Human Reproduction, Vol. 15, No. 3, 567-571,
March 2000
© 2000 European Society of Human Reproduction and Embryology
Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: competence to undergo maturation, embryogenesis, and development to term
The Jackson Laboratory, Bar Harbor, Maine 04609, USA
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Abstract |
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Only primordial and primary follicles of frozen–thawed mouse ovaries survive after grafting to the ovarian bursa; large secondary follicles and antral follicles together with the oocytes contained in them degenerate. This study was undertaken to determine whether fully grown oocytes isolated from the antral follicles of frozen–thawed mouse ovaries are viable and can be rescued to undergo maturation, fertilization, and embryo development in vitro. Ovaries were cryopreserved after removal from 22-day-old (C57BL/6JxSJL/J)F1 mice, with or without prior priming with equine chorionic gonadotrophin, and fresh non-frozen ovaries were used as controls. Only cumulus cell-denuded oocytes were recovered from frozen unprimed ovaries while both cumulus cell-enclosed and denuded oocytes were retrieved from frozen primed ovaries. Oocytes from both groups of frozen–thawed ovaries were able to undergo maturation, fertilization, and development to the blastocyst stage in vitro, though at lower percentages than oocytes from control unfrozen ovaries. Moreover, 19% of 2-cell stage embryos derived from frozen–thawed primed ovaries, compared with 42% of embryos derived from control primed ovaries, developed to term after transfer to pseudopregnant foster mothers (not significantly different). Therefore, fully grown oocytes in antral follicles survive the cryopreservation protocol, as demonstrated by maturation, fertilization and embryo development in vitro, and development to term after embryo transfer.
Key words: cryopreservation/mouse/oocyte maturation in vitro/ovary
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Technology for cryopreservation of gametes will find important application in the human clinic, agriculture, and wildlife preservation. Moreover, the preservation of germplasm of the inevitable vast resource of induced mouse mutations looms as a critical important objective. One approach to cryopreservation of female gametes is to freeze intact pieces of ovaries. Significant success has been achieved in producing live offspring by natural matings after transfer of cryopreserved mouse ovaries to the ovarian bursa of ovariectomized recipient females (Gunasena et al., 1997a
,b; Sztein et al., 1998, 1999). However, when cryopreserved ovaries were surgically implanted, only the small primordial and primary follicles survived and underwent further development (Sztein et al., 1998). Antral follicles did not persist after transfer probably because of failure to vascularize appropriately before onset of follicular atresia. Moreover, because of the size and cellular complexity of antral follicles, it is also possible that follicular somatic cells or oocytes could become damaged by incomplete permeation of cryoprotectants. However, if oocytes of antral follicles could be rescued from cryopreserved ovaries before transplantation and matured and fertilized in vitro as it was previously suggested (Harp et al., 1994), they could provide additional opportunities for producing offspring and would not be wasted. In this study, fully-grown germinal vesicle (GV) stage oocytes were isolated from antral follicles of thawed cryopreserved ovaries and matured in vitro. They were assessed for progression of meiosis, competence to undergo preimplantation development after fertilization, and development to term after transfer to foster mothers.
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Materials and methods |
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Ovaries were isolated from 22-day-old (C57BL/6JxSJL/J)F1 mice; half of the mice were injected i.p. with 5 IU equine chorionic gonadotrophin (eCG) 48 h before removing the ovaries. Ovaries were cut in half and cryopreserved in cryotubes (Nunc 1.8 ml; Roskilde, Denmark) containing 1.5 mol/l dimethyl sulphoxide (DMSO; Sigma Chemical Co, St Louis, MO, USA) in M2 medium (Sigma Chemical Co) supplemented with 10% fetal calf serum (FCS; Sigma Chemical Co.) using a programmed slow freezing cooling (–0.5°C per minute to –80 °C before being transferred to liquid nitrogen) as described previously (Sztein et al., 1998
). For each of three independent experiments, the ovaries from two animals for each group, freshly isolated or cryopreserved, eCG-primed or unprimed, were used. Thawing cryopreserved ovaries was carried out at room temperature (23°C) until the ice was melted. After thawing, the cryoprotectant was immediately removed and replaced with M2 medium and allowed to equilibrate for 10 min before being transferred to maturation medium.
Oocytes were isolated by puncturing the antral follicles with 26 gauge needles and both the cumulus cell-enclosed oocytes (CEO) and the oocytes that emerged from the follicles denuded of cumulus cells (DO) were collected with micropipettes. CEO were matured in Waymouth Medium MB752/1 (GIBCO-BRL, Gaithersburg, MD, USA) supplemented with 5% fetal bovine serum (FBS) as described in detail (Eppig and Telfer, 1993; O'Brien et al., 1993; Eppig, 1999). DO were matured in Eagle's minimal essential medium supplemented with 10% FBS as described (Schroeder and Eppig, 1984) except that the medium was also supplemented with 0.01 mmol/l tetrasodium EDTA (Sigma Chemical Co). All groups of oocytes were matured for 16–17 h at 37°C in modular incubation chambers (Billups Rothenberg, Del Mar, CA, USA) infused with 5% O2, 5% CO2, 90% N2. After maturation, the progression of meiosis, as indicated by the presence or absence of either the germinal vesicle (GV) or a polar body, was assessed. Mature oocytes with a polar body were designated as metaphase II-arrested oocytes and those that had undergone germinal vesicle breakdown (GVB) without forming a polar body were designated as metaphase I-arrested oocytes. Then the oocytes that had undergone GVB, both metaphase I- and metaphase II-arrested oocytes, were inseminated and those that cleaved to the 2-cell stage 24 h later are referred to as fertilized. The 2-cell stage embryos were cultured as described previously (Eppig and Wigglesworth, 1994; Ho et al., 1995; Eppig, 1999). The percentage of embryos developing to the expanded blastocyst stage within 5 days of insemination was scored upon examination with a stereo microscope.
Statistical analysis
Groups of interest were compared using 
2 analysis; P < 0.05 was considered to be statistically significant.
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Results |
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Three-fold more oocytes were isolated from fresh ovaries than from those that were cryopreserved (Table I
). Of the oocytes isolated from fresh unprimed ovaries, 60% were denuded of their cumulus cells while 36% were denuded when the ovaries were isolated from fresh primed ovaries. In all, 79% of the oocytes collected from frozen–thawed primed ovaries were denuded. All the oocytes from the unprimed frozen–thawed ovaries were denuded; no CEO were recovered from these ovaries. Most of the DO collected from the frozen–thawed ovaries appeared normal upon inspection by light microscopy (Figure 1); approximately the same percentage (2%) of degenerated oocytes was collected from all groups before and after maturation in vitro (Table II). However, 12% of the CEO from the cryopreserved primed ovaries were degenerated upon collection and an additional 5% were degenerated after maturation (Table II). Thus 17% of the CEO from frozen–thawed primed ovaries became degenerated during maturation. However, little degeneration was seen in the oocytes of the other groups (Table II).
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After culture for 16–17 h, >95% of the oocytes underwent GVB and then progressed to either metaphase I or metaphase II (Table I
). Similar percentages of DO (56–67%) progressed to metaphase II, whether isolated from fresh or frozen–thawed ovaries (Table I). However, only 46% of the CEO from the primed frozen–thawed ovaries matured to metaphase II, whereas 78% of the oocytes from fresh CEO matured to metaphase II.
Oocytes matured after isolation from cryopreserved ovaries were competent to undergo fertilization and preimplantation development, but at a lower frequency than oocytes isolated from fresh ovaries (Table III). For example, the frequency of development to the 2-cell stage was twice as high after maturation of oocytes from fresh ovaries versus frozen–thawed ovaries (P < 0.01 for both primed and unprimed). Nevertheless, the frequency of development to the 2-cell stage was the same after maturation of CEO from fresh and cryopreserved primed ovaries, 60 versus 64% respectively. The frequency of development from the 2-cell stage to blastocyst was also similar between these two groups, 80 versus 83% respectively. However, since the number of oocytes was much lower in the cryopreserved group (Table I), the total number of blastocysts derived from CEO was much lower (Table III). When data from both CEO and DO were pooled, the number of blastocysts produced from cryopreserved primed ovaries was ~15% that of the fresh primed ovaries (Table III). Nevertheless, 74 blastocysts were obtained after maturation of oocytes from frozen–thawed primed ovaries; 19% of the total matured oocytes.
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In a separate experiment, 19% (five out of 26) of 2-cell stage embryos derived from frozen/thawed primed ovaries, compared with 42% (11 out of 26) of embryos derived from fresh primed ovaries (not significant), developed to term after transfer to pseudopregnant foster mothers. Thus, fully-grown oocytes rescued from frozen–thawed mouse ovaries and matured in vitro have full developmental competence.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Previous studies have shown that cryopreserved mouse ovaries can function to produce offspring after surgical implantation within the ovarian bursa (Gunasena et al., 1997a
,b; Sztein et al., 1998, 1999). These offspring were probably derived from oocytes of preantral follicles since the antral follicles undergo degeneration shortly after surgery. The data presented here demonstrate that oocytes of antral follicles can be rescued by isolating them from the cryopreserved ovaries, then maturing and fertilizing the oocytes in vitro. Since ~20% of these oocytes matured in vitro are competent to develop to the 2-cell stage, and ~20% of these can develop to term, this is a feasible, and perhaps practical, protocol for the production of live offspring from cryopreserved mouse ovaries. Thus, maturation of fully-grown oocytes in vitro in combination with surgical implantation of the remaining ovarian tissue to the ovarian bursa may provide the highest probability of producing young from cryopreserved mouse ovaries. The best results using frozen–thawed ovaries were achieved using primed ovaries. No CEO were obtained from unprimed ovaries. Presumably because the complexes of unprimed mice are so much more delicate, even without cryopreservation, the cumulus cells are more easily stripped from the oocytes by mechanical forces of isolation. Even with fresh ovaries, a higher proportion of the isolated oocytes from unprimed ovaries was denuded of cumulus cells than from primed ovaries. Probably the association of the cumulus cells with the oocyte becomes even more tenuous as a result of the freeze–thaw protocols.
Previous studies have indicated that only primordial, or perhaps primary, follicles survive cryopreservation and grafting procedures, though the surviving small follicles can undergo subsequent development after grafting (Candy et al., 1997; Sztein et al., 1998). Indeed, it should be expected that both procedures could have deleterious effects, owing to the size and complexity of the tissue, probable differential penetration of cryoprotectants during the freezing protocol, and rates of vascularization after grafting. Here it is shown that many fully-grown oocytes in antral follicles survive the cryopreservation protocol, as demonstrated by maturation, fertilization and embryo development in vitro. It is possible that the fully-grown oocytes in antral follicles do, in fact, sustain some damage inflicted by the freeze–thaw protocols. If so, either the damage is not serious, or serious damage is repaired during maturation in vitro. That this is possible was demonstrated in studies in which oocytes isolated from mouse ovaries several hours after death of the female recovered from post-mortem degenerative damage during oocyte maturation in vitro (Schroeder et al., 1991).
Fewer oocytes were recovered from cryopreserved ovaries than from fresh ovaries. Although most of the oocytes recovered from the cryopreserved ovaries appeared in good morphological condition, it must be assumed that the cryopreservation protocol in fact damaged many fully-grown oocytes to the extent that they could not be recovered. Interestingly, more degenerated oocytes were actually recovered from the primed than the unprimed ovaries. Most of the degenerated oocytes were enclosed by cumulus cells, which could have protected them from complete fragmentation during the isolation procedure. Since the percentage of oocytes that completed maturation and preimplantation development was always lower in the cryopreserved than the fresh group, it must be assumed that many oocytes that appeared morphologically normal must have been inflicted with developmental lesions by the cryopreservation protocol.
The cryopreservation of portions of human ovaries is indicated under several circumstances (Donnez and Bassil, 1998). It is clear that primordial follicles survive cryopreservation protocols (Newton et al., 1996; Oktay et al., 1997), though it is far less certain that approaches described here to rescue the fully grown oocytes of large antral follicles will find application in the human clinic, at least in the near future (Oktay et al., 1998). Competence to complete nuclear and cytoplasmic maturation is acquired only during the final stages of antral follicle development (Eppig and Schroeder, 1989; Pavlok et al., 1992; Lonergan et al., 1994; De Smedt et al., 1994; Crozet et al., 1995; Cognie et al., 1998), a time when the large size of human antral follicles may be prohibitive to successful cryopreservation. However, eventually it may be possible to grow human oocytes from the much smaller primordial, primary, or even secondary follicle stages in vitro, as reported for the mouse (Eppig and O'Brien, 1996), after cryopreservation. Indeed promising results have already been obtained using partially isolated fresh human follicles (Hovatta et al., 1999; Wright et al., 1999). It will be important to establish whether this success can be achieved with cryorpreserved material, although ultimate success seems likely.
Fully-grown mouse oocytes can be isolated and cryopreserved either before or after maturation (Whittingham, 1977; Schroeder et al., 1990; Carroll et al., 1993; Candy et al., 1994). Thus, there are several options available to make efficient use of limited numbers of ovaries that might be available for cryopreservation. Fully-grown oocytes, both cumulus-enclosed and denuded, could be isolated before cryopreservation of residual ovarian tissue containing primordial and preantral follicles. The oocytes could be cryopreserved either before or after maturation in vitro. However, in anticipation of possible need for efficiency in both personnel effort and storage facilities required for cryopreservation of large numbers of ovaries produced in induced mutagenesis programmes, the simplest protocol is initial ovarian cryopreservation followed by oocyte isolation and maturation as described here.
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Acknowledgments |
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This research was performed as part of the National Cooperative Program on Nonhuman In vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development (NICHD) to M.J.O. and J.J.E. through Cooperative Agreement HD21970. Additional support to J.M.S., J.S.F., and L.E.M. was provided by grants RR01262 and RR11081 from NCRR and CA34196 from NCI. The scientific services of the Jackson Laboratory receive support from a Cancer Center Core Grant (CA34196) from the National Cancer Institute. We are grateful to Drs. Wes Beamer, Keith Latham, Randy Prather, and Andy Watson for their helpful suggestions in the preparation of this manuscript.
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Notes |
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1 To whom correspondence should be addressed
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Submitted on July 2, 1999; accepted on November 25, 1999.
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