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Regular Article |
a Infertility Center, Department of Obstetrics and Gynecology and
b Department of Gynecologic Oncology, Ghent University Hospital, B-9000 Ghent, Belgium
ABSTRACT
The objectives of the present study were to achieve 1) oocyte maturation, 2) oocyte competence of fertilization, and 3) oocyte competence of embryogenesis with oocytes from primordial follicles obtained from cryopreserved newborn mouse ovaries by using a two-step method. In the first step, frozen-thawed newborn mouse ovaries were transplanted under the kidney capsule of recipients for the initiation of growth from the primordial follicle stage on. In the second step, growing preantral follicles in the ovarian grafts were recovered and cultured. The results demonstrated that primordial follicles were able to be recruited to preantral follicles during the period of transplantation, and preantral follicles could be mechanically isolated from ovarian grafts. Under the present in vitro culture conditions, 85.8% of the isolated follicles (n = 332) from ovarian grafts survived the 12-day in vitro culture process, 84.9% of the recovered oocytes (n = 285) were germinal vesicle breakdown (GVBD)-competent, and 76% of the oocytes that underwent GVBD (n = 242) developed to the metaphase II (MII) stage. In the in vitro fertilization experiments, 75.4% of 142 inseminated MII oocytes underwent fertilization and cleavage to the 2-cell stage. Subsequently, 79.7% of the 2-cell-stage embryos (n = 69) progressed to the late morula-early blastocyst stage. Transfer of late morula-early blastocyst embryos resulted in the production of live offspring. From our experiments, it may be concluded that in vivo maturation by grafting followed by in vitro maturation of frozen-thawed primordial follicles can restore fertility in mice. This model could be useful for a similar application in the human.
fertilization, follicular development, oocyte development, ovary
INTRODUCTION
The pool of primordial follicles represents the definitive number of female gametes for the entire reproductive life span. Primordial follicles can survive the cryopreservation process at a rate of 70–80% [1–3] and constitute a potential source for preservation and later use. For conservation of their fertility potential, patients could be presented the option of cryopreservation of ovarian tissue before cancer treatment [4, 5]. Culture systems for preantral follicles and oocyte granulosa cell complexes from the preantral stage have been successfully established in mice [6–10]. Although promising progress has been made in humans [11, 12], primary and primordial follicles cannot yet be brought to full maturation by using the same culture system [13]. Therefore, patients cannot yet be given a realistic prospect of restoration of their fertility by in vitro oocyte maturation of primordial follicles of cryopreserved ovarian tissue [14–16].
Fertility can be restored by auto- or allografting and xenografting in a number of species [17–23]. The survival and follicular growth of primordial follicles following grafting of ovarian tissue is higher than what can be obtained by in vitro culture of individual primordial follicles or ovarian cortical slices. While the results of autografting experiments are encouraging, the most important proviso remains that ovarian tissue from cancer patients must not harbor malignant cells that could reintroduce cancer after transplantation [24]. This risk would be reduced or eliminated by complete in vitro maturation of isolated ovarian follicles. The sequential combination of both grafting and in vitro maturation could exploit the advantages of both procedures. We have, therefore, established a two-step strategy for complete primordial follicle maturation by combination of in vivo transplantation and in vitro culture in a mouse model [25]. The objective of the present study was to achieve complete development of the oocytes from primordial follicles present in cryopreserved newborn mouse ovaries. The developmental capacity of primordial oocytes was evaluated with respect to oocyte maturation, oocyte competence of fertilization, and embryogenesis.
MATERIALS AND METHODS
Animals and Experimental Groups
C57Bl/6j x CBA/Ca F1 hybrid mice used in this study were housed and bred in the Central Animal House of the Ghent University Hospital. Ten- to twelve-week-old F1 female mice were used as recipients for ovarian transplantation. One-day-old F1 females were used for the collection of newborn mouse ovaries. The F1 females (2 wk) were used as controls for the isolation of early preantral follicles from their ovaries. The F1 females (10–12 wk) were used for collection of in vivo-matured oocytes. Sperm from F1 males (10–12 wk) were used for in vitro fertilization, and CD1 vasectomized males (8–10 mo) were used for mating studies. Newborn ovarian tissue collected from 1-day-old F1 female mice was cryopreserved prior to transplantation. Two weeks after ovarian transplantation, recruited preantral follicles were isolated from grafted ovaries and put to culture in the experimental group. Preantral follicles isolated from 2-wk-old F1 females were cultured in vitro serving as the in-vitro control group. Ten- to twelve-week-old F1 female mice were superovulated with 5 IU eCG (Folligon; Intervet, Turnhout, Belgium) followed by 5 IU hCG (Chorulon; Intervet) given 48 h later. Cumulus-enclosed oocytes collected 14 h after hCG injection underwent in vitro fertilization serving as the in vivo control group. Ethical approval for this study was obtained from the Animal Research Ethical Committee, Ghent University Hospital.
Cryopreservation of Newborn Mouse Ovaries
Ovaries from 1-day-old F1 females were slowly cryopreserved using a modification of the method of Gosden et al. [26]. Briefly, whole ovaries were suspended in a cryoprotectant medium of Leibovitz 15 (L-15), 10% (v/v) heat-inactivated fetal bovine serum (FBS; Life Technologies, Merelbeke, Belgium) and 1.5 M dimethylsulfoxide (DMSO; Sigma, Bornem, Belgium) on crushed ice. Each ovary was drawn up into the middle of a 0.25-ml plastic freezing straw (type-2A 175; Industrie de la Médicine Vétérinaire, L'Aigle, France) in a volume of 
100 µl of cryoprotectant medium. The straws were sealed with polyvinyl chloride powder and held on ice for 20 min before they were placed in a programmable biological freezer (Planer Biomed, Sunbury, Middlesex, UK) precooled to 0°C. The straws were cooled at -2°C/min to -7°C and held for 5 min, seeded manually, held at -7°C for a further 10 min, cooled to -40°C at -0.3°C/min, further cooled to -140°C at a cooling rate of -10°C/min, and finally transferred to liquid nitrogen (LN2) for storage. For thawing of the ovaries, the straws were removed from LN2, held in air for 20 sec, and transferred to a water bath at room temperature for 10–20 sec. The contents of straws were emptied into L-15 medium with 10% (v/v) heat-inactivated FBS. The cryoprotective agent was removed by repeated rinsing. The mouse ovaries were kept in this medium at room temperature till transplantation.
Transplantation Procedure
The recipient mice were anesthetized with i.p. injection of 100 µl of sodium pentobarbital (Nembutal; Sanofi, Brussels, Belgium) diluted 1:4 in PBS (Sigma). The transplantation procedure used was a modification of the method of Gosden et al. [18]. Briefly, the left kidney was exteriorized through a dorso-horizontal incision. A small hole was torn in the kidney capsule using fine watchmakers' forceps. Two whole frozen-thawed newborn mouse ovaries were inserted through the small hole underneath the renal capsule of each recipient mouse. Each recipient's own ovaries were removed by cautery at the top of the uterine horns.
Isolation and In Vitro Maturation of Preantral Follicles
Two weeks after transplantation, recipient mice were sacrificed by cervical dislocation, and ovarian grafts were aseptically removed from the kidney and dissected from other tissue in L-15 medium supplemented with 10% (v/v) heat-inactivated FBS, 100 IU/ml penicillin-100 µg/ml streptomycin (Roche Diagnostics, Brussels, Belgium) (designated L-15* medium). Mechanical isolation of ovarian follicles from the grafts was done using 25 5/8-gauge needles (Becton Dickinson, Erembodegem, Belgium). Selected early preantral follicles that had centrally located oocytes with two to four layers of granulosa cells were washed in L-15* medium three times. The follicles were put individually in 10-µl culture medium droplets under mineral oil (Sigma) in 60- x 15-mm tissue culture dishes (Falcon; VEL, Leuven, Belgium). The follicle culture system used was a modification of the method of Cortvrindt et al. [8]. On Day 2, an additional 10 µl of medium was added to each droplet. From Day 4 onward, medium replacement was done every other day by changing half of the droplet (10 µl). The culture medium was 
-minimal essential medium (MEM-glutamax; Life Technologies) enriched with 5% (v/v) heat-inactivated FBS, 5 µg/ml insulin, 10 µg/ml transferrin, 5 ng/ml selenium (ITS; Boehringer Mannheim, Mannheim, Germany), 100 mIU/ml recombinant FSH (Puregon; Organon, Oss, the Netherlands), and 0.1% (w/v) fetuin (Life Technologies). The culture dishes were incubated at 37°C at 100% humidity and 5% CO2 in air. At Day 12 of culture, final oocyte maturation was induced by addition of 2.5 IU/ml hGC (Pregnyl; Organon) to the in vitro culture droplets. Mucified oocyte-cumulus complexes were collected for in vitro fertilization 14–16 h later. Age-matched ovaries were obtained from 14-day-old mice. Early preantral follicles were isolated from these ovaries and cultured in vitro in the same conditions as described above. This group of follicles served as the in vitro control group for oocyte maturation, fertilization, and embryogenesis.
Fertilization In Vitro
Sperm preparation The caudae epididymis were removed from 10- to 12-wk-old F1 males and placed into 1-ml drops of potassium simplex optimized medium (KSOM) supplemented with 0.4% (w/v) BSA (Sigma) (designated KSOM + BSA, Table 1) under oil in sperm dispersion dishes (#3037; Falcon). Epididymal contents were carefully squeezed out and the residual caudal tissue was discarded. The dispersion dishes were placed in a 6% CO2:6% O2 incubator for 20 min to allow the sperm to disperse. Ten microliters of sperm suspension was added to 90-µl drops of KSOM + BSA in the fertilization dishes (#3002; Falcon). Capacitation was allowed to proceed for 45–60 min at 37°C in the incubator. The dispersion and fertilization dishes were prepared the day before the start of each trial and were equilibrated overnight in the incubator. After capacitation, oocyte-cumulus complexes were transferred to the 100-µl fertilization droplets (10 µl sperm suspension + 90 µl KSOM + BSA).
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Oocyte collection Mucified oocyte-cumulus complexes from in vitro-cultured follicles from 2-wk-old mouse ovaries or 2-wk-grafted ovaries were collected. Approximately 20 complexes were transferred to each fertilization droplet. Superovulated females were killed 14–16 h post-hCG administration, and the entire oviduct was dissected out to L-15* medium at 37°C. The ampullae were opened by tearing with the 25-gauge needle and cumulus-oocyte masses were released. Two cumulus-oocyte masses were transferred per fertilization droplet and served as control group of in vivo-matured oocytes.
Fertilization In vitro fertilization (IVF) was carried out in 100-µl droplet of KSOM + BSA as described above. Incubation was allowed to proceed for 4 h at 37°C in a 6% CO2:6% O2 atmosphere. At the end of this period, the cumulus cells and attached sperm were removed from the oocytes by drawing the oocytes in and out of a fine-drawn pipette. Following five times rinsing, about 10 inseminated oocytes were incubated in 20-µl drops of KSOM + BSA in culture dishes (#3001; Falcon). Oocyte maturation status was scored as GV when the germinal vesicle (GV) was present, as GVBD (germinal vesicle breakdown) when the GV had broken down, and as MII (metaphase II) when the first polar body had been extruded. Some oocytes were mechanically damaged with the fine pipettes during denudation.
Embryo Culture and Transfer
Fertilized oocytes were cultured in 20-µl drops of KSOM + BSA under 2 ml mineral oil in culture dishes (#3001; Falcon) for approximately 72 h without changing the medium. The dishes were prepared the day before the start of culture and equilibrated overnight in a 6% O2:6% CO2 atmosphere. Fertilization was scored as the percentage of cleavage to the 2-cell-stage on Day 2 (insemination day defined as Day 1). Embryo development was determined at the late morula-early blastocyst stages at Day 4. Pseudopregnant adult F1 females were produced by mating hyperstimulated females with vasectomized CD1 males. Hyperstimulation was accomplished with the same regimen as described above. When a vaginal copulatory plug was present in the morning after mating, that day constituted Day 1 of pseudopregnancy. Late morula-early blastocyst embryos were transferred to the uteri of Day 3 pseudopregnant mice with a glass pipette, five or six embryos to each uterine horn. Foster mothers were allowed to deliver and raise pups.
Assessment of Steroid Production in In Vitro Follicle Culture
Every other day from Day 4 of follicle culture onward, culture medium was refreshed with retrieving and adding 10 µl of the medium. The 10-µl samples of retrieved conditioned medium from surviving follicles were pooled together per culture dish. Estradiol production was measured with an RIA from Clinical Assay (Estradiol-2; DiaSorin, Brussels, Belgium). The sensitivity of this assay is below 5 pg/ml at 95% confidence limit and its total precision is <10% (% coefficient of variation, CV). Progesterone production was measured with an in-house developed RIA (Department of Endocrinology, Ghent University Hospital [27]), with a sensitivity of 0.05 ng/ml and a total precision <9% (%CV).
Statistical Methods
Follicle survival data and nuclear maturity were analyzed by chi-square analysis for trend. The proportions of oocytes that underwent fertilization and late morula-early blastocyst formation were compared by contingency table analysis followed by chi-square testing. Hormone concentrations in function of culture time were analyzed by one-way analysis of variance (Friedman test). Student's t-test was used for comparison of means of hormone concentrations between the groups of cultured follicles from 2-wk-old mouse ovaries and from 2-wk-grafted ovaries. P < 0.05 was considered statistically significant.
RESULTS
All grafted newborn mouse ovaries (n = 46) were recovered 2 wk after transplantation from 23 recipients in five separate experiments. A total of 332 early preantral follicles that had two to four layers of granulosa cells were mechanically isolated from ovarian grafts. For the in vitro control group, a total of 575 preantral follicles were isolated from 14-day-old mouse ovaries (n = 22). These preantral follicles were cultured individually in vitro for a total of 12 days (Fig. 1). The majority (85.8%) of follicles from ovarian grafts survived the process of in vitro culture; this was evidenced by proliferation of granulosa cells and sustenance of oocytes within their companion granulosa cells (Table 2). A few oocytes degenerated. In the in vitro control group, the follicle survival rate was 92.5%, which is significantly higher than the percentage obtained in the experimental group (P = 0.002).
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After addition of the hCG stimulus on Day 12 in the experimental group, 15.1% of all surviving oocytes remained blocked in the GV stage, and 84.9% of recovered oocytes underwent GVBD. Progress to MII was achieved by 76.0% of GVBD oocytes (Table 2). The remaining GVBD oocytes (24%) were blocked at metaphase I. Compared with the in vitro control group, there was a significant difference in GVBD rate (90.8% in control, P = 0.02), but not in MII formation rate (71.0% in control, P = 0.2).
To assess the capacity of the matured oocytes produced from ovarian transplantation and follicle culture, matured oocytes from the experimental group and the in vitro control group were fertilized in vitro and cultured in KSOM + BSA (Fig. 2). In vivo-matured oocytes from superovulated mice were fertilized in vitro under the same condition (in vivo control group). Zygotes were examined for cleavage to the 2-cell stage 24 h after fertilization; for late morula-early blastocyst formation and embryo developmental competence in vivo by transfer to foster females at Day 4. The results are summarized in Table 3. In the experimental group, a total of 142 oocytes were fertilized in three separate experiments; 107 of 142 cleaved to the 2-cell stage (75.4%). Sixty-nine 2-cell-stage embryos were maintained in culture (the remaining 38 2-cell embryos were processed for a cytological study, data not shown); 55 of 69 formed late morula-early blastocysts from 2-cell embryos (79.7%). In the in vitro control group, a total of 162 oocytes were inseminated in three separate experiments; 77 of 162 cleaved (47.5%), and 62 of 77 formed late morula-early blastocysts from 2-cell embryos (80.5%). In the in vivo control group, a total of 254 in vivo-matured oocytes retrieved after superovulation were fertilized in six separate experiments; 222 of 254 cleaved (87.4%), 196 of 222 formed late morula-early blastocysts from 2-cell embryos (88.3%) at 72 h after insemination. The fertilization rate in the control group of in vivo-matured oocytes was significantly higher than that in both the experimental and the in vitro control groups (P = 0.004, P < 0.001, respectively). Of interest is the lower fertilization rate observed in the in vitro control group compared with the experimental group (P < 0.001). No significant difference was observed in the yield of formation of late morula-early blastocysts from 2-cell embryos among the three groups.
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Late morula-early blastocyst embryos at Day 4 of culture were transferred in groups of five or six to the uteri of Day 3 pseudopregnant foster mothers. The results are summarized in Table 4. A total of five live pups (two were cannibalized) were produced in the experimental group. The yield from the experimental and the in vitro control groups appeared to be similar, but it was lower in both cases than the yield from the in vivo control group.
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The secretion profiles of estradiol and progesterone from in vitro-cultured follicles of 2-wk-old grafts or ovaries are given in Figure 3. Average basal progesterone concentrations remained at a level of 0.5 ng/ml up to Day 10 in both groups. From Day 10 onward, luteinization of granulosa cells occurred as evidenced by the increase of progesterone levels to approximately 1.3 ng/ml. After the hCG stimulus on Day 12, a substantial progesterone output was observed in the experimental and in vitro control groups (Friedman test, P < 0.001). Estradiol (E2) production continued to increase progressively up to Day 12. The production of E2 reached 1800 pg/ml and 7000 pg/ml in experimental and in vitro control groups, respectively (t-test, P < 0.001).
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DISCUSSION
Cryopreserved ovaries from newborn mice were thawed and transplanted under the kidney capsule of recipients to initiate follicular growth from the primordial stage. Preantral follicles from the ovarian grafts were recovered and cultured. This procedure yielded mature oocytes that could be fertilized in vitro and gave rise to live birth. This is the first study to show that mouse oocytes from cryopreserved primordial follicles can be developed into fertilizable mature oocytes by using a combination of in vivo transplantation of cryopreserved newborn mouse ovaries and in vitro culture of follicle-enclosed oocytes.
Primordial follicles are the most abundant stage of ovarian follicles present in the ovary at every age, and tens to hundreds of these small follicles can be obtained using a simple biopsy procedure. Undifferentiated primordial oocytes are relatively quiescent, are small, and do not have a zona pellucida and cortical granules. Due to these characteristics, they tolerate freezing and thawing much better than mature oocytes [28]. Live births were derived from frozen-thawed individual primordial mouse follicles after orthotopic replacement [29]. However the isolation of primordial follicles is still difficult, especially from fibrous ovarian tissue. Primordial follicles can be cryopreserved and stored in slices of ovarian cortex in LN2 for as long as required. Freezing immature primordial follicles in situ in slices of ovarian cortex could serve as an alternative strategy to oocyte and embryo freezing and could eventually be used to preserve and restore fertility, e.g., in cancer patients. Under optimal conditions more than 70–80% of the estimated original population of follicles can survive [1–3].
The complete development of mouse oocytes in vitro from the primordial follicle stage has been reported by only one group [30]. Intact ovaries of newborn mice, containing only primordial follicles, were cultured for 8 days, producing preantral follicles with two layers of granulosa cells. The preantral follicles were enzymatically isolated from the cultured ovaries and subsequently developed in a collective in vitro culture system. The percentage of the oocytes that acquired competence to undergo GVBD was 32%, and 22% of GVBD oocytes progressed to the mature stage (MII). Forty-two percent of the mature oocytes were fertilized, and less than 2% of the 2-cell-stage embryos developed to the blastocyst stage. Two mouse pups (one surviving) were born after transfer of 190 2-cell-stage embryos to pseudopregnant females. Complete in vitro systems are of interest for developing a method for restoring fertility in patients after cancer treatment as they avoid the need for transplantation, which could reintroduce cancerous cells to the patients. The method used by Eppig and O'Brien [30] is interesting as it permits follicle growth from primordial to mature stage in vitro; however, the yield of fertilizable and developmentally competent oocytes is low.
Although substantial progress has been made in the field of ovarian cryobiology, in vitro culture and maturation technology for primordial follicles within cortical slices of ovaries is still in its infancy [13, 14, 31, 32]. To grow isolated individual follicles or follicles in ovarian slices over extended periods in vitro has proved to be very difficult [16, 32, 33]. Fertility can be restored to adult mice by transferring fresh or frozen-thawed primordial follicles isolated from immature donor organs to the periovarian capsule [29, 34]. Ovarian cortex transplantation may therefore provide a practical alternative for the restoration of fertility after ovarian cryopreservation because isolation of primordial follicles is still difficult. Autologous and allogeneic orthotopic transplantation of cryopreserved ovarian tissue resulted in steroidogenic activity [35], live births in mice [19, 36, 37], and resumption of follicular growth, ovulation [22], and delivery of a lamb [17] in sheep. Recently, ovarian follicle growth could be established after transplantation of cryopreserved ovarian tissue in the human [38]. Heterotopic transplantation (e.g., under the kidney capsule or subcutaneously) of fresh or frozen-thawed ovarian tissue resulted in folliculogenesis, steroidogenesis, and ovulation in mice [19, 39] and in sheep [22]. Xenografts to immunodeficient mice have been successfully used as an experimental model for evaluation of viability of ovarian tissue in vivo [18, 40]. Human primordial follicles in ovarian xenografts retain their developmental potential and form antral follicles following gonadotropin stimulation [21, 23]. In summary, primordial follicles can survive and initiate maturation after transplantation. The ovarian slices are well suited for grafting because most of the primordial follicles are present in the periphery and are the first to benefit from revascularization. In addition, ovarian follicles may be resistant to ischemia, as they normally develop within an avascular epithelium and a relatively hypoxic environment [41]. Primordial follicles are more resistant to the effect of ischemia than growing follicles presumably because they are dormant and have a low metabolic rate. Approximately 50% of the primordial follicle population survives in isologous grafts in mice [42].
Follicle growth and differentiation processes during culture might be reflected by the secretion of steroids. Estradiol production, which continued to increase up to Day 12 of culture, indicated the progressive proliferation and aromatase activity of granulosa cells. Basal luteinization occurred from Day 10 on, in particular after hCG stimulation at Day 12, as evidenced by the increase of progesterone production. It also indirectly demonstrated the expression of LH receptors on the differentiated granulosa cells. In vivo, LH receptor expression in mural granulosa cells starts to appear only shortly before the follicles reach the preovulatory stage [43]. The luteinization of granulosa cells in vitro might reflect the maturity of follicles [44]. The E2 secretion in the conditioned medium production by the follicles from 2-wk-grafted cryopreserved ovaries was significantly lower compared with that in the in vitro controls (follicles from 2-wk-old mouse ovaries). This might indicate that the number of granulosa cells is reduced, possibly as a result of granulosa cell death during cryopreservation and/or impaired proliferation of granulosa cells during transplantation. That the granulosa cell function might also be affected by cryopreservation and transplantation is less likely because it has been demonstrated that steroid production by cryopreserved granulosa cells is not different from that of nonfrozen ones [45].
The developmental potential of embryos produced in our study was assessed by transferring the late morula-early blastocyst embryos into the uteri of stimulated pseudopregnant mice. We used gonadotropin-stimulated mice for producing pseudopregnant mice. The offspring rate of 25% found in the in vivo control group was lower than that found by other investigators when embryos were transferred to untreated pseudopregnant females (70%) [46]. This low rate is thought to be caused by low embryo quality, or low endometrial receptivity, or by a combination of both. Ovarian hyperstimulation with exogenous gonadotropin may elevate serum E2 levels that might impair the developmental capacity of embryos and result in low implantation and pregnancy rates [47, 48]. Anyhow, the yields of live young obtained from follicles in 2-wk ovaries or grafted ovaries were lower than that in the in vivo control group. In the mouse, blastocyst formation occurs after five or six rounds of cell division and results in the formation of the first two lineages, the inner cell mass (ICM) and trophectoderm (TE). The regulation of cell division, differentiation, and cell death in embryos is likely to be critical for later development. Unregulated cell death in the ICM would compromise later development, as a critical number of ICM cells are required for normal postimplantation development. It is necessary to investigate the quality of embryos derived from the two-step strategy in terms of total embryo cell number, cell allocation to ICM and TE, and occurrence of apoptosis in blastomeres.
In conclusion, a combination of in vivo transplantation and in vitro follicle culture can sustain full oocyte maturation in the mouse system. This approach might be a useful paradigm for the development of a method to restore fertility in the human by cryopreservation of ovarian tissue.
ACKNOWLEDGMENTS
The authors acknowledge the supportive role of Dr. B. Desmet in breeding the mice needed for this study in the Central Animal House of the Ghent University Hospital (UZG) Medical Campus. Ms. V. David is acknowledged for her help in taking care of the mice. Ms. L. Verdonck (Department of Endocrinology, Ghent University Hospital) is thanked for her technical help in performing the hormone measurements.
FOOTNOTES
1 Supported by a research grant from the Bijzonder Onderzoeksfonds of Ghent University, Belgium (grant BOF01112199). 
2 Correspondence: Jun Liu, Infertility Center, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium. FAX: 32 9 240 4972; jun.liu{at}rug.ac.be
Accepted: August 17, 2000.
Received: June 6, 2000.
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