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Human Reproduction vol.8 no.8 pp. 1163 -116 7, 1993

Transplantation of frozenthawed mouse primordialfollicles

John Carroll1 and Roger G.Gosden2Medical Research Council, Experimental Embryology andTeratology Unit, St George's Hospital Medical School, CranmerTerrace, London SW17 ORE and 2Department of Physiology,University Medical School, Teviot Place, Edinburgh EH8 9AG,UK'To whom correspondence should be addressedPrimordial follicles were isolated from juven ile m ouse ovariesand cryopreserved by slow freezing with dlmethylsulphoxideas the cryoprotectant. After thawing, - 8 0 % of the oocytesand 65% of the somatic cells excluded Trypan Blue dye,indicating that cell membranes were still intact.Frozenthawed cells were suspended in plasma clots andtransplanted to the ovarian bursas of host animals that hadbeen sterilized by oophorectomy. The grafts offrozenthawed cells reorganized into morphologicallydistinguishable ovaries which produced signs of oestrogenicactivity. After natural mating, host females produced normaloffspring that were demonstrated by genetic markers to bederived from the transplanted frozen-thawed primordialfollicles.Key words: cryopreservation/development/mouse/primordialfollicle/transplantation

IntroductionCryopreservation of germ cells is a valuable procedure for theconservation of rare and endangered species, manipulation ofanimal breeding, and in the treatment of certain types of hum aninfertility. Spermatozoa are available for storage in their millionsand successful techniques have been available for many years(Polge et al., 1949). Howe ver, the ease and success of artificialinsemination techniques contrasts markedly with the problemsassociated with freezing the mammalian oocyte.

The earliest attempts to preserve mammalian oocytes involvedthe freezing of fragments of ovarian tissue. These experimentsresulted in the birth of offspring after orthotopic transplantationof frozen-thawed ovarian tissue (Parrott, 1960). Success withwhole or parts of ovaries was limited and the 5 % of the oocytepopulation surviving was confined to small follicles (Deanesly,1954; Green et al., 1956). Further attempts to preserve themature oocyte were limited by difficulties of dehydrating theselarge cells. Despite initial problems, the development of methodsfor 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

minimal loss of viability (Carroll et al., 1993).A major limitation of preserving oocytes at the pre-ovulatorystage is that only a tiny fraction of the original population re achesthis final stage of maturation. The possibility of storing oocytesat early stages of development when they are more abundant istherefore very attractive. Recently we demonstrated that matureoocytes obtained after culturing and transplanting frozenthawedprimary ovarian follicles remained viable (Carroll et al., 1990).Storage of the primordial or 'resting' follicle stages is potentiallybetter because these germ cells are more abundant, have a paucityof potentially sensitive organelles, are less metabolically activeand their small size renders them less susceptible to adverseeffects of low-temperature storage.

The popu lation of primordial follicles around the time of birthis maximal and represents an irreplaceable source of potentialgametes for the entire reproductive lifespan of the femalemammal. It is timely to apply cryopreservation technologybecause efficient methods are available for isolating primordialfollicles from enzymatically dissociated ovaries and subsequentlytransplanting them to sterilized host animals (Gosden, 1990). Theaim of this study was to devise a successful cryopr eservationprotocol for m ouse follicles and test the fertility of frozenstoredoocytes after transplantation to suitably prepared host females.

Materials and methodsIsolation of primordial folliclesThe m ethods for isolating primordial follicles have been describedelsewhere (Gosden, 1990). Briefly, ovaries of 48-day-oldC57BL/6 mice homozygous at the Gpi-lsb locus were dissectedfree and transferred to 2 ml of HEPES-buffered medium M 2(Fulton and Whittingham, 1978) containing 5% fetal calf serum(M2+FCS). The ovaries were bisected and incubated at 37Cfor 2030 min in M2+FCS containing collagenase (1.5 mg/ml;type 1; Sigma Chemical Co., London, UK). The fragments ofovary were transferred to fresh M2+FCS and pipetted to obtaina disaggregation of single cells. The cells were washed twiceby centrifugation (80 g) and re-suspended in fresh M2 + FCS.Cryopreservation of primordial folliclesAfter centrifugation and removal of the supernatant the cells werere-suspended in M2+FCS containing 1.5 M dimethylsulphoxide(M2 + DMSO) at 4C. A small volume of M2 +D M SO wasdrawn into a plastic freezing straw followed by an air bubbleand finally 0.2 ml of cell suspension (equivalent to four to fiveovaries). The straws were sealed with haematocrit putty andplaced in a programmable cell freezer (Planar) pre-cooled to 0C;10 min after addition of M 2+ DM SO the straws were cooled at

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2C/min to 7C. After 5 min, pre-chilled forceps were usedto induce ice-formation (seeding) in the fraction of M2 + D M S 0at the top of the straw. After a further 5 min the straws werecooled slowly (0.3C/min) to -40C before rapid cooling(10C /min) to - 150C and transfer to liquid nitrogen (LN2) forstorage. For thawing and transplantation of primordial folliclesthe straws were transported in LN 2 by British Rail toEdinburgh.

For thawing, the straws were removed from LN 2, held in airfor 30 s and transferred to a water bath at 30C for 5 10 s.The contents of two to four straws w ere emptied into 10 mlcentrifuge tubes and the cryoprotectant w as diluted w ith a volumeof M 2 + F C S (0.8 ml/straw). After 10 min the volume wasdiluted by a factor of tw o, and 5 min later the cells werecentrifuged (80 g) and resuspended in fresh M2+FCS.Assessm ent of cell viability after cryopreservationAfter the cells were washed a sample was taken to assess viabilityby the Trypan Blue dye exclusion test. Preliminary studies werecond ucted to assess the ability of frozenthawed cells to growin culture. Cells obtained from frozenthawed dissociatedovaries were cultured in minimal essential medium (MEM;Earle's salts) containing 5% FCS (Flow Laboratories, Irvine,Scotland, UK) at 37C in a humidified atmosphere of 5% CO 2in air. After 24 h in culture, cell attachment and growth in thedishes was compared with non-frozen control tissue.Transp lantation of primor dial folliclesThe cells were transferred to 0.4 ml microcentrifuge tubes (AlphaLaboratories, Eastleigh, UK). To prepare the cells in plasmaclots, they were centrifuged (80 g) and resuspended in 0.1 mlof fresh m ouse venous plasma. Clotting was induced by touchingthe surface with a sterile needle, after which the tubes wereincubated at 37 C for 1 5 - 3 0 min. The clots containing the dis-aggregated ovarian tissue were transferred to Medium 199containing 10% FCS, L-glutamine, pyruvate and antibioticsand cultured for 12 days as described previously (Torranceetal., 1989; Gosden, 1990). A single clot was transferredto each ovarian bursa of virgin 6 8-week-old C57BL/6(Gpi-ls^/Gpi-ls") female m ice (Fig ure 1), as describedpreviously (Gosden, 1990). Host ovaries were removedimmediately before transplantation and bleeding was controlledby light cauterization, taking care to avoid damage to theFallopian tubes. Tw o series of transplants were performed. Thefirst series consisted of 10 clots that represented a total of 45pairs of donor organs and they were transferred to five hosts;the second comprised eight clots from 55 pairs which weretransferred to four hosts.Assessm ent of function of transplan ted graftsTo check that the removal of the host ovaries was complete andthat the grafts becam e functionally active, the vagina of the hostwas examined daily. Closure of the introitus was taken as anindication of oestrogen deficiency and re-opening as a sign thatoestrogenic follicles had emerged in the graft. After vaginalopening o r 3 weeks post-operation (whichever was earlier), hostfemales were paired with fertile males C57BL/6 (Gpi-l^/Gpi-ls*).The hosts w ere inspected daily for signs of mating (vaginal plug).1164

Donor C57BL/6 Gpi-1sbIdisaggregate ovaries1Freeze/thaw1

suspend tissue in fibrin clot

1culture for 24-48h1Recipient C57BL/6 Gpi-1sa1

assess for oestrogenic activity1mate with C57BL/6 Gpi-1sb malei Ihistology offspring

Fig . 1. Experimental approach for the production of offspring fromfrozenthawed transplanted primordial follicles.

Those that had mated were allowed to litter. At 6- 1 2 weeksafter transplantation the hosts were autopsied, the status of thereproductive tract was examined, and the graft from the ovariancapsule was removed and fixed in Bouin 's fluid, paraffin-embedded, sectioned and stained with haematoxylin and eosin.Some of the grafts together with liver tissue from pups and fetusesand blood from host animals were stored at - 2 0 C for glucosephosphate isomerase (GPI)-1 analysis.Analysis of glucose phosphate isomerase (GPI-1)The use of different Gpi-ls genotypes allowed the derivation ofoffspring from oocytes in the grafts to be verified and thepossibility of incomplete oophorectomy to be excluded (seeFigure 1). GPI-1 activity was assessed by cellulose acetateelectrophoresis and scanning densitometry. Specimens were runon Helena Titan HI electrophoresis plates in a Trisglycinebuffer (pH 8.5) according to Eicher and Washburn (1978). After

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Cryopreservation of primordial follicles

Fig . 2. Photomicrograph of froz en-th awe d cells after 48 h culture, (a) Somatic cells form a monolayer. X50 0. (b) Intact oocytes coveredwith follicular cells , and others free of follicular cells can be seen. X1 100.

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

ResultsSurvival of cells after cryopreservationAbout 80% of oocytes and 65% of somatic cells survivedcryopreservation as determined by the Trypan Blue exclusion test.The preliminary studies demonstrated that the majority of thesesomatic cells, whether frozen-thawed or not, attached, spreadand multiplied on culture dishes. The pre-granulosa cells ofprimordial follicles also attached and grew but the normal three-dimensional integrity was always lost and the oocyte sometimesemerged free at the surface (Figure 2).Function of the graftsThe fibrin clots contracted during overnight culture and the cellsand follicles formed a dense mass of tissue. The functional activityof the grafts is summarized in Table I. A fter transp lantation, allof the hosts had closed vaginas, confirming that oophorectomyhad been com plete in every case. Within 7 days of the operation,eight of the nine recipients showed vaginal opening. Five of therecipients mated and two litters, of three and two pups,respectively, were pro duced. At the time of culling an additionaltwo pregnant recipients had two implants (one of which wasresorbing) and five implants, respectively. GPI-1-typing of thelive-born pups confirmed they had originated from germ cellsin the grafted material (GPI-1B ), but the tissue from the fetuseswas not tested. Grafts that were recovered from host femalesshowed triple banding, suggesting that they were chimaeric asa result of invasion by vascular tissues from the host.

At autopsy , the grafts were histologically indistinguishable fromnormal ovaries and contained a spectrum of follicle sizes withone or more generation of corpora lutea (Figure 3). The ovariesresembled those of older animals because primordial and small

Table I.

Series 1Series 2Total

Outcome of transplantedNo. ofgrafts

108

18*Live-born.

No. ofanimals

549

grafts containingNo. of animalsshowingoestrogenicactivity448

primordialNo. ofanimalsthatmated325

folliclesNo. ofpregnancies(size oflitter)2 (3*,2)2 (2*,5)4 ( 1 2 )

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

DiscussionThis study shows for the first time that it is possible to restorefertility to sterile animals by the transplantation of froze n thaw edprimordial follicles. In addition we confirmed, using geneticmarkers, that the offspring produced by bulk transfer ofprimordial follicles arose from transplanted cells rather thanincomplete removal of host organs.

Several problems are associated w ith the cryopreservation andsubsequent transplantation of mammalian tissues. The mostserious is achieving adequate permeation of cryoprotectant intointact organs or pieces of tissue. This was demonstrated in theearly attempts to preserve fragments of ovarian tissue in whichonly 5 % of the population of gametes survived the procedure(Green et al., 1956). In this study and our previous one (Carrollet al., 1990) the problem was overcom e by dissociating the tissueinto its constituent cells before cryopreservation. The ability ofthe dispersed o varian cells to reorganize into a functioning organpermits the restoration of fertility after transplantation.Alternatively the oocytes may be grown, matured and fertilizedin extra-ovarian conditions (Carroll et al., 1990) or entire ly1165

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v V

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

in vitro (Eppig and Schroeder, 1989) and the resultant embryostransferred to the foster mother.In a complex mixture of cells there is the potential problemthat different cell types may have different cryopreservationoptima. The ability of frozenthawed ovarian tissue to formfunctional ovaries capable of normal folliculogenesis andsteroidogenesis, ovulation and corpus luteum formation suggeststhat all major cell types, or their stem cells , were able to survivefreezing and thawing by our current methods. The single mostimportan t limitation of the transplantation technique was the largereduction in the numbers of follicles remaining in the grafts aftertransplantation and the observed loss of long-term reproductivecapacity. Clearly, follicles may be lost at each stage in theprocedure, but since a similar deficiency was obtained in acomp arable study (Gosden, 1990), it would appear that most ofthe losses are not accounted for by cryopreservation.The successful preservation of primordial follicles allows thebanking of large numbers of potentially viable female gametes.Cryo preservation of mature mouse oocytes has been successfulfor so me y ears (W hittingham, 1977) but is limited by the numberof oocytes available from an individual donor, even with the useof superovulation techniques. In addition, there are some concernsthat the organization of the meiotic spindle may be disrupted bycooling (Pickering and Johnson, 1987) and by cryoprotectants(Johnson and Pickering, 1987), which can lead to aneuploidemb ryos . W hile these concerns appear largely unfounded in themouse (Glenister et al., 1987; Bouquet et al., 1992; Carroll et al.,

1993), there are differences in the cytoskeletal organization of

the human oocyte that may render them more vulnerable tocooling (Pickering et al., 1988). Attempts to cryop reserve fullygrown, cumulus-intact oocytes have met with little success andas yet no fetuses have been produced (Schroeder et al., 1990).Larger numbers of oocytes are available in the form of pre-antralfollicles from the ovaries of juvenile mice, and these stages havebeen successfully cryopreserved (Carroll etal., 1990).

Although we have emphasized the advantages of storingisolated follicles at low temperatures, the feasibility of usingwhole organs or slices of ovarian cortex should not be over-looked. This approach can be successful in small animals (Par rott,1960) and may be the only practicable method for human follicleswhich are scattered throughout dense connective tissue. Thelocation of primordial follicles in the ovarian cortex, which ispenetrated first by cryoprotectants, probably contributed to thesuccess. As yet there has been little effort to replicate Parrott'swork, but given the advances in knowledge of cryobiology(M azur, 1970), small slices of tissue promise to provide anotherstrategy for storing female gametes.The possibility of the allograft reaction is still a major limitationfor tissue transplantation, and current evidence sug gests that theovary is not a particularly privileged organ in this respect(Gosden, 1992). While murine ovarian grafts can restore fertilityto sterilized syngenic hosts, there will be few occasions whengrafting is either feasible o r required between monozygotic humantwins. Nevertheless, frozen banking of ovarian tissue is anattractive possibility for children and young women who are at

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abdominal radiation. The tissue could be returned to the bodyafter cessation of treatment and full remission from disease. Itis likely that if ovary cryopreservation is to play any role inreproductive medicine, these patients will be the first to benefitbecause no immunological or ethical problems are raised.

AcknowledgementsWe thank Helen Taylor for assistance with the electrophoresis, ProfessorD.G.Whittingham for support and discussion and Dr Maureen Woodand Chris Candy for comments on the manuscript. This work was fundedby the Medical Research Council (J.C.) and a Wellcome trust projectgrant awarded to R.G.G.

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