively (Fig. 4). The number of oocytes able to resume meiosis and develop to MII decreased with the increasing period of antral follicle culture in an approximately linear response ({chi}21 = 36.2, p < 0.001). The cell cycle stage of oocytes after follicle culture and subsequent maturation is shown in Figure 5. The oocyte group classified as intermediate includes all of the oocytes morphologically classified as between GV stage and MII, and those that had degenerated.

Embryo Development after Antral Follicle Culture

The development of oocytes cultured for 24 h within intact antral follicles prior to maturation, fertilization, and culture was compared to that of control oocytes matured directly after aspiration. The results from this comparison are summarized in Table 3; 68.6% of control oocytes had developed to the 4+-cell stage 48 h after fertilization compared to 54.6% of oocytes after antral follicle culture. Statistical analysis showed that there was a significant difference ({chi}21 = 7.2, p < 0.01) in the cleavage rates between the control and follicle culture oocytes. The number of blastocyst-stage embryos produced after 8 days culture in vitro was 45 (32.8%) and 25 (48.4%) for the control and follicle cultured groups, respectively. Statistical analysis of these data showed a significant difference ({chi}21 = 109.8, p < 0.001) between these two groups. There were no significant differences in terms of embryo quality based on total cell numbers, which were 144.6 ± 7.28 and 152.0 ± 8.44 in the control and treatment groups.


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  		TABLE 3. Development of antral follicle cultured (24 h) and control oocytes following maturation and fertilization in vitro.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of these studies on the role of follicle wall in the maintenance of the GV stage in bovine oocytes is supported by recent published data [23–25, 36]. In the present study, the percentage of oocytes that were maintained at the GV stage was significantly greater when oocytes were cultured within intact antral follicles. The majority (96.8%) of the oocytes obtained after 24-h follicle culture were arrested in GV stage; in contrast, culture of oocytes in hemisections or attached to part of the follicle wall maintained 62.7% and 24.6% of oocytes at the GV stage, respectively. The nuclear morphology of oocytes from each method was assessed microscopically. Oocytes derived from follicle wall or hemifollicle cultures showed a range of degrees of abnormal chromatin condensation, from partially to totally condensed, while maintaining an intact nuclear envelope. The same abnormalities were observed when intact antral follicles were cultured submerged in the culture medium. Oocyte viability assessed by trypan blue exclusion indicated that these oocytes were dying at early stages of maturation. In this population of oocytes, okadaic acid, which is a potent inhibitor of protein phosphatases [37] and accelerates GVBD in cattle oocytes [32], did not induce GVBD. There are a few reports [11, 38] regarding the quality of meiotically arrested oocytes and their subsequent ability for maturation, fertilization, and development in vitro. In these previously reported studies, meiotic arrest was induced using a range of chemical inhibitors including cycloheximide, 6-dimethylaminopurine, or vanadate. Following these treatments, a significantly lower frequency of development to the blastocyst stage was obtained than in control oocytes matured directly after aspiration.

During the studies reported here, it was observed that when oocytes are cultured either attached to part of the follicle wall or inside hemisections of follicles, the volume of culture medium is critical. When a small volume was used, changes in pH appeared to adversely affect the oocytes; although the oocytes remained at the GV stage, an abnormal chromatin morphology was observed. In contrast, when a large volume of culture medium was used, a lower percentage of oocytes remained arrested at the GV stage. This may reflect the dilution of one or more inhibitory factors produced by the follicular components.

When oocytes were cultured in intact antral follicles for various time periods, it was shown that shorter periods (24 and 48 h) resulted in significantly higher rates of GV arrest and subsequent maturation than for follicles maintained for longer periods. In addition, during prolonged follicle culture, oocytes resumed meiosis within the follicle. The reasons for this are presently unknown; however, one possibility is that the follicles themselves may degenerate, releasing the oocytes from the inhibitory effects of follicular components, allowing the resumption of meiosis. Figure 6 shows an example of an oocyte with expanded cumulus layers following culture within an intact follicle for 3 days. In these experiments there was no supplementation of the culture medium with specific hormones or growth factors that have previously been reported to be necessary for follicular viability and growth in vivo, i.e., gonadotropins [39–42], estradiol [43], insulin-like growth factor-1 [44, 45], epidermal growth factor [41, 46, 47], or basic fibroblast growth factor [48].



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  		FIG. 6. Example of an oocyte recovered after 3 days of antral follicle culture, showing expansion of the cumulus layer and detachment from the follicle wall. x400.

The data presented here suggest that the culture of intact bovine antral follicles is an efficient method for the maintenance of meiotic arrest for at least 24 h. In addition, oocytes derived after 24 h of antral follicle culture have significantly higher developmental competence than oocytes aspirated directly from follicles 3–8 mm in diameter. This suggests that during the culture period, follicle-enclosed oocytes acquire a greater developmental competence. However, when the proportion of the oocytes that developed to the blastocyst stage was calculated, there were no significant differences between the two groups (24% vs. 26%). This observation is in agreement with recently published data [49] demonstrating that oocytes acquire developmental competence prior to maturation. An explanation of these observations is that during the period of follicle culture, the oocytes are able to translate or posttranslationally modify essential proteins or to transcribe mRNAs essential for further embryonic development. In the present study, the percentage of embryos that developed to 4 cells or more during the first 48 h of culture was greater in the control group (68.6%) than in the treatment group (54.6%). The higher cleavage rate in the control oocytes may arise from the process of selection of directly aspirated oocytes prior to maturation. Oocytes from nonatretic follicles were aspirated and selected on morphological criteria for culture. In contrast, the selection for the treatment group was imposed at the follicle culture stage. Although only healthy, nonatretic follicles were isolated for culture, it seems that judgment based solely on follicle morphology may not be a precise method for selection of nonatretic oocytes. There are many apparently normal follicles that may include atretic oocytes. In addition, the process of atresia itself consists of different stages [50]. Further research on antral follicle culture and the role of specific factors that support follicle growth is necessary. In conclusion, intact antral follicle culture is an efficient method for maintaining bovine oocytes in meiotic arrest for periods up to 48 h. The results suggest that during antral follicle culture, the enclosed oocytes may acquire a greater developmental competence than do oocytes matured directly following aspiration.


   ACKNOWLEDGMENTS
 
The authors would like to thank Professor R.W. Webb, University of Nottingham, coordinator of grant DS0206, and Dr. T. Kruip, Institute for Animal Science and Health (ID-DLO), The Netherlands.


   FOOTNOTES
 
1 This work was supported by Ministry of Agriculture, Fisheries and Food (MAFF grant #DS0206). A.F. was funded by a Ph.D. studentship grant from Ministry of Culture and Higher Education, Islamic Republic of Iran. Back

2 Correspondence. FAX: 0131 440 0434; akbar.fouladi{at}bbsrc.ac.uk Back

Accepted: March 12, 1998.

Received: October 6, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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