BIOLOGY OF REPRODUCTION 58, 1071-1074 (1998)
Cryopreservation and Orthotopic Transplantation of Mouse Ovaries: New Approach
in Gamete Banking'
Jorge Sztein,2 Hope Sweet, Jane Farley, and Larry Mobraaten
The Jackson Laboratory, Bar Harbor, Maine 04609
plantation, utilizing genetically distinguishable histocompatible recipients, that makes this approach a practical option for gamete preservation.
ABSTRACT
Mouse half ovaries were cryopreserved and orthotopically
transplanted into ovariectomized recipients genetically identical
to ovary donors except for the coat color gene. Fertility was
reestablished in 57% of the female recipients, which became
pregnant in an average of 40 days after transplantation of frozen-thawed half ovaries. These experiments demonstrate that
ovary cryopreservation can be a very useful option for banking
mouse germplasm, or managing subfertile animal colonies,
when embryo or sperm freezing cannot be used or is not cost
effective.
MATERIALS AND METHODS
Animals
Inbred female C57BL/6J (black) and C57BL/6-Aw-J
(white-bellied agouti) mice, 21-40 days old, were obtained
from the Animal Resources colonies of the Jackson Laboratory and were maintained under standard conditions. A
cycle of 14L:10OD was maintained, and food (Jax Lab Diet
96W-A; Old Guilford, Guilford, CT) and acidified water
(HC1, pH 2.8-3.1) were provided ad libitum.
INTRODUCTION
Ovarian tissue transplantation was utilized as a research
tool by many pioneers studying reproductive physiology.
Most of the studies regarding the physiology of the ovary
were carried out by transplanting the grafts to heterotopic
sites and analyzing gonadotropin production.
The first report of a successful orthotopic ovary transplantation in the mouse was by Robertson in 1940 [1]. A
few years later, Russell and Hurst [2], from the Jackson
Laboratory, published their work on maintaining inbred
strains by ovary transplantation. Later, Stevens [3] published a modification of the technique utilizing half an ovary, a variation that overcame most of the grafting problems
experienced in earlier investigations. Since then, more than
50 mouse strains with breeding difficulties have been maintained at the Jackson Laboratory using this technique.
Cryopreservation of the ovary has been studied in many
different species, but production of live offspring from
treated ovaries has been reported only from sheep and mice
[4-7]. In 1960, Parrot [5] reported live offspring after orthotopic transplantation of a mouse ovary slice that had
been frozen to -79 0 C. This investigator found, however,
that the ovary could not be preserved for more than 44
days at that temperature. Since then, many attempts have
been made to improve the methodology for ovary cryopreservation; however, the surgical procedure for orthotopic
transplantation of the frozen-thawed ovary used in mouse
studies impaired full appreciation of the reproductive potential of the preserved organ.
In previous studies of mouse ovary cryopreservation, the
size of the graft varied from that of a fetal gonad [8] to
that of a whole mature ovary [6, 9]. Difficulties in the transfer procedure related to the graft size might explain some
of the differences in the results reported. In this study we
describe a successful combination of appropriate techniques
for ovary cryopreservation coupled with half-ovary transAccepted December 1, 1997.
Received August 28, 1997.
'This research was funded by NIH grants RR01181, RR09781,
RR01262, and CA34196.
2Correspondence: Jorge Sztein, Cryobiology Lab, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609. FAX: (207) 288-6149; email: [email protected]
Ovaries
Female mice, 21-30 days old, were killed by cervical
dislocation and the ovaries were aseptically removed. The
ovaries were placed in a 35 x 10-mm disposable petri dish
(Falcon 1008; Becton Dickinson, Lincoln Park, NJ) containing 2 ml of M2 medium (Sigma, St. Louis, MO) at
room temperature (23°C). Dissection of the ovary from the
fat pad and the bursa was done using a stereo microscope
at x 2 magnification with fiber optic illumination. Both ovaries were sectioned into halves.
Freezing and Thawing
The protocol described is a modification from Harp et
al. [9]. The four pieces from the two ovaries of each female
mouse were transferred into a cryotube (1.8 cc; Nunc Cryotube, Roskilde, Denmark) containing 200 pI of cryoprotectant (CPA) solution consisting of 1.5 M dimethylsulfoxide (DMSO; Sigma) in M2 medium (Sigma) supplemented with 10% fetal bovine serum (Sigma).
Each sample already in CPA was kept at 23°C (room
temperature) for 10 min and then placed on ice (0-0.5°C)
for approximately 45 min. After that time the samples were
placed in an ice-salt bath at -6°C for 5 min, and ice crystallization was induced (seeded) on the surface of the medium with the aid of a pasteur pipette containing a small
amount of frozen medium in the tip cooled at - 100 C. After
this procedure the vials were placed in a controlled-rate
freezer (Cryomed 1010 with model 8024 freezing chamber;
Cryomed-Forma Scientific, Marietta, OH), preequilibrated
at -6°C, and then cooled at -0.5°C per minute to -80°C
before being transferred into liquid nitrogen (-196°C) for
storage. The minimum storage time was 2 wk.
Thawing was performed at room temperature (23 0C)
(slow thawing) until the ice was melted. After thawing, the
cryoprotectant was immediately removed and replaced with
200 pul of M2 medium. The tissue was allowed to equilibrate in the new medium for 10 min before ovary transplantation.
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SZTEIN ET AL.
Ovary Transplantation
The technique used here has been previously described
in detail by Stevens [3] and Cunliffe-Beamer [10]. Host
females chosen as recipients for the ovary transplant were
40-day-old C57BL/6J (black) or C57BL/6-A -J (agouti)
mice. These mice were genetically identical to ovary donors except for the coat color gene. This genetic difference
was used to determine whether or not progeny derived from
the transplanted ovary.
Recipients were anesthetized with 0.2 ml/10 g BW of
2.5% tribromo-ethanol (Aldrich, Milwaukee, WI) and tertamyl alcohol (Aldrich) solution. The surgical field was prepared for aseptic surgery, and a single transverse incision
of the skin at dorsal, across the lumbar area, gave access
to the ovaries on both sides. Both native ovaries were removed, and a transplant consisting of a half piece of ovary
was placed into the right bursa [11]. All recipients were
mated with donor-strain males, carrying the distinguishing
donor coat color gene, 7 days after the surgery.
A comparison group for our experiments on reproductive
capacity of frozen-thawed ovary consisted of 30 female recipients of nonfrozen-ovary transplants taken from mutant
C57BL/6-Lepob/Lep°b mice routinely maintained by this
procedure at the Jackson Laboratory.
Histology
All ovary samples obtained at various stages of the experiment were fixed in Bouin's solution for 45 min and then
transferred to 70% ethanol for storage until processing for
paraffin embedding. The 3-txm serial sections were made
from paraffin blocks and stained by standard protocols with
hematoxylin and eosin. Microphotography slides were obtained using a Leica (Deerfield, IL) DMRXE upright light
microscope with magnification x20 and X40. Slides were
scanned and processed by digital imaging.
Statistical Analysis
Because groups compared were not from the same population, the nonparametric Mann-Whitney statistical analysis and two-way contingency table test by Abacus Concepts, Statview 4.5 software (Abacus Concepts, Berkeley,
CA) were used for analysis.
RESULTS
Surgical Transplantation
The data on reproductive activity of females that received frozen-thawed ovaries are summarized in Table 1.
The implantation of frozen-thawed ovaries was effective in
57% of the experimental females, compared with a 70%
success rate observed for nonfrozen-ovary transfer mice.
The difference was not significant. The interval between
mating after surgery and first litter did not significantly differ between the two groups (40.1 days for the frozenthawed group and 45.7 days for the group used as control).
Only three frozen-thawed ovary recipients produced more
than two litters, one giving five litters. The average number
of pups was 3.2 per litter. Four females had delivery problems apparently caused by early fetal death or by excessively large fetuses. Four recipients failed to become pregnant, and another two females produced only host-derived
progeny.
Of 64 animals born, in litters with at least one donorderived pup, 23 originated from the recipients' ovaries.
TABLE 1. Transplantation statistics of experimental frozen/thawed ovaries and representative nonfrozen ovaries.
Parameter
Number of recipients
Number of successful ovary
transplant takes
Mean number of donor-type
pups per recipient (SE)
Mean number of litters per
recipient (SE)
Mean interval (days) to first litter (SE)
Frozen/thawed
ovariesa
Nonfrozen
ovariesb
23
30c
13
(57%)
3.2 (+ 0.4)
1.5 (+ 0.3)
40.1 (+ 4.9)
21
(70%)c
11.2 (+ 2.1 )d
2.4 (+ 0.4) C
45.7 (+ 2.5)'
and C57BL6J-AwJ ovaries into C57BL/6J-AwJ and C57BL/6J
female recipients, respectively.
b Mutant C57BL6J-Lepob/Lepb ovaries transplanted into C57BL6J-AwJ recipients; data collected from mutant ovary transplant program of the Jackson Laboratory Animal Resources.
C No significant difference (p > 0.05).
aC57BL/6J
d p < 0.01.
This observation underscores the importance of having a
different coat color or a molecular marker to distinguish the
ovary-donor from the recipient strains.
Histology
The microphotographs shown in Figure 1 are from ovary
samples obtained before and after surgical transplantation.
Ten minutes after thawing (not transplanted), the classical
degeneration and coagulative necrosis caused by freezing
damage was observed (Fig. 1A); this has also been described by other authors [5, 9, 12]. The large follicles containing multiple layers of cumulus cells or theca follicular
attached to the oocyte were the most affected.
Twenty-four hours after implantation, the tissue appeared hemorrhagic, perhaps as a result of passive infiltration of blood collected in the bursa during the surgery (Fig.
IB). There were no major changes in the general cellular
morphology as compared to that observed 10 min after
thawing. Even though the stroma still appeared hemorrhagic at 3 days, signs of cell division could be observed in the
cumulus cells (Fig. D).
On the fifth day posttransplantation, the small follicles
appeared to have activity. The previously observed blood
infiltration tended to disappear, and simultaneously, early
evidence of neovascularization processes was observed (not
shown). Finally, at 30 days the transplanted ovary was totally functional, as evidenced by the presence of a clearly
visible corpus luteum surrounded by some primary and
preantral follicles (Fig. 1E). A similar pattern was found at
4 mo postsurgery, at which time the histology revealed a
normally functioning ovary with graafian follicles at different stages of development (Fig. IF).
DISCUSSION
The first report of mouse ovary freezing was by Parrot
[5], and the procedure involved soaking a slide of ovary
tissue in 12% glycerol for 40 min before slow freezing to
-79°C. Although live mouse pups were obtained from 1
pregnancy out of 10, the complication of sterilizing the recipients by x-irradiation, plus the limited period of time
during which the tissue survived storage at -79°C, made
this method inadequate for routine banking requirements.
A few years ago, Harp et al. [9], described a slow freezing method in straws containing the whole ovaries in 1.4
M DMSO as cryoprotectant cooled in liquid nitrogen vapor.
MOUSE OVARY CRYOPRESERVATION
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FIG. 1. A) Histology of a half ovary 10
min after thawing and before transplantation. The classic freezing injury affected
the large follicles and cumulus cells. B-F)
Histology of a frozen-thawed half ovary at
various periods of time after surgical transfer. Twenty-four hours (B) after surgery, tissue appears hemorrhagic. At 3 days (C)
the epithelial cells from the theca follicular start mitosis (arrow). The nuclear chromatin appears decondensed. At 5 days
(D), the frozen-thawed ovary has a normal
aspect. Large and small follicles developed
normally. Thirty days (E) after implantation,
corpus luteum formation demonstrated full
ovarian activity that was maintained till 4
mo (F) after surgery. Pr, primary; P, primordial; PA, preantral; A, antral; CL, corpus
luteum; GF, graafian follicle.
This newer cryobiological technique was unfortunately
combined with an autologous surgical transfer that resulted
in no pregnancies. Even though ovarian functionality was
observed by vaginal smears and histology, this negative
result was perhaps a consequence of transferring the whole
ovary. Cox et al. [8] described the cryopreservation of fetal
ovaries using the same cryoprotectant at 1.5 M in a slow
freezing protocol using a programmable freezer. Considering the difficulty in collecting and manipulating a 16-day
fetal mouse ovary of 1 square millimeter in size, the practical application of this method is limited.
Two more recent publications on mouse ovary cryopreservation by Gunasena et al. [6, 7] reported live pups after
autologous orthotopic transplantation of a cryopreserved
whole ovary. The first study, with a 63% pregnancy rate,
was accomplished by reimplanting the frozen-thawed ovary
back to the same female, and thus it was not possible to
distinguish whether the progeny were derived from the frozen-thawed ovary or the residual ovary tissue present in the
bursa after the ovariectomy. Nevertheless, statistical analysis led to the conclusion that pups were derived from the
frozen-thawed transplant. The second study, using immunodeficient mice as recipients, resulted in a pregnancy rate
of 1 in 4 among the female hosts. The freezing methodology was the same for both studies: a slow freeze in cryovials containing 1.4 M DMSO with a slight modification of
the cooling rate from that used by Harp et al. [9], combined
with rapid thawing.
In our study, we took advantage of a well-established
routine procedure at the Jackson Laboratory for half-ovary
transfer [3] that has been demonstrated to be more successful than use of the whole gonad, and froze the ovary
in halves. The freezing method, a slight modification from
Harp et al. [9], consisted of the same slow cooling program
to -80 0 C used for routine freezing of eight-cell mouse embryos. This procedure facilitated the freezing of embryos
and vials of ovaries at the same time.
In previous reports describing ovarian histology after
cryopreservation, it was concluded that the large follicles
are the most affected by freezing injury, and only the small
immature follicles survived [5, 8, 9, 12-14]. The number of
large follicles that survived the freezing has also been estimated to be about 5% of the total number of small follicles [15]. A more recent analysis on the effect of cryoprotectants on follicle survival after freezing demonstrated that
81-94% of primordial follicles survived when DMSO was
used as cryoprotectant [16]. The histological findings in this
study coincided with previous observations that freezing
damage occurred in most of the large follicles; however,
we have preliminary evidence that some of these follicles
indeed survive, because oocytes were rescued and matu-
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SZTEIN ET AL.
rated in vitro. Those oocytes were fertilized and developed
to blastocysts (data not shown).
Our observations from orthotopic transplantation show
that the frozen histocompatible half ovary recovered the
capacity for endocrine regulation approximately 10 days
after transfer. This conclusion, confirmed by vaginal plug
observation, was previously reported [9], although no pregnancies were obtained in that work.
The histological observation of mitotic processes in the
frozen-thawed samples obtained after transplantation clearly showed that by the third day, the ovary was overcoming
the freezing injuries. This could be an indication of the
reason the interval between surgery and first litter was not
significantly different from that in transfers done with nonfrozen half ovaries. However, in comparison with the nonfrozen group, the average number of pregnancies and pups
produced by a female host of a frozen ovary was lower,
perhaps due to the amount of tissue injured from freezing.
The causes of the delivery problems and early fetal death
could be related to the small size of the graft (25% of the
normal amount), which may have resulted in poor endocrine production. If the fertilized oocyte was from the residual ovary tissue, and the piece of ovary implanted did
not ovulate, the corpus luteum that was created could have
been too small to support the gestation. In previous studies,
this phenomena was also observed; Parrot [5] reported that
two pregnancies resulted in fetal reabsorptions, and Gunasena [6] cited one abortion on the 19th day of gestation and
cannibalism of the progeny.
The application of this methodology will complement
embryo and sperm freezing as a way to bank valuable genetic material from mouse models for human diseases.
ACKNOWLEDGMENTS
The authors thanks Dr. Wesley Beamer for guidance and helpful comments on the manuscript and the Animal Resources Mutant Section of the
Jackson Laboratory for providing data on mutant ovary transplantation.
REFERENCES
1. Robertson GAG. Ovarian transplantation in the house mouse. Proc
Soc Exp Biol Med 1940; 44:302-304.
2. Russell W, Hurt J. Pure strain mice born to hybrids mother following
ovarian transplantation. Proc Natl Acad Sci USA 1945; 31:267-273.
3. Stevens LC. A modification of Robertson's technique of homoiotopic
ovarian transplantation in mice. Transplant Bull 1957; 4:106-107.
4. Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to
oophorectomized sheep by ovarian autografts stored at -196°C. Hum
Rep 1994; 9:597-603.
5. Parrot DM. The fertility of mice with orthotopic ovarian grafts derived
from frozen tissue. J Reprod Fertil 1960; 1:230-241.
6. Gunasena KT, Villines PM, Critser ES, Critser JK. Live births after
autologous transplant of cryopreserved mouse ovaries. Hum Reprod
1997; 12:101-106.
7. Gunasena KT, Lakey JRT, Villines PM, Critser ES, Critser JK. Allogeneic and Xenogeneic transplantation of cryopreserved ovarian tissue
to athymic mice. Biol Reprod 1997; 57:226-231.
8. Cox SL, Shaw J, Jenkin G. Transplantation of cryopreserved fetal
ovarian tissue to adult recipients in mice. J Reprod Fertil 1996; 107:
315-322.
9. Harp R, Leibach J, Black J, Keldahl C, Karow A. Cryopreservation
of murine ovarian tissue. Cryobiology 1994; 31:336-343.
10. Cunliffe-Beamer TL. Biomethodology and surgical techniques. In:
Foster HL, Small JD, Fox JG (eds.), The Mouse in Biomedical Research, Vol 3. New York: Academic Press; 1983: 402-430.
11. Wiebold J, Becker W. Inequality in function of the right and left ovaries and uterine horns of the mouse. J Reprod Fertil 1987; 79:125134.
12. Smith AU. The viability of the frozen ovarian tissue. In: Barcroft H,
Davinson H, Paton WDM (eds.), Biological Effects of Freezing and
Supercooling. Monographs of the Physiological Society. London: E.
Arnold Publishers; 1961: 166-196.
13. Gosden RG. Transplantation of fetal germ cells. J Assist Reprod Genet
1992; 9:118-123.
14. Candy C, Wood M, Whittingham D. Follicular development in cryopreserved marmoset ovarian tissue after transplantation. Hum Reprod
1995; 10:2334-2338.
15. Green SH, Smith AV, Zukerman S. The numbers of oocytes in ovarian
autografts freezing and thawing. J Endocrinol 1956; 13:330-334.
16. Candy CJ, Wood MJ, Whittingham DG. Effect of cryoprotectants on
the survival of follicles in frozen mouse ovaries. J Reprod Fertil 1997;
110:11-19.