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BOR - Papers in Press, published online ahead of print September 3, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.021238
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BIOLOGY OF REPRODUCTION 70, 83–91 (2004)
DOI: 10.1095/biolreprod.103.021238
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

In Vitro Growth and Development of Bovine Oocyte-Granulosa Cell Complexes on the Flat Substratum: Effects of High Polyvinylpyrrolidone Concentration in Culture Medium1

Yuji Hirao2, Takehiro Itoh, Manabu Shimizu, Kosuke Iga, Kazushige Aoyagi, Masato Kobayashi, Masayuki Kacchi, Hiroyoshi Hoshi, and Naoki Takenouchi

Department of Animal Production and Grasslands Farming,3 National Agricultural Research Center for Tohoku Region, Iwate 020-0198, Japan Research Institute for the Functional Peptides,4 Yamagata 990-0823, Japan Yamagata Agricultural Research and Training Center,5 Yamagata 996-0041, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 µm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 µg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360 000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.

follicle, gamete biology, granulosa cells, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian ovary contains a large number of follicles in various growth stages, the majority of which are at the primordial stage. Nevertheless, most of these follicles will eventually be lost in ovaries, and only a small proportion will produce oocytes competent to undergo successful maturation. To utilize the potential female gametes stored in ovaries, it will be important to develop a technology that provides oocytes with suitable culture conditions before they undergo degeneration.

Studies of oocyte growth in vitro have been conducted largely with mouse oocytes. Emphasis has been put on the crucial role in the coupling between oocytes and their companion granulosa cells, because the provision of nutrition from granulosa cells to oocytes is essential to their growth and functioning (reviewed in [1, 2]). The oocyte and its attached layer of granulosa cells are therefore the functional core units of cultures. Three typical systems have been described that support oocyte development from the midgrowth stage to the end of the growth phase in mouse preantral follicles (reviewed in [3, 4]). In the most widely used system, preantral follicles [5–7] or oocyte-granulosa cell complexes prepared from preantral follicles [8, 9] have been cultured on a flat substratum such as a microporous insert membrane [8, 9] or the surface of the culture dish [5–7]. In this situation, complexes adhere to the substratum and create somewhat flat organization with a gentle swelling in the center, where the core unit is located. Another system is characterized by follicular growth, as its spherical shape is sustained on the dish surface [10–12]. In the third system, a three-dimensional matrix supports a spherical structure of follicles [13–15]. The first system differs from the others in that the contents of the follicles are exposed to the culture medium.

In contrast to the variety of systems established for mouse oocytes, there are few good choices for culturing growing bovine oocytes. The available systems sustain a spherical shape either on the dish surface [16–19] or within the three-dimensional matrix [20, 21]. This limited availability may partly explain why the focus of this research has been mostly on the growth of follicles and not on the oocytes. In one case with a three-dimensional matrix system [22], a bovine oocyte grown for 14 days, starting with materials isolated from early antral follicles, acquired competence to undergo embryo development to term. Nevertheless, the overall efficiency of oocyte recovery and subsequent development was low. If bovine oocytes were able to grow in a substratum-adherent manner, more attention would be paid to the quality of oocytes. In addition, the oocyte microenvironment could be manipulated more easily than in the current systems because the oocytes would have immediate access to nutrients and oxygen.

Other attempts to culture follicles of large animals on a flat substratum have been made (see a very brief description in a review [22]), but the publication of details of such attempts has been rare. The larger size of the bovine oocyte (approximately 125 µm in diameter compared with 75 µm for a fully grown mouse oocyte) may make it difficult to apply the mouse system. However, that the larger size probably is not the sole factor that has made accurate application difficult. In studies with ovine oocyte-granulosa cell complexes [23], the complexes that had lost their spherical shape spread out, and these cells migrated away from the oocytes. These findings and our preliminary experiments suggest that it may be more difficult to maintain the proper association between the oocyte and granulosa cells on a flat substratum in large animal species than in the mouse. A similar problem used to exist in cultures of growing mouse oocytes [24] but was solved by improvement of the culture medium [25] and the use of an appropriate substratum [8]. However, difficulties in organizing oocyte-granulosa cell complexes in other species remain.

We have investigated the effects of various macromolecular supplementations to the culture medium, because these supplements could affect the adhesion properties of granulosa cells to the substratum, to oocytes, and to one another. Altered adhesion properties would have a crucial effect on the organization and behavior of the complexes. Supplemented macromolecules also could affect the diffusion of paracrine or autocrine factors in the culture medium. Both oocytes and granulosa cells produce and secrete paracrine factors essential to normal oocyte growth and development [26, 27]. These factors are usually macromolecules, and their movement is affected by interactions with other macromolecules, such as proteoglycans in the extracellular matrix in vivo. Thus, medium abundantly supplemented with macromolecules might provide an environment in which paracrine factors become less mobilized, thus enhancing the opportunities for these factors to encounter the cell-surface receptors. To test whether these presumptive effects could help to overcome the problem of organizing bovine oocyte-granulosa cell complexes, we sought to determine the effects of a high concentration of polyvinylpyrrolidone (PVP), a synthetic polymer, on the organization of the complexes and oocyte development. PVP was chosen because of its excellent solubility, preferable pH, well-known properties, and relatively homogenous size that enabled us to modify the characteristic of the medium precisely.

As starting materials for oocyte growth in vitro, bovine oocytes with a mean diameter of about 95 µm were obtained from early antral follicles, because the growth stage of these oocytes corresponds to that of mouse oocytes of about 56 µm in preantral follicles that largely populate the ovaries of 12-day-old mice [8, 9]. The stages of growing follicles are categorized broadly into two kinds by the histological criterion of before or after the formation of antral cavities. This crucial divergence point, however, does not mark the same stage of oocyte growth among different species, including mice and cattle. Growing mouse oocytes and bovine oocytes of 56 µm and 95 µm, respectively, both represent about 75%–76% of their maximum diameters. The mouse oocytes at this stage are not competent to resume meiosis [8, 28, 29]. Likewise, bovine oocytes of about 95 µm isolated from follicles 0.4–0.7 mm in diameter also are incompetent [20, 30]. Recent studies involving a nuclear transfer technique suggested that nuclei of mouse and bovine oocytes that are <60 µm and <100 µm, respectively, are not equipped with the genomic modification necessary to support development to term [31, 32]. As growth proceeds, the majority of oocytes become competent to complete the first meiotic division when their size reaches 65 and 110 µm in mice and cattle, respectively [28, 33].

Complexes consisting of a growing bovine oocyte and its companion cells were cultured on two types of flat substratum as described for mouse oocytes [8, 9, 34], and the developmental abilities of the resulting oocytes were examined. Our results clearly demonstrate that bovine oocytes can grow in a substratum-adherent manner as maintained in the complex that developed organization similar to that observed in studies with mouse oocyte-granulosa cell complexes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Oocyte-Granulosa Cell Complexes

Bovine ovaries were obtained at a local abattoir and transported to the laboratory in calcium- and magnesium-free Dulbecco PBS (Nissui Pharmaceutical, Tokyo, Japan). From the surface of the ovaries, early antral follicles 0.4–0.7 mm in diameter (Fig. 1A) were isolated. Oocyte-cumulus/granulosa cell complexes (Fig. 1B) were dissected from the follicles with fine forceps [30]. Neither theca cells nor basal laminae were included in the complexes. The cumulus cells and pieces of mural granulosa cells were apparently distinctive, but structural distinction between these became obscure within a day after transfer to a collagen-coated substratum. Therefore, oocyte-cumulus/granulosa cell complexes are hereinafter referred to as oocyte-granulosa cell complexes or simply complexes. Collected complexes were each transferred to a microdrop of Hepes-buffered TCM199 (Sigma, St. Louis, MO) under paraffin oil. Only oocytes with a healthy appearance were used for culture. The diameter of oocytes, exclusive of the zona pellucida, was measured to the nearest 0.5 µm with an ocular micrometer attached to an inverted phase-contrast microscope. These oocytes were not competent to resume meiosis during 24 h of maturation culture under the conditions described below (Table 1).



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  		FIG. 1. A) Bovine follicles at the early antral stage. Bar = 500 µm. B) Isolated oocyte-cumulus/granulosa cell complexes Bar = 200 µm


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  		TABLE 1. Meiotic competence of growing bovine oocytes isolated from early antral follicles 0.4–0.7 mm in diameter and fully grown oocytes cultured for 24 h

Culture Medium for Oocyte Growth In Vitro

The culture medium was TCM199 (Nissui) supplemented with 0.1 mg/ml sodium pyruvate (Wako, Osaka, Japan), 0.08 mg/ml kanamycin, 0.1 µg/ml estradiol-17ß (Sigma), 4 mM hypoxanthine (Gibco-BRL, Grand Island, NY), and 5% fetal bovine serum (FBS; HyClone, Logan, UT). PVP (molecular weight 360 000; Sigma) was supplemented at concentrations of 0%, 2%, or 4% (w/v). In preliminary experiments, this type of PVP exhibited a greater effect than did a lower molecular weight (40 000) type. Concentrations >4% were also tested, but the viscosity of the culture medium increased sharply in an accelerated manner. The approximate viscosity was 1, 6, 14, 25, 50, 82, and 133 mPa·s at 25°C in medium supplemented with 0%, 2%, 4%, 6%, 8%, 10%, and 12% PVP, respectively. The highly viscous medium made the vacuum filtration very inefficient and the handling of complexes difficult. For this reason, concentrations >4% were not used. PVP was dissolved in high-purity water by gentle stirring. The culture medium prepared with this PVP solution and/or with the pure water was filtered (Falcon 7111; Becton Dickinson Labware, Bedford, MA) before being stored at 4°C. Because the culture medium was supplemented with 5% serum, even the control medium should have contained a base level of macromolecules for oocyte culture medium. Hypoxanthine was added to prevent premature meiotic resumption [8, 25] and to support the integrity of oocyte-granulosa cell complexes [25, 30, 35] during the growth period.

Culture of Complexes on the Substratum

Two different types of flat substratum were used. One substratum was a microporous insert membrane (Biocoat Cell Culture Inserts, Falcon 35444; Becton Dickinson) with a pore size of 0.45 µm that fit in a 24-well culture plate. The inserts were prepared according to the manufacturer's instructions. The collagen matrix was rehydrated with medium for 0.5 h, and then the medium was replaced with 0.5 ml of fresh culture medium. Before the inserts were fit to the plates, 0.6 ml of medium was added to each well. Three or four complexes were cultured on each insert. Oocytes were viewable clearly under the light microscope unless they were confined deeply in the mass of granulosa cells. The other type of substratum was the bottom surface of the wells in 96-well culture plates (Biocoat Collagen I Cellware, Falcon 354407; Becton Dickinson), which were also coated with type I collagen matrix. In this case, complexes were cultured individually with 200 µl of culture medium. All cultures were housed in an incubator maintained at 38.5°C under an atmosphere of 5% CO2 in air. The day the complexes were isolated was designated Day 0. The cultures were fed on Days 4, 8, and 12 by removing half of the culture medium and adding the same amount of fresh medium. At the end of a 14-day culture period, oocytes and associated cells were removed from the substratum using a fine pipette and transferred to the maturation culture medium without FSH for washing. Only oocytes with a normal appearance had their diameters measured and were used for further experiments.

Oocyte Maturation and Fertilization In Vitro

For in vitro maturation, oocytes were cultured in microdrops of medium (5–10 complexes/100 µl) made in culture dishes covered with paraffin oil. The culture medium was TCM199 (Gibco-BRL) supplemented with 0.1 mg/ml sodium pyruvate, 0.08 mg/ml kanamycin sulfate, 5% FBS, and FSH (20 mIU/ml; Denka, Japan; or 100 ng/ml; NIDDK, Washington, DC). Oocytes were cultured at 38.5°C under an atmosphere of 5% CO2 in air for 23–24 h. For in vitro fertilization, frozen semen in straws was thawed in water and washed twice by centrifugation in a modified BO solution [36]. Prior to insemination, oocytes were washed twice in BO solution with 7.5 µg/ml heparin, 5 mM caffeine, and 10 mg/ml BSA; oocytes were then transferred into drops of sperm suspension for insemination. The final volume of microdrops for insemination was 100 µl. The oocytes in the sperm suspension were maintained for 6 h at 38.5°C in 5% CO2 in air and then denuded completely by being drawn in and out of a small-bore pipette. Cumulus-free oocytes (one-cell embryos) were transferred to a six-well culture plate (Repro C-1 plate; Research Institute for the Functional Peptides, Yamagata, Japan) [37] containing 0.25 ml serum-free medium (IVD101; Research Institute for the Functional Peptides), covered with oil, and then cultured at 38.5°C in an incubator equilibrated with 5% CO2, 5% O2, and 90% N2. Embryo cultures were continued for up to 9 days.

Four blastocysts derived from oocytes grown in 96-well culture plates were transferred to four recipient cattle. All procedures and care of the animals were in accordance with the guide for the use of agricultural animals at the Yamagata Agricultural Research and Training Center.

Histology

Oocyte-granulosa cell complexes grown on the insert membrane were fixed overnight in 3.7% formaldehyde in PBS and then dehydrated with ethanol and embedded in a methacrylate resin (JB-4; Polysciences, Warrington, PA) according to the manufacturer's instructions. Serial 3-µm sections were cut, reacted with periodic acid-Schiff, and stained by Mayer hematoxylin.

Data Presentation and Statistical Analysis

The distribution of oocyte size in the figures is shown using notched boxes and whisker plots prepared with Statview software (Abacus Concepts, Berkeley, CA), as described by Eppig and O'Brien [9]. Comparisons of oocyte diameter between groups were performed with a t-test. Other data of oocyte recovery and subsequent development are shown as mean percentages of a minimum of three independent experimental replicates, and the variation between experiments is shown with the SEM. For statistical analysis of the percentage, data were subjected to arcsine transformation and ANOVA. The groups were compared using the Fisher protected least significant difference posthoc test om Statview. Difference were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Oocyte Growth and Development on Insert Membranes

Granulosa cells that came in contact with the membrane promptly adhered to it and started to grow outward. By the next day, the complexes had formed a gently conical (almost flat) organization with an oocyte at the center (Fig. 2, A and B). On Day 7, the subpopulations were evident again in the control medium, but the reconstituted cumulus layers were unusually bulky (Fig. 2C). However, in medium supplemented with 4% PVP, the architecture of the complexes was essentially the same as it had been on Day 1, except for a general growth due to cell proliferation (Fig. 2D). By the end of a 14-day culture period in the control medium, the mass of granulosa cells had grown further, which made oocyte observation difficult (Fig. 2E). In medium with 4% PVP, the oocyte existed inside of a domelike structure (Fig. 2F). Characteristics of the complex organization are shown by simplified schematic drawings of cross sectional views (Fig. 2, G and H).



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  		FIG. 2. Morphology of oocyte-granulosa cell complexes developed on the insert membrane on Day 1 (A and B), Day 7 (C and D), and Day 14 (E and F). Complexes were cultured in control medium (A, C, and E) or medium with 4% PVP (B, D, and F). Simplified schematic cross sectional views show complexes cultured in medium containing 0% (G) or 4% (H) PVP. Bar = 200 µm

A group of complexes was planted in a closer formation at the beginning of culture. The typical morphologies developed by complexes on Days 7 and 14 are shown in Figure 3. There was a remarkable enlargement of complexes in the presence of 4% PVP, including the movement of the core units in the same direction (Fig. 3, B and D).



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  		FIG. 3. Morphology of oocyte-granulosa cell complexes cultured on the insert membrane in a close formation on Day 7 (A and B) and Day 14 (C and D). Complexes were cultured in control medium (A and C) or medium with 4% PVP (B and D). In medium with 4% PVP (B), the complexes enlarged in the direction shown by arrows. Bars = 500 µm

After the 14-day culture period, oocytes with their associating cells were removed from the membrane by a mechanical method. Many oocytes grown in control medium were still confined in a somewhat large cell mass (Fig. 4A). However, a proportion of these oocytes became denuded easily by gentle pipetting, suggesting weak contacts between cells. In contrast, oocytes grown in medium with 4% PVP were typically enclosed within a single layer of granulosa cells (Fig. 4, B and C). The physical association between these cells was firm, as indicated by how difficult it was to tear the cells apart by pipetting. Histological observations also revealed that the innermost cell layer was arranged closely along with the oocyte surface in medium with 4% PVP, but other cells made contact with neighboring cells more randomly (Fig. 4, D and E).



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  		FIG. 4. Morphology of oocyte-granulosa (cumulus) cell complexes recovered from the insert membrane after a 14-day culture period (A–C). Complexes were grown in control medium (A) and in medium supplemented with 4% PVP (B), and the same complexes are shown at larger magnification (C). Histological sections of a complex were examined after growth culture on the insert membrane (D and E), with an overview (D) and a higher magnified view of the oocyte in the same section (E). Note the single layer of cells arranged along with the oocyte. One of these cells is indicated by an arrow. Bars = 200 µm (A and B) and 50 µm (C–E)

The percentage of oocytes recovered completely enclosed within granulosa cells was higher in medium with 4% PVP (88% ± 6%) than in medium with 2% PVP (69% ± 5%) or 0% PVP (58% ± 5%) (Fig. 5).



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  		FIG. 5. Percentage of oocytes recovered completely enclosed with granulosa (cumulus) cells from the insert membrane after a 14-day culture period. Data are expressed as the mean (±SEM) percentage of complexes. An asterisk indicates a significant difference (P < 0.05)

An increase in oocyte size in vitro, from 95 µm before culture, was evident in all three groups (Fig. 6). The mean diameter attained in vitro was 105, 108, and 113 µm in medium with 0%, 2%, and 4% PVP, respectively (shaded boxes in Fig. 6). Oocytes grew to a larger size in medium with 4% PVP than in the other two media (P < 0.05), but these oocytes were still smaller than those grown in vivo (Fig. 6).



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  		FIG. 6. Comparison of oocyte diameter after growth on the insert membrane for a 14-day culture period. The white notched box on the left represents the diameter of oocytes isolated from early antral follicles (0.4–0.7 mm). The shaded boxes indicate the diameter of oocytes cultured for 14 days in medium supplemented with 0%, 2%, and 4% PVP. The white box on the right represents the diameter of fully grown oocytes obtained from antral follicles >3 mm. Numbers above the box plots indicate mean diameters (µm). Different letters beside the mean diameters indicate significant differences (P < 0.05). The results of the experiments shown in Figure 5 are included

During the maturation culture, expansion of oocyte-associated granulosa cells was obvious. When a portion of the oocytes were fixed and examined for maturation in preliminary trials, oocytes <115 µm almost never progressed to metaphase II, but 6 of 10 oocytes >=115 µm reached the metaphase II stage. After in vitro fertilization, cleavage was observed in 14%, 28%, and 47% of oocytes that had been grown in medium supplemented with 0%, 2%, and 4% PVP, respectively, and 2%, 7%, and 14% developed to the blastocyst stage, respectively (Table 2). In comparisons of the frequencies of blastocyst formation from the number of oocytes initially used for culture, the percentage formed from oocytes derived from the medium with 4% PVP was significantly higher than that from oocytes derived from medium with 0% PVP but was lower (although not significantly) than that from in vivo grown oocytes (Table 2).


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  		TABLE 2. Developmental competence of bovine oocytes grown on the insert membrane with culture medium supplemented with different concentrations of PVP

Oocyte Growth and Development in 96-Well Culture Plates

Complexes also were cultured in a combination of another substratum and medium with 4% PVP. Oocytes had been maintained enclosed with a thin layer(s) of granulosa cells. However, the domelike structure was not developed. A common organization was a two-dimmensional rimmed pattern of granulosa cell proliferation (Fig. 7). The oocyte and the peripheral rim area and the ridge(s) of cells connecting them created cavities, the number and shape of which were not consistent (Fig. 7, A–D).



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  		FIG. 7. Morphology of oocyte-granulosa cell complexes cultured individually on the bottom surface of the well in 96-well culture plates in the presence of 4% PVP. The basic architecture was essentially the same among complexes, although oocytes with companion granulosa cells (arrows) were surrounded by various numbers of cavities (arrowheads); one (A), two (B), three (C), or more (D) cavities. Bars = 200 µm

After growth in vitro, 51.0% ± 4.6% oocytes were recovered enclosed by cell layer(s). Oocytes <110 µm were discarded at this stage. The remaining oocytes, with a mean (± SEM) diameter of 118.3 ± 0.9 µm, were examined for their ability to undergo preimplantation development. When these complexes (n = 69) were used for in vitro maturation and fertilization, 61.4% ± 4.2% cleaved and 21.9% ± 4.5% developed to the blastocyst stage, which combined represented 8.8% ± 2.1% of the oocytes originally used for the growth culture (n = 173).

Four fresh blastocysts were transferred to four recipient cows, and the pregnancy of one of these cows was confirmed. A male calf weighing 32 kg was delivered on April 15, 2003 (Day 272).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The studies presented here were performed using a culture model in which bovine oocyte-granulosa cell complexes develop an organization similar to that of the rodent models [5–9, 35, 38, 39]. A high concentration of PVP exerted a profound influence on the morphology of this organization. Oocytes that were grown in our systems acquired functional competence, as determined by the incidence of oocytes undergoing maturation, fertilization, and preimplantation development in vitro. Furthermore, the birth of live young derived from the oocytes suggests that the genomic modification, which had only partially been made when isolated from early antral follicles [32], was established during the culture period.

When compared with the follicles of mice and rats, bovine follicles become extremely large. It has been suspected that the large size of these follicles restrains the effectiveness of culture systems in which the spherical shape of follicles is sustained [4]. The establishment of such systems in which the factors in the medium are directly accessible to oocytes and companion cells has been sought for large animal species [4]. The systems developed in the present study would essentially meet these conditions. The overall efficiency of oocyte recovery after growth culture and the ability of the oocytes to undergo further development were greatly improved, as compared with studies in which the successful production of a calf from oocytes of a similar growth stage was also accomplished [20]. The frequency of producing blastocysts from the oocytes originally used for growth culture was 1%–2% in the system with a three-dimensional cell matrix [20], whereas that in the present studies was about 10%. Presumably, more oxygen and nutrients might have reached these oocytes than reached oocytes enclosed by follicular walls, because the oxygen that entered intact follicles should be mostly metabolized by the mural granulosa cells before reaching the oocytes [40]. However, oocytes cultured in control medium in our system may have experienced hypoxic conditions because they were confined in a large cell mass.

Successful growth of oocytes in reorganized complexes was realized because of a modification of the culture medium. PVP and polyvinylalcohol are frequently added to medium as a substitute for macromolecules in serum, which provide some colloid osmotic pressure and are useful for preventing loss of oocytes/embryos due to sticking to the glass or plastic surface. Nevertheless, the general concentration of these additives to oocyte culture medium is not >0.4% (w/v). The control medium containing 5% serum apparently did not support appropriate complex reorganization. Therefore, a high concentration of PVP may support function(s) different from those customarily associated with PVP. For example, the addition of a high concentration of hyaluronan is beneficial in promoting preimplantation development of bovine embryos [41], indicating the importance of medium viscosity. Viscosity was a property obviously altered by the addition of PVP. Thus, its addition could have influenced the health and function of individual cells, as seen in the embryos [41], and the assemblage of such affected cells could have been reflected by the characteristic reorganization of complexes.

The modification of culture medium also could have affected the interactions of cells. Bidirectional interactions between oocytes and granulosa cells [26, 42] are crucial for both oocytes and complexes to grow normally [43–45]. In follicles, oocytes secrete essential factors that are delivered to somatic cells in a paracrine fashion [27]. An example of such a regulation is the differentiation of granulosa cells into cumulus cells and mural granulosa cells [44, 46]. In the culture systems described here, as pointed out by Eppig et al. [3], factors secreted from cells could diffuse into culture medium before they come in contact with cells in the target. The complexes were isolated from early antral follicles, and thus there were histological subpopulations from the beginning of culture. However, the divergence may not necessarily be maintained under suboptimal conditions, as suggested by the morphology of the complexes in control medium. By contrast, in medium with 4% PVP, complexes were reorganized, and cumulus cells were clearly distinguished from mural granulosa cells. A plausible explanation for this difference is that more factors were localized near the complexes because of restricted diffusion. However, it is not known why a high concentration of macromolecules is not necessary in mouse or rat models that have a culture system with the same architecture. It is important to determine the requirements specific to each species to improve culture systems.

In a different experiment (not shown), the concentration of PVP was raised as high as 12% (w/v) with adjusted osmotic pressure (295 mOsm). The viscous medium somehow impaired the growth of complexes, the appearance of which was not healthy. However, three of four oocytes were recovered apparently alive enclosed in complexes after a 14-day culture period. One of those even extruded a polar body during the subsequent maturation culture. Therefore, although 12% PVP in medium seems to have some deleterious effects on the complexes, the medium is not lethal in itself and can permit complexes to survive for 14 days.

On both the insert membranes and the bottom surfaces of the 96-well plates, complexes were highly organized in the presence of 4% PVP. A domelike structure, previously reported for mouse cultures [5, 6], was typical on the insert membrane. In 96-well culture plates, however, such a structure did not appear. Instead, a two-dimensional antrumlike structure was notable. This morphology was similar to that observed in studies with rats [38, 39]. Both substrata had the collagen matrix, and thus the difference in morphology probably was not due to the manner of cell attachment to the substratum. The major difference was the free diffusion of culture medium from under the microporous membrane in the insert system, which might have provided an improved physiological environment for granulosa cells and thus induced their vigorous proliferation. When the concentration of estradiol was reduced to <=5 ng/ml from our standard concentration of 0.1 µg/ml, the domelike structure did not appear, even on the insert membrane (not shown). However, cell proliferation was not so active in 96-well culture plates, as suggested by the fact that nearly half of the oocytes had become denuded or degenerated during the 14-day culture period. The development rate of such oocytes was about 10% in the insert system. Perhaps further induction of mitotic proliferation would be desirable in 96-well culture plates. Gonadotropin, which was not added to our medium for oocyte growth, may be an option. However, when FSH was added with insulin to the medium for mouse oocyte growth, deleterious effects on embryonic development were evident [47]. Thus, supplementation with FSH should be considered carefully. Inversely, the vigorous proliferation of granulosa cells alone was not sufficient to organize the domelike structure, as suggested by the aberrant organization of complexes in control medium. Oocyte-induced differentiation of granulosa cells might be a prerequisite for a domelike structure to appear. The domelike structure is not an indispensable architecture for the oocytes to acquire the competence to undergo preimplantation development to the blastocyst stage.

Culture systems that can support bovine oocyte growth from still earlier stages are desirable. Our finding that bovine oocytes can be grown on a flat substratum is important for determining the direction of future improvement of culture systems. The overall yield of blastocysts was similar between the systems with the insert membranes (12%) and the 96-well culture plates (9%). This study is only the first step, and substantial improvement is necessary. For example, the choice of culture medium has a major impact on the results of oocyte growth in vitro [48]. Our experiments using Waymouth medium suggest that results similar to those presented here can be obtained with different media (not shown). Extensive studies on the effects of other basal media will be necessary. The precise role of PVP in the development of complexes remains to be determined. It will be important to resolve whether the characteristic complex organization would primarily depend on the medium viscosity. The effects of other macromolecules, which may add some viscosity to the medium, also should be a subject of investigation.


   ACKNOWLEDGMENTS
 
We thank Dr. John J. Eppig for helpful suggestions in the preparation of this manuscript and Dr. A.F. Parlow and The National Hormone and Pituitary Program of the NIDDK for providing the FSH. We also thank Dr. Tomotada Ono for his assistance in the measurement of medium viscosity.


   FOOTNOTES
 
1 This research was supported by a grant for the Pioneer Research Project (12072 to Y.H.) from the Ministry of Agriculture, Forestry and Fisheries of Japan and also by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences of Japan (to H.H.). Back

2 Correspondence: Yuji Hirao, Animal Breeding and Reproduction Laboratory, Department of Animal Production and Grasslands Farming, National Agricultural Research Center for Tohoku Region, 4 Akahira, Shimo-kuriyagawa, Morioka, Iwate 020-0198, Japan. FAX: 81 19 643 3542; yujih{at}affrc.go.jp Back

Received: 14 July 2003.

First decision: 30 July 2003.

Accepted: 18 August 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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