Human Reproduction vol.8 no.8 pp. 1163-1167, 1993

Transplantation of frozen—thawed mouse primordial follicles

John Carroll1 and Roger G.Gosden2

'To whom correspondence should be addressed

Primordial follicles were isolated from juvenile mouse ovaries and cryopreserved by slow freezing with dlmethylsulphoxide as the cryoprotectant. After thawing, - 8 0 % of the oocytes and 65% of the somatic cells excluded Trypan Blue dye, indicating that cell membranes were still intact. Frozen—thawed cells were suspended in plasma clots and transplanted to the ovarian bursas of host animals that had been sterilized by oophorectomy. The grafts of frozen—thawed cells reorganized into morphologically distinguishable ovaries which produced signs of oestrogenic activity. After natural mating, host females produced normal offspring that were demonstrated by genetic markers to be derived from the transplanted frozen-thawed primordial follicles. Key words: cryopreservation/development/mouse/primordial follicle/transplantation

Materials and methods Introduction Cryopreservation of germ cells is a valuable procedure for the conservation of rare and endangered species, manipulation of animal breeding, and in the treatment of certain types of human infertility. Spermatozoa are available for storage in their millions and successful techniques have been available for many years (Polge et al., 1949). However, the ease and success of artificial insemination techniques contrasts markedly with the problems associated with freezing the mammalian oocyte. The earliest attempts to preserve mammalian oocytes involved the freezing of fragments of ovarian tissue. These experiments resulted in the birth of offspring after orthotopic transplantation of frozen-thawed ovarian tissue (Parrott, 1960). Success with whole or parts of ovaries was limited and the 5 % of the oocyte population surviving was confined to small follicles (Deanesly, 1954; Green et al., 1956). Further attempts to preserve the mature oocyte were limited by difficulties of dehydrating these large cells. Despite initial problems, the development of methods for freezing preimplantation embryos (Whittingham et al., 1972) led to success with mature oocytes (Tsunoda et al., 1976; Whittingham, 1977) which can now be cryopreserved with © Oxford University Press

Isolation of primordial follicles The methods for isolating primordial follicles have been described elsewhere (Gosden, 1990). Briefly, ovaries of 4—8-day-old C57BL/6 mice homozygous at the Gpi-lsb locus were dissected free and transferred to 2 ml of HEPES-buffered medium M2 (Fulton and Whittingham, 1978) containing 5% fetal calf serum (M2+FCS). The ovaries were bisected and incubated at 37°C for 20—30 min in M2+FCS containing collagenase (1.5 mg/ml; type 1; Sigma Chemical Co., London, UK). The fragments of ovary were transferred to fresh M2+FCS and pipetted to obtain a disaggregation of single cells. The cells were washed twice by centrifugation (80 g) and re-suspended in fresh M2 + FCS. Cryopreservation of primordial follicles After centrifugation and removal of the supernatant the cells were re-suspended in M2+FCS containing 1.5 M dimethylsulphoxide (M2 + DMSO) at 4°C. A small volume of M2+DMSO was drawn into a plastic freezing straw followed by an air bubble and finally —0.2 ml of cell suspension (equivalent to four to five ovaries). The straws were sealed with haematocrit putty and placed in a programmable cell freezer (Planar) pre-cooled to 0°C; 10 min after addition of M2+DMSO the straws were cooled at 1163

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Medical Research Council, Experimental Embryology and Teratology Unit, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE and 2Department of Physiology, University Medical School, Teviot Place, Edinburgh EH8 9AG, UK

minimal loss of viability (Carroll et al., 1993). A major limitation of preserving oocytes at the pre-ovulatory stage is that only a tiny fraction of the original population reaches this final stage of maturation. The possibility of storing oocytes at early stages of development when they are more abundant is therefore very attractive. Recently we demonstrated that mature oocytes obtained after culturing and transplanting frozen—thawed primary ovarian follicles remained viable (Carroll et al., 1990). Storage of the primordial or 'resting' follicle stages is potentially better because these germ cells are more abundant, have a paucity of potentially sensitive organelles, are less metabolically active and their small size renders them less susceptible to adverse effects of low-temperature storage. The population of primordial follicles around the time of birth is maximal and represents an irreplaceable source of potential gametes for the entire reproductive lifespan of the female mammal. It is timely to apply cryopreservation technology because efficient methods are available for isolating primordial follicles from enzymatically dissociated ovaries and subsequently transplanting them to sterilized host animals (Gosden, 1990). The aim of this study was to devise a successful cryopreservation protocol for mouse follicles and test the fertility of frozen—stored oocytes after transplantation to suitably prepared host females.

J.Carroll and R.G.Gosden

Donor C57BL/6 Gpi-1sb

I disaggregate ovaries

1 1 1 1

Freeze/thaw

suspend tissue in fibrin clot

Assessment of cell viability after cryopreservation After the cells were washed a sample was taken to assess viability by the Trypan Blue dye exclusion test. Preliminary studies were conducted to assess the ability of frozen—thawed cells to grow in culture. Cells obtained from frozen—thawed dissociated ovaries were cultured in minimal essential medium (MEM; Earle's salts) containing 5% FCS (Flow Laboratories, Irvine, Scotland, UK) at 37°C in a humidified atmosphere of 5% CO2 in air. After 24 h in culture, cell attachment and growth in the dishes was compared with non-frozen control tissue.

culture for 24-48h

Recipient C57BL/6 Gpi-1sa

1 1

Transplantation of primordial follicles The cells were transferred to 0.4 ml microcentrifuge tubes (Alpha Laboratories, Eastleigh, UK). To prepare the cells in plasma clots, they were centrifuged (80 g) and resuspended in —0.1 ml of fresh mouse venous plasma. Clotting was induced by touching the surface with a sterile needle, after which the tubes were incubated at 37°C for 15-30 min. The clots containing the disaggregated ovarian tissue were transferred to Medium 199 containing 10% FCS, L-glutamine, pyruvate and antibiotics and cultured for 1—2 days as described previously (Torrance etal., 1989; Gosden, 1990). A single clot was transferred to each ovarian bursa of virgin 6 —8-week-old C57BL/6 (Gpi-ls^/Gpi-ls") female mice (Figure 1), as described previously (Gosden, 1990). Host ovaries were removed immediately before transplantation and bleeding was controlled by light cauterization, taking care to avoid damage to the Fallopian tubes. Two series of transplants were performed. The first series consisted of 10 clots that represented a total of 45 pairs of donor organs and they were transferred to five hosts; the second comprised eight clots from 55 pairs which were transferred to four hosts.

assess for oestrogenic activity

mate with C57BL/6 Gpi-1sb male

i

I

histology

offspring

Fig. 1. Experimental approach for the production of offspring from frozen—thawed transplanted primordial follicles.

Those that had mated were allowed to litter. At 6 - 1 2 weeks after transplantation the hosts were autopsied, the status of the reproductive tract was examined, and the graft from the ovarian capsule was removed and fixed in Bouin's fluid, paraffinembedded, sectioned and stained with haematoxylin and eosin. Some of the grafts together with liver tissue from pups and fetuses and blood from host animals were stored at -20°C for glucose phosphate isomerase (GPI)-1 analysis.

Assessment of function of transplanted grafts To check that the removal of the host ovaries was complete and that the grafts became functionally active, the vagina of the host was examined daily. Closure of the introitus was taken as an indication of oestrogen deficiency and re-opening as a sign that oestrogenic follicles had emerged in the graft. After vaginal opening or 3 weeks post-operation (whichever was earlier), host females were paired with fertile males C57BL/6 (Gpi-l^/Gpi-ls*). The hosts were inspected daily for signs of mating (vaginal plug). 1164

Analysis of glucose phosphate isomerase (GPI-1) The use of different Gpi-ls genotypes allowed the derivation of offspring from oocytes in the grafts to be verified and the possibility of incomplete oophorectomy to be excluded (see Figure 1). GPI-1 activity was assessed by cellulose acetate electrophoresis and scanning densitometry. Specimens were run on Helena Titan HI electrophoresis plates in a Tris—glycine buffer (pH 8.5) according to Eicher and Washburn (1978). After

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2°C/min to —7°C. After 5 min, pre-chilled forceps were used to induce ice-formation (seeding) in the fraction of M2 + DMS0 at the top of the straw. After a further 5 min the straws were cooled slowly (0.3°C/min) to -40°C before rapid cooling (10°C/min) to - 150°C and transfer to liquid nitrogen (LN2) for storage. For thawing and transplantation of primordial follicles the straws were transported in LN2 by British Rail to Edinburgh. For thawing, the straws were removed from LN2, held in air for 30 s and transferred to a water bath at 30°C for 5 —10 s. The contents of two to four straws were emptied into 10 ml centrifuge tubes and the cryoprotectant was diluted with a volume of M2+FCS (0.8 ml/straw). After 10 min the volume was diluted by a factor of two, and 5 min later the cells were centrifuged (80 g) and resuspended in fresh M2+FCS.

Cryopreservation of primordial follicles

~ 1 h at 200 V the plates were stained and the proportions of GPI-1 allozymes were quantified.

Table I. Outcome of transplanted grafts containing primordial follicles No. of grafts

No. of animals

No. of animals showing oestrogenic activity

No. of animals that mated

No. of pregnancies (size of litter)

10 8 18

5 4 9

4 4 8

3 2 5

2 (3*,2) 2 (2*,5) 4(12)

Results Survival of cells after cryopreservation About 80% of oocytes and 65% of somatic cells survived cryopreservation as determined by the Trypan Blue exclusion test. The preliminary studies demonstrated that the majority of these somatic cells, whether frozen-thawed or not, attached, spread and multiplied on culture dishes. The pre-granulosa cells of primordial follicles also attached and grew but the normal threedimensional integrity was always lost and the oocyte sometimes emerged free at the surface (Figure 2).

Series 1 Series 2 Total *Live-born.

growing follicles were scarce, though no attempt was made to estimate their numbers.

Function of the grafts

Discussion

The fibrin clots contracted during overnight culture and the cells and follicles formed a dense mass of tissue. The functional activity of the grafts is summarized in Table I. After transplantation, all of the hosts had closed vaginas, confirming that oophorectomy had been complete in every case. Within 7 days of the operation, eight of the nine recipients showed vaginal opening. Five of the recipients mated and two litters, of three and two pups, respectively, were produced. At the time of culling an additional two pregnant recipients had two implants (one of which was resorbing) and five implants, respectively. GPI-1-typing of the live-born pups confirmed they had originated from germ cells in the grafted material (GPI-1B), but the tissue from the fetuses was not tested. Grafts that were recovered from host females showed triple banding, suggesting that they were chimaeric as a result of invasion by vascular tissues from the host. At autopsy, the grafts were histologically indistinguishable from normal ovaries and contained a spectrum of follicle sizes with one or more generation of corpora lutea (Figure 3). The ovaries resembled those of older animals because primordial and small

This study shows for the first time that it is possible to restore fertility to sterile animals by the transplantation of frozen—thawed primordial follicles. In addition we confirmed, using genetic markers, that the offspring produced by bulk transfer of primordial follicles arose from transplanted cells rather than incomplete removal of host organs. Several problems are associated with the cryopreservation and subsequent transplantation of mammalian tissues. The most serious is achieving adequate permeation of cryoprotectant into intact organs or pieces of tissue. This was demonstrated in the early attempts to preserve fragments of ovarian tissue in which only — 5 % of the population of gametes survived the procedure (Green et al., 1956). In this study and our previous one (Carroll et al., 1990) the problem was overcome by dissociating the tissue into its constituent cells before cryopreservation. The ability of the dispersed ovarian cells to reorganize into a functioning organ permits the restoration of fertility after transplantation. Alternatively the oocytes may be grown, matured and fertilized in extra-ovarian conditions (Carroll et al., 1990) or entirely 1165

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Fig. 2. Photomicrograph of frozen-thawed cells after 48 h culture, (a) Somatic cells form a monolayer. X500. (b) Intact oocytes covered with follicular cells, and others free of follicular cells can be seen. X1100.

J.Carroll and R.G.Gosden

v

V

in vitro (Eppig and Schroeder, 1989) and the resultant embryos transferred to the foster mother. In a complex mixture of cells there is the potential problem that different cell types may have different cryopreservation optima. The ability of frozen—thawed ovarian tissue to form functional ovaries capable of normal folliculogenesis and steroidogenesis, ovulation and corpus luteum formation suggests that all major cell types, or their stem cells, were able to survive freezing and thawing by our current methods. The single most important limitation of the transplantation technique was the large reduction in the numbers of follicles remaining in the grafts after transplantation and the observed loss of long-term reproductive capacity. Clearly, follicles may be lost at each stage in the procedure, but since a similar deficiency was obtained in a comparable study (Gosden, 1990), it would appear that most of the losses are not accounted for by cryopreservation. The successful preservation of primordial follicles allows the banking of large numbers of potentially viable female gametes. Cryopreservation of mature mouse oocytes has been successful for some years (Whittingham, 1977) but is limited by the number of oocytes available from an individual donor, even with the use of superovulation techniques. In addition, there are some concerns that the organization of the meiotic spindle may be disrupted by cooling (Pickering and Johnson, 1987) and by cryoprotectants (Johnson and Pickering, 1987), which can lead to aneuploid embryos. While these concerns appear largely unfounded in the mouse (Glenister et al., 1987; Bouquet et al., 1992; Carroll et al., 1993), there are differences in the cytoskeletal organization of 1166

the human oocyte that may render them more vulnerable to cooling (Pickering et al., 1988). Attempts to cryopreserve fully grown, cumulus-intact oocytes have met with little success and as yet no fetuses have been produced (Schroeder et al., 1990). Larger numbers of oocytes are available in the form of pre-antral follicles from the ovaries of juvenile mice, and these stages have been successfully cryopreserved (Carroll etal., 1990). Although we have emphasized the advantages of storing isolated follicles at low temperatures, the feasibility of using whole organs or slices of ovarian cortex should not be overlooked. This approach can be successful in small animals (Parrott, 1960) and may be the only practicable method for human follicles which are scattered throughout dense connective tissue. The location of primordial follicles in the ovarian cortex, which is penetrated first by cryoprotectants, probably contributed to the success. As yet there has been little effort to replicate Parrott's work, but given the advances in knowledge of cryobiology (Mazur, 1970), small slices of tissue promise to provide another strategy for storing female gametes. The possibility of the allograft reaction is still a major limitation for tissue transplantation, and current evidence suggests that the ovary is not a particularly privileged organ in this respect (Gosden, 1992). While murine ovarian grafts can restore fertility to sterilized syngenic hosts, there will be few occasions when grafting is either feasible or required between monozygotic human twins. Nevertheless, frozen banking of ovarian tissue is an attractive possibility for children and young women who are at risk of iatrogenic ovarian failure during chemotherapy or

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Fig. 3. (a and b) Histological sections of grafts recovered 6 weeks after transplantation. Note the presence of antral follicles and corpora lutea. X125.

Cryopreservation of primordial follicles

abdominal radiation. The tissue could be returned to the body after cessation of treatment and full remission from disease. It is likely that if ovary cryopreservation is to play any role in reproductive medicine, these patients will be the first to benefit because no immunological or ethical problems are raised. Acknowledgements We thank Helen Taylor for assistance with the electrophoresis, Professor D.G.Whittingham for support and discussion and Dr Maureen Wood and Chris Candy for comments on the manuscript. This work was funded by the Medical Research Council (J.C.) and a Wellcome trust project grant awarded to R.G.G.

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