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a Infertility Center, Department of Obstetrics and Gynecology and
b Department of Gynecologic Oncology, Ghent University Hospital, B-9000 Ghent, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MEM supplemented with 100 mIU rFSH and 5% fetal bovine serum for 12 days under an atmosphere of 5% CO2 in air at 37°C. By Day 12 of culture, 66.5% of follicles retained their oocytes in the grafting group versus 97.5% in the control group (P < 0.001). Final oocyte maturation was induced by addition of 2.5 IU/ml hCG. At 14–16 h post-HCG, the percentages of oocytes showing germinal vesicle breakdown and polar body extrusion were significantly higher in the control group (90.6% and 82.8%) compared to the grafting group (60% and 45%). The mean diameter of the mature oocytes of the grafting group (69.9 ± 4.45 µm) was similar to that of oocytes from the control group (70.5 ± 2.35 µm). Our results suggest that maturation of mouse primordial follicles is feasible by combination of in vivo transplantation and in vitro culture. This two-step strategy may be an attractive model for promoting the growth and maturation of primordial follicles from other species.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Frozen-stored primordial follicles within the ovarian cortex can be used in different ways to restore fertility. Studies on autologous or allogeneic orthotopic transplantation of fresh or cryopreserved ovarian tissue have shown that the recovery of folliculogenesis, steroidogenesis, ovulation, and fertilization followed by the production of embryos or the delivery of live young is possible [10–16]. Heterotopic and xenogeneic transplantation of ovarian tissue into a different in vivo environment is an alternative option. Murine, ovine, and primate ovarian tissues were found to revascularize after transplantation with follicle growth being initiated [13, 17, 18]. Furthermore, primordial follicles within human ovarian cortex grafted under the kidney capsule of hypogonadal SCID mice grew to the antral stage after 6 wk of exogenous FSH stimulation [19]. In vitro growth of isolated follicles to maturity is another attractive method for restoration of fertility. During the last decade, various in vitro culture systems for murine ovarian preantral follicles and oocyte-granulosa cell complexes prepared from preantral follicles have been successfully established. Oocytes recovered from these culture systems can be fertilized, and live young can be obtained [20–22]. However, the recovery and reproducible in vitro development of primordial follicles to maturity, in which oocytes acquire complete competence to undergo maturation, fertilization, and embryonic development, are still a considerable challenge. Presently little is known about the signals that initiate the growth of primordial follicles [23]. The complete in vitro oocyte development from primordial mouse oocytes to the MII stage has been successfully reported by only one group [24]. In their study, primordial follicles were grown to the preantral follicle stage in organ cultures of whole newborn mouse ovaries. The preantral follicles were further matured after enzymatic isolation, and two mouse pups (one surviving) were born after in vitro fertilization (IVF) and transfer of 190 two-cell-stage embryos to recipients.
The aim of the present study was to achieve initial growth and complete in vitro maturation of primordial follicles present in newborn mouse ovaries. A two-step strategy was used. The first step was to initiate the transition from primordial to preantral follicles by in vivo grafting. The preantral follicles were then recovered, and an established culture system [25] was used to support the follicles to maturity. In our experiment, newborn mouse ovaries that contained only primordial follicles were allotransplanted beneath the kidney capsule of ovariectomized recipients. Next, the growing preantral follicles were mechanically isolated from the ovarian grafts and cultured further in vitro to complete oocyte maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female F1 hybrid (C57BL/6j x CBA/Ca) mice housed and bred in the Central Animal House of the Ghent University Hospital according to national standards were used throughout the study. Approval for the study was obtained from the Animal Research Ethical Committee of the Ghent University Hospital. Newborn mouse ovaries were allotransplanted under the kidney capsule of adult ovariectomized female recipients. Fourteen days after transplantation, ovarian grafts were freed from kidney and connective tissue. Preantral follicles were mechanically released and cultured individually. Preantral follicles isolated from ovaries of 14-day-old mice were also cultured as a control group. The competence of oocyte nuclear maturation was evaluated after 12 days of culture in vitro.
Heterotopic Allografting Procedure
Newborn mice were decapitated, and the abdomen was opened. The ovaries (n = 26) were dissected free and transferred to Leibovitz L-15 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin (referred to subsequently as L-15* medium; all products obtained from Life Technologies, Merelbeke, Belgium). Recipient mice (8–12 wk old) were anesthetized by i.p. injection of 100 µl of sodium pentobarbital (Nembutal; Sanofi, Brussels, Belgium) that had been diluted 1:4 in PBS. Surgery was carried out under strictly aseptic conditions. The left kidney was exteriorized through a small dorso-horizontal incision. A small hole was torn in the kidney capsule using a pair of fine watchmakers' forceps, and a whole newborn mouse ovary was inserted beneath the capsule. The kidney was returned to the body cavity, and the recipient was ovariectomized bilaterally by means of cautery. Finally, the body wall incision and skin were closed.
In Vitro Growth of Preantral Follicles
Two weeks after transplantation, 26 recipient mice were killed by cervical dislocation, and the grafts were aseptically removed from the left kidney and collected in L-15* medium. Four grafts were processed for histology. Follicles were mechanically isolated from the remaining 22 grafts using 25–1/2-gauge needles (Becton Dickinson, Erembodegem, Belgium). The preantral follicles included in the study had a round follicular structure. They were composed of round and centrally located oocytes surrounded by a thin zona pellucida and 2–4 layers of granulosa cells enclosed by a basal membrane to which some theca cells were attached. This description corresponds to the type 4 or type 5a follicles according to Pedersen and Peters' classification [26]. The selected follicles were cultured individually in 20 droplets per dish (Falcon: 60 x 15-mm culture dish; Becton Dickinson), with 10 µl medium in every droplet. The culture medium consisted of -minimal essential medium (-MEM) (Life Technologies) supplemented with 5% FBS and 100 mIU/ml recombinant human FSH (rFSH; Puregon: Organon, Oss, The Netherlands). Follicle diameters were measured at Day 1 of culture. Measurement of follicle diameter was performed with a caliper in the eyepiece of an inverted microscope. From two perpendicular diameters (the length a and the width b), the mean diameter (D) was estimated by means of the formula D = (a + b)/2. At Day 2 of culture, 10 µl fresh -MEM medium was added to each droplet. From Day 4 onwards, the medium was refreshed by exchanging half of the droplet volume every other day. Follicles were cultured under this condition for 12 days in an incubator at 37°C, 100% humidity, and 5% CO2 in air. Preantral follicles from six ovaries of 14-day-old mice were cultured under the same conditions and served as control group.
Induction of Final Nuclear Maturation
At Day 12 of culture, final oocyte maturation was induced by addition of 2.5 IU/ml hCG (Pregnyl: Organon) to the in vitro culture droplets. After 14–16 h of incubation for oocyte maturation, the cumulus cells were removed from the oocytes by drawing the mucified cumulus-oocyte complexes in and out of a mouth-controlled fine pipette. The cumulus cell-free oocytes were scored as germinal vesicles (GV) when the GV was present in the oocytes, as GVBD when the GV had broken down, and as MII oocytes when the first polar body had been extruded. The diameters of cultured oocytes, which were denuded of their companion cumulus cells, were measured as described above for the follicles. The diameters of oocytes were measured excluding the zona thickness.
Histology
Ovarian grafts were removed from 4 recipient mice on Day 14 following transplantation beneath the renal capsule. Ovaries obtained from newborn mice as well as 14-day-old mice were processed for histological examination for subsequent comparison with grafted ovaries. The grafts and ovaries were fixed in Bouin's fluid for 24 h, embedded in paraffin, serially sectioned at 6 µm, and stained with hematoxylin and eosin. The sections were examined for the presence of follicles, and their developmental stage was determined. Follicles were classified as follows: primordial follicles, having one layer of flattened pregranulosa cells surrounding the oocyte; primary follicles, having one layer of cuboid granulosa cells; preantral follicles, having two or more layers of granulosa cells but not showing an antrum; and antral follicles, showing an antral cavity.
Statistics
Percentages such as those of oocytes at various stages of maturation were calculated from at least three independent replicate experiments. The percentages were compared between groups by chi-square analysis using data pooled from all experiments. Oocyte diameters, presented as the mean ± SD (standard deviation), were compared using Student's t-test. When P 
0.05, the difference was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All grafts of whole newborn mouse ovaries (n = 26) were recovered 14 days after transplantation (100% recovery). The grafts were easily identified under the kidney capsule and freed from kidney tissue. Histological examination of recovered grafts (n = 4) revealed that follicles had proceeded to preantral and early antral follicle stages (Fig. 1A). Development at stages reached by follicles grown in ovarian grafts was comparable to the developmental stages reached by follicles grown in vivo in 14-day-old mice (Fig. 1B). The sections showed also that some follicles in the grafts were abnormal in terms of partial disconnection between oocytes and their companion granulosa cells (Fig. 1A).
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Follicle Yield
Isolation by mechanical dissection of 14-day-old ovarian grafts yielded 248 preantral follicles from 22 grafts with an average of 11 preantral follicles per graft. The mean diameter of the cultured follicles (n = 248) was 114.4 ± 13.6 µm (mean ± SD) with a range of 92.5–162.5 µm (median = 112.5 µm). Isolation by mechanical dissection of six ovaries from 14-day-old prepubertal control mice yielded 197 preantral follicles with an average of 33 preantral follicles of a similar size per ovary. Partial disconnection between oocytes and their companion granulosa cells was more often observed in follicles recovered from ovarian grafts than in control follicles (Fig. 2, A and B).
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Follicle Development In Vitro
On Days 4–8 of culture, the follicles became attached to the bottom of the dish, and a monolayer was fully formed around the follicles. Granulosa cells proliferated and grew through the basal membrane to form the diffuse appearance. During the whole in vitro culture period, follicle degeneration occurred by showing either spontaneous release of the oocytes or failure of further proliferation of granulosa cells. By Day 12 of culture, 66.5% and 97.5% of the follicles retained their oocytes inside the granulosa cells in grafting and control groups, respectively (P < 0.001, Fig. 3). And, respectively, 94% and 99.5% of surviving follicles became diffuse shaped in the transplanted and control groups (P < 0.002). An antral-like cavity, a translucent area in the granulosa cell mass around the oocyte, was formed on Day 12 in about 19.4% and 18.0% of surviving transplant and control follicles, respectively (P = 0.785). At 14–16 h after hCG stimulation, mucification of the cumulus-oocyte complexes was recognized under the stereomicroscope as expansion of the cumuli oophori. Mucification was induced in all diffusing follicles in both the grafting and the control groups.
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Oocyte Growth In Vitro
During handling of cumulus-oocyte complexes for oocyte recovery in the experimental group, 14 out of 165 oocytes were damaged because of drawing in and out of fine mouth-controlled pipettes. Oocytes recovered from developing follicles from grafts reached a diameter of 69.9 ± 4.45 µm (mean ± SD, median = 70 µm; n = 151) after a 12-day culture period. The diameter of these oocytes was similar to the diameter of oocytes from in vitro-grown follicles in the control group (70.5 ± 2.35 µm, median = 70 µm; n = 192). Mean diameters of oocytes recovered from cultured preantral follicles in both experimental and control groups were smaller than the diameters of in vivo-matured oocytes (75 µm in diameter; Fig. 4).
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Maturation of In Vitro-Grown Oocytes
After final oocyte maturation induced by hCG stimulation, 60% of the oocytes from grafts underwent GVBD, and 45% of these produced a polar body (Fig. 2C). There were significant differences between the grafting and control groups in the percentages of the oocytes acquiring competence to GVBD (control: 90.6%; P < 0.001) and in the percentages of MII oocytes (control: 82.8%; P < 0.001; Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The transplantation of newborn mouse ovarian grafts can restore cyclicity in ovariectomized mice by about 11 days after transplantation [28]. The primordial follicle population appears to be more resistant to the effects of ischaemia than follicles in the growing stages, presumably by virtue of being dormant and having a low metabolic rate [29]. The kidney capsule, which is highly vascularized, provides an ideal site for rapid revascularization. Both ovary and kidney capsule are rich in angiogenic factors, such as vascular endothelial growth factor, which further enhance survival and revascularization [13, 30]. The ovariectomized recipients provided an endocrine milieu with high concentrations of circulating gonadotrophins, and low concentrations of estrogen and inhibin, which can promote the development of newborn mouse ovarian grafts [13].
Despite such natural advantages, tissue ischaemia can still be an important problem for implants, as the process of revascularization can take more than one day to complete [29]. In a previous study, approximately 50% of the primordial follicles survived in isologous grafts in mice [31]. In the present study, the yield of preantral follicles (11 follicles on average) dissected mechanically from ovarian grafts was clearly less than the number from 14-day-old mouse ovaries (33 follicles on average). Moreover, the morphology of some preantral follicles in ovarian graft sections showed poor connections between the oocytes and their companion granulosa cells. This phenomenon was confirmed when the isolated follicles were observed under the inverted microscope with a Hoffman contrast-modulation system (Hoffman Modulation Optics, Inc., Greenvale, NY). The less organized follicle units may give rise to lower follicle survival rate, GVBD rate, and MII oocyte formation rate, since communication between oocytes and their granulosa cells is essential for proliferation and differentiation of granulosa cells as well as oocyte development [32–34].
Methods for the long-term culture of intact individual preantral follicles or oocyte-granulosa cell complexes enzymatically isolated from preantral follicles were developed in mice [20–22]. Oocytes grown in vitro by these culture systems can undergo GVBD, fertilization, cleavage, blastocyst formation, and implantation, and the methods can lead to delivery of live young. Nevertheless, these in vitro methods have had very limited success for primordial and primary follicles. The factors that initiate the growth of primordial follicles are currently unknown, although the size of the remaining stockpile influences the rate at which follicles enter the growing pool in rats [35], and the ovarian stroma may have a restraining influence in cattle [36]. In rats, neurotransmitters are involved in the cytodifferentiation process by which newly formed ovarian follicles acquire responsiveness to gonadotropins [23]. Eppig and O'Brien [24] reported that mouse primordial follicles within newborn mouse ovaries could initiate growth and develop to preantral follicles by means of an organ culture for 8 days. The oocytes of growing preantral follicles were subsequently developed in a collective in vitro culture system. It therefore seems feasible that an appropriate combination of methods will permit follicle growth from the primordial to the mature stage in vitro.
It is clear that the safeguarding of fertility in the human by ovarian cryopreservation is a long-term objective. None of the current existing approaches, whether transplantation (in vivo development) or full in vitro development, have been shown to be successful in restoration of fertility in the human. Although far from superior to the full in vitro or in vivo development approaches, the two-step method described here offers the possibility of combining the advantages of both types of strategy. On the one hand, the transplantation strategy offers the possibility of initiating growth of primordial follicles, which so far has been unsuccessful in vitro. On the other hand, the in vitro culture of isolated early preantral follicles in the second step allows circumvention of the problem of transmission of malignant cells to the recipients [37].
The two-step strategy used in this study shows that follicle transition from the primordial stage to the preantral follicle can be initiated by grafting of newborn mouse ovaries. Subsequent development of recovered follicles with oocyte growth and complete nuclear maturation up to MII can be achieved by an in vitro culture system. The final oocyte volume, however, had a value of only 81% of that of in vivo-matured oocytes. Nevertheless, by use of IVF of oocytes following in vitro maturation of preantral follicles isolated from 14-day-old mice, hatching blastocysts and live young were obtained [22, 25] with oocytes that had a volume comparable to that of the oocytes in the present study. The expectation that MII oocytes matured in vitro from ovarian grafts in our culture will have the competence for fertilization and early embryogenesis is therefore warranted.
A necessary prerequisite for collecting an optimal number of MII oocytes for embryo culture is to find the optimal point in follicle culture at which final oocyte maturation can be induced by the hCG stimulation. Recent studies have demonstrated an effect of culture time on meiotic progression. In the study by Johnson et al. [38], when follicles initially measuring around 200 µm were cultured for more than 3 days, meiotic progression fell. In a study by Cortvrindt et al. [39], the optimal point for inducing meiosis was Day 12 of culture: the highest rate of MII oocytes was induced, spontaneous GVBD was still at an acceptable level, and intrafollicular oocyte degeneration had not yet happened. In the present study, the preantral follicles isolated from ovarian grafts as well as from 14-day-old mouse ovaries were cultured in vitro for 12 days before hCG stimulation. The follicles from both groups had a similar diameter of 114.4 ± 13.6 µm with a range of 92.5–162.5 µm, which was comparable to the follicle size (100–130 µm) in the Cortvrindt et al. study [39].
Further studies are needed to investigate the developmental potential of in vitro-grown oocytes derived from ovarian grafts. IVF experiments are presently being carried out to investigate the developmental capacity of retrieved MII oocytes. The final validity of this two-step method will be established by the transfer of embryos to pseudopregnant recipients. In order to enhance follicle survival after transplantation, measures should be taken to reduce ischemic damage by reactive oxygen species, such as chilling of the ovarian tissue. To improve the quality of isolated follicles, the ovarian grafts may be kept for a longer time in the grafting site (e.g., 2.5–3 wk) for full early folliculogenesis.
In conclusion, the results reported here show that initiation of growth and maturation of mouse primordial follicles are possible through a combination of in vivo grafting and in vitro culture of recovered preantral follicles. Until now, in vitro culture of individual intact primordial follicles as well as the isolation of primordial follicles has been very difficult to achieve. Moreover, the mechanism involved in the recruitment of primordial follicles into the preantral follicle stage is unknown and may require the presence of ovarian factors external to the follicle itself [24]. The two-step system presented here requires optimization at both the in vivo and in vitro steps to improve oocyte development. Nevertheless, this two-step method may be a potential attractive model to promote the transition from primordial to preantral follicles during the in vivo grafting period, after which isolated preantral follicles can be further supported by an in vitro culture system to achieve complete development of oocyte competence.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a research grant from the Bijzonder Onderzoeksfonds of the Ghent University, Belgium (grant No.: BOF 01112199). 
2 Correspondence: Jun Liu, Infertility Center, Department of Obstetrics and Gynecology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium. FAX: 32 9 240 4972; jun.liu{at}rug.ac.be
Accepted: December 29, 1999.
Received: July 21, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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