The Jackson Laboratory, Bar Harbor, Maine 04609, USA
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Abstract |
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Key words: cryopreservation/mouse/oocyte maturation in vitro/ovary
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Introduction |
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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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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 1617 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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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 frozenthawed 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 frozenthawed primed ovaries; 19% of the total matured oocytes.
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Discussion |
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The best results using frozenthawed 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 freezethaw 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 freezethaw 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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Notes |
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References |
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Candy, C.J., Wood, M.J., Whittingham, D.G. et al. (1994) Cryopreservation of immature mouse oocytes. Hum. Reprod., 9, 17381742.[Abstract]
Carroll, J., Wood, M.J. and Whittingham, D.G. (1993) Normal fertilization and development of frozenthawed mouse oocytes: protective action of certain macromolecules. Biol. Reprod., 48, 606612.[Abstract]
Cognie, Y., Benoit, F., Poulin, N. et al. (1998) Effect of follicle size and of the Fec(B) Booroola gene on oocyte function in sheep. J. Reprod. Fertil., 112, 379386.[Abstract]
Crozet, N., Ahmed-Ali, M. and Dubos, M.P. (1995) Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro. J. Reprod. Fertil., 103, 293298.[Abstract]
De Smedt, V., Crozet, N. and Gall, L. (1994) Morphological and functional changes accompanying the acquisition of meiotic competence in ovarian goat oocyte. J. Exp. Zool., 269, 128139.[ISI][Medline]
Donnez, J. and Bassil, S. (1998) Indications for cryopreservation of ovarian tissue. Hum. Reprod. Update, 4, 248259.
Eppig, J.J. (1999) Mouse oocyte maturation, fertilization, and preimplantation development in vitro. In Richter, J.D. (ed.), A Comparative Methods Approach to the Study of Oocytes and Embryos. Oxford University Press, Oxford, UK, pp. 39.
Eppig, J.J. and O'Brien, M.J. (1996) Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod., 54, 197207.[Abstract]
Eppig, J.J. and Schroeder, A.C. (1989) Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation and fertilization in vitro. Biol. Reprod., 41, 268276.[Abstract]
Eppig, J.J. and Telfer, E.E. (1993) Isolation and culture of oocytes. Methods Enzymol., 225, 7784.[ISI][Medline]
Eppig, J.J. and Wigglesworth, K. (1994) Atypical maturation of oocytes of strain I/LnJ mice. Hum. Reprod., 9, 11361142.[Abstract]
Gunasena, K.T., Lakey, J.R.T., Villines, P.M. et al. (1997a) Allogeneic and xenogeneic transplantation of cryopreserved ovarian tissue to athymic mice. Biol. Reprod., 57, 226231.[Abstract]
Gunasena, K.T., Villines, P.M., Critser, E.S. and Critser, J.K. (1997b) Live births after autologous transplant of cryopreserved mouse ovaries. Hum. Reprod., 12, 101106.[ISI][Medline]
Ho, Y., Wigglesworth, K., Eppig, J.J. and Schultz, R.M. (1995) Preimplantation development of mouse embryos in KSOM: Augmentation by amino acids and analysis of gene expression. Mol. Reprod. Dev., 41, 232238.[ISI][Medline]
Hovatta, O., Wright, C., Krausz, T. et al. (1999) Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation. Hum. Reprod., 14, 25192524.
Lonergan, P., Monaghan, P., Rizos, D. et al. (1994) Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro. Mol. Reprod. Dev., 37, 4853.[ISI][Medline]
Newton, H., Aubard, Y., Rutherford, A. et al. (1996) Low temperature storage and grafting of human ovarian tissue. Hum. Reprod., 11, 14871491.
O'Brien, M.J., Wigglesworth, K. and Eppig, J.J. (1993) Mouse oocyte and embryo culture. In Chapin, R.E. and Heindel, J. (eds), Methods in Reproductive Toxicology. Academic Press, New York, USA, pp. 128141.
Oktay, K., Nugent, D., Newton, H. et al. (1997) Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil. Steril., 67, 481486.[ISI][Medline]
Oktay, K., Newton, H., Aubard, Y. et al. (1998) Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology? Fertil. Steril., 69, 17.[ISI][Medline]
Pavlok, A., Lucas-Hahn, A. and Niemann, H. (1992) Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles. Mol. Reprod. Dev., 31, 6367.[ISI][Medline]
Schroeder, A.C., Champlin, A.K., Mobraaten, L.E. and Eppig, J.J. (1990) Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J. Reprod. Fertil., 89, 4350.[Abstract]
Schroeder, A.C., Johnston, D. and Eppig, J.J. (1991) Reversal of postmortem degeneration of mouse oocytes during meiotic maturation in vitro. J. Exp. Zool., 258, 240245.[ISI][Medline]
Schroeder, A.C. and Eppig, J.J. (1984) The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal. Dev. Biol., 102, 493497.[ISI][Medline]
Sztein, J., Sweet, H., Farley, J. and Mobraaten, L. (1998) Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking. Biol. Reprod., 58, 10711074.[Abstract]
Sztein, J.M., McGregor, T.E., Bedigian, H.J. and Mobraaten, L.E. (1999) Transgenic mouse strain rescue by frozen ovaries. Lab. Anim. Sci., 49, 99100.[Medline]
Whittingham, D.G. (1977) Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at 196°C. J. Reprod. Fertil., 49, 8994.[Abstract]
Wright, C.S., Hovatta, O., Margara, R. et al. (1999) Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles. Hum. Reprod., 14, 15551562.
Submitted on July 2, 1999; accepted on November 25, 1999.