ILAR Journal Online, Volume 41(4) 2000: Cryobiology of Embryos, Germ Cells, and Ovaries

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ILAR Journal V41(4) 2000
Cryobiology of Embryos, Germ Cells, and Ovaries

Cryopreservation of Oocyte and Ovarian Tissue
Yuksel Agca

Yuksel Agca, D.V.M., Ph.D., is a Research Associate in the Cryobiology Research Institute, Department of Pediatrics, Indiana University Medical School, Indianapolis, Indiana.

Abstract

Cryopreservation of reproductive cells (i.e., oocytes, spermatozoa) and tissues (i.e., ovarian and testicular tissue) is a developing technology that has tremendous implications for rapid advancement of biomedical research. Since the early 1980s, advances have been made in establishing optimal conditions for in vitro oocyte maturation, fertilization, and embryo culture. These in vitro systems have contributed significantly to utilization of thawed cells and tissues and have made it possible to evaluate protocols designed to cryopreserve such biomaterials more effectively. Although cryopreservation of preimplantation embryos from various species including mouse, human, and farm animals has been successful, cryopreservation of oocytes and ovarian tissue from most mammalian species has been more challenging due to their extreme sensitivity to suboptimal conditions during the cryopreservation process. Cryopreservation of mouse oocytes has been well documented and has resulted in greater success than studies with other mammalian species. Ovarian tissue cryopreservation and transplantation techniques have recently received much scientific and public attention due to their great potential for use in human infertility treatment, in safeguarding the reproductive potential of endangered species, and in genome banking of genetically important laboratory animal strains. A review of past and current research in the field of oocyte and ovarian tissue cryopreservation and transplantation and a discussion of possible strategies for oocyte and ovarian tissue banking are provided.

Key words: cryopreservation; oocyte; ovarian tissue; primordial follicle; transplantation

Introduction

The effects of subzero temperatures on survival of both animal and human cells and tissues have long been the subject of much interest. In the context of reproduction, restoring fertility from cryopreserved reproductive cells (i.e., oocytes, spermatozoa) and tissues (i.e., ovarian and testicular tissue) has been a long-term goal of physicians and researchers (Nugent et al. 1997). Recently, there has been a rapid increase in the number of genetically engineered mice and rats, and production of these animals provides a powerful model to explore the regulation of gene expression and cellular and physiological processes. However, the high costs of producing and maintaining these animals demands reliable cryopreservation protocols to preserve specific strains without perpetuating them through continuous live breeding (Carl 1994). The ability to preserve oocytes and ovarian tissues in a healthy state for a desired period of time would have tremendous application in the field of biomedical research.

For decades, researchers have attempted to cryopreserve oocytes and ovaries; and significant improvements have resulted in many assisted reproductive techniques, such as in vitro oocyte maturation, fertilization, culture, and intracytoplasmic sperm injection (Leibfried-Rutledge et al. 1997). Ovarian tissue banking is being considered as an option for banking of mouse genomes, particularly for subfertile animal colonies, when gamete and embryo freezing cannot be utilized or is not cost effective (Sztein et al. 1999). Furthermore, ovarian tissue banking in humans is also being considered in the hope of restoring fertility to patients who lose ovarian function due to chemo- or radiotherapy during cancer treatment (Newton 1998; Paynter et al. 1997).

Methods developed for oocyte and ovarian tissue cryopreservation must protect structural and functional viability. To date, several strategies have been suggested to restore fertility using cryopreserved oocyte or ovarian tissue (Figure 1). These strategies describe cryopreservation of oocytes at either an immature (isolated from the ovary) or a mature (after ovulation) state. In regard to ovarian tissue, one strategy involves autologous orthotopic or xenogeneic transplantation of the frozen-thawed ovaries and monitoring the developmental potential and follicular dynamics in vivo (Aubard et al. 1998; Gosden et al. 1994a,b; Gunasena et al. 1997a,b; Harp et al. 1994; Oktay et al. 1998b; Weissman et al. 1999). The second strategy incorporates in vitro maturation, fertilization, and culture of primordial follicles isolated from frozen-thawed ovaries (Carroll et al. 1990; Oktay et al. 1998a; Sztein et al. 2000).

This article provides a review of past and current research in the field of oocyte and ovarian tissue cryopreservation and a discussion of possible alternative strategies for oocyte and ovarian tissue banking. Although primary emphasis is on mice and rats, studies from other species used as research models are discussed in the context of ovarian function after xenogeneic transplantation.

Structure and Function of the Mammalian Ovary and Oocytes

The general anatomical and functional description of the mammalian ovary has been well documented (Mossman and Duke 1973). The size of adult mouse and rat ovaries are approximately 2 to 5 mm³, and, at the onset of puberty, they contain approximately 3000 and 5000 primary follicles, respectively (Peters and McNatty 1980). The ovary is generally divided into two major regions (Figure 2), the outer cortex and the inner medulla. In the adult mouse and rat ovary, the outermost layer, termed tunica albuginea, is composed of fibrous connective tissue, and the surface of the ovary has a layer of flattened cells or germinal epithelium. Whereas the cortex region contains the majority of the functional units such as follicles, corpora lutea, and interstitial tissue, the medulla consists mainly of blood, lymph vessels, and connective tissues. Although the growing follicles may lie close to the ovarian periphery, in the context of cryopreservation, the cortex region of the ovary has more significance because of the presence of abundant numbers of primordial follicles, which are the target structures of cryopreservation efforts. The cortex is the site of both egg formation and hormone production. Because the ovary is a dynamic organ in which primordial follicles are constantly developing into preovulatory follicles, it contains different structures at various stages of development. A primordial follicle consists of an oocyte surrounded by one layer of flattened cells; and as oocyte maturation progresses, the diameter of the follicles increases. Besides egg formation, the ovary has the ability to make steroid hormones such as progesterone and estrogen, which are secreted by particular cell types (i.e., theca and granulosa cells). The levels of hormones produced are determined by the stage of the estrous cycle. The surrounding cells also increase in number and shape and are vascularized as folliculogenesis progresses. Metabolic and structural integrity of these various tissues after freezing and thawing is crucial for normal folliculogenesis.

The oocyte is the largest (~80-120 mm diameter) single mammalian cell. Fully developed mouse and rat oocytes consist of a plasma membrane (oolemma) with a clear ooplasm. The oolemma is surrounded by the zona pellucida (extracellular glycoprotein matrix), which is surrounded by several layers of corona cumulus cells. Immature oocytes are present in the antral follicle of ovaries and arrested in the prophase of the first meiotic division (Figure 3A). These cells have a distinct large prophase nucleus called the germinal vesicle (GV¹). After gonadotropin stimulation, the GV breaks down, a metaphase plate forms, and the oocyte is arrested at the metaphase of the second meiotic division (MII¹) characterized by the presence of the first polar body (Figure 3B). Fundamental changes in the structure and function of oocytes occur as they develop from the GV stage (in which the chromosomes are well protected in their condensed form) to the MII stage (when the chromosomes and connected spindle fibers exist freely in the cytoplasm). The other important components are the cytoskeletal elements (microtubules and microfilaments) and cortical granule vesicles in the cytoplasm. Maintenance of structural and functional integrity of oolemma, cortical granule vesicles, meiotic spindles, and other cytoplasmic organelles (e.g., mitochondria, golgi, endoplasmic reticulum) are crucial for successful fertilization and further cell division. Although cortical granule exocytosis and subsequent zona hardening are crucial steps to prevent polyspermic fertilization, disruption of the meiotic spindle can cause chromosomal abnormalities in the resulting embryos.

Fundamentals of Cryobiology

Cryopreservation is a multistep procedure that involves an initial exposure of cells and tissues to cryoprotective agents (CPAs¹), cooling to subzero temperatures, storage, thawing, and finally, dilution and removal of the CPA with return to physiological environment allowing further development. The cells or tissues must maintain their structural integrity throughout these procedures. Slow cooling (equilibrium freezing) involves equilibrating the cells in relatively low CPA concentrations (~1.5M) and dehydration during cooling (0.3-2°C/min). This method was the one first used successfully to cryopreserve mouse embryos (Whittingham et al. 1972).

During the freezing process, at least two types of damage can occur to biological materials in the solution: (1) intracellular ice formation, when the sample is cooled "too quickly" causing water to be "trapped" inside the cells; and (2) damage due to the high-solute concentrations generated when water precipitates as ice (Karllsson et al. 1996; Mazur 1977; Mazur et al. 1984). This second type of damage occurs when cells are cooled "too slowly," which will cause the cells to be exposed to high-solute concentrations, which form when water precipitates as ice and concentrates the solutes (e.g., electrolytes) in the unfrozen channels (Mazur et al. 1972). In addition, cells that are cooled slowly are potentially affected by chilling injury (Mazur et al. 1992). The major rate-limiting factor that governs the slow cooling procedure is the rate of passage of water and CPA through the cell membrane. This rate depends on (1) membrane composition, (2) temperature-dependent permeability characteristics of the cell plasma membrane to water and CPAs (Mazur and Schneider 1986), and (3) the surface-to-volume ratio (Leibo 1980); Mazur and Schneider 1986). These parameters permit the calculation of optimal methods for (1) the addition and removal of CPAs, and (2) cooling and warming rates necessary to avoid intra-cellular ice formation and so-called "solution effects" (Karlsson et al. 1996; Mazur 1990).

Quasiequilibrium (rapid cooling, ~200°C/min) and non-equilibrium freezing (ultrarapid cooling, ~2500°C/min) are alternatives to equilibrium freezing (Mazur 1990). Slow cooling involves the precipitation of water as ice, resulting in the separation of water from the solutes collectively composing the solution. Vitrification is the transition of aqueous solutions from the liquid to the glass state (solid), bypassing the crystalline solid state (Fahy et al. 1984). To achieve vitrification in the context of cell and tissue cryopreservation, the solutions in which the cells or tissue are suspended must have a high concentration of CPAs (often 7-8 M). Equilibrating oocytes with these high concentrations of CPAs has been both biologically problematic and technically difficult. Furthermore, in practice, for the vitrification of biological samples, there are both a practical limit to achievable cooling rates and a biological limit to the concentration of CPAs that the cells will tolerate. However, this method of cryopreservation does not require programmable freezing machines, which permits utilization of this technique in field conditions. Furthermore, ultrarapid cooling may be helpful to cryopreserve some cell types that are extremely sensitive to low temperature (Mazur et al. 1992).

Review of Ovarian Tissue Cryopreservation

The idea of ovarian tissue transplantation after cryopreservation is not new. Historically, ovarian tissue transplantation studies using either fresh or cryopreserved tissue have basically been divided into two time periods. Most of the early studies were performed in the 1950s, when glycerol was the main component of the freezing solutions used as a CPA. Parkes and Smith (1954) attempted to cryopreserve rat ovarian tissue slices (1-mm³ pieces) using slow cooling to -79°C in the presence of 15% glycerol. They were able to obtain partial restoration of cyclic function. Using the same protocol, Deanesly (1954) cryopreserved relatively larger (one half of a rat ovary) tissue and observed that the central region did not survive as well as the peripheral region. Most other studies during the 1950s focused on graft establishment rates, duration and interval of the estrous cycle after exposure of various concentrations of glycerol at various exposure times (without freezing), and various cooling rates (Deanesly 1957; Green et al. 1956).

Robertson (1940) and Krohn (1958) were able to obtain live offspring after orthotopic transplantation of fresh mouse ovaries, which was an important step forward for ovarian tissue cryopreservation technology. This achievement led to the delivery of live offspring after orthotopic transplantation of frozen mouse and hamster ovaries using 15% glycerol (Parrott 1959, 1960). A total of 80% of the follicles were destroyed using this procedure.

Interest in investigating ovarian tissue cryopreservation did not receive additional attention until the 1990s, due, in part, to poor rates of cryosurvival and lack of reproductive techniques that would support utilization of this technology. In the 1990s, other CPAs such as dimethyl sulfoxide (DMSO¹), ethylene glycol (EG¹), and propylene glycol (PG¹) were found to have cryoprotective properties. Among these, DMSO has been the most commonly used CPA to cryopreserve various reproductive cells. There is also general agreement in the literature that slow cooling procedures appear to be the optimal method to cryopreserve ovarian tissues from a wide ranges of species (Shaw et al. 2000). Most of the studies conducted on ovarian tissue cryopreservation in the 1990s have used 1.5 M DMSO followed by slow cooling (0.3-0.5°C/min) procedures and plunging into liquid nitrogen between -55 and -140°C (Shaw et al. 2000). Besides frozen-thawed ovary transplantation and subsequent natural mating, some studies have taken different approaches toward banking ovarian follicles. Some studies aimed to cryopreserve follicles after they were isolated from freshly dissected ovaries, and others aimed to grow follicles isolated from previously frozen ovaries.

Gosden and colleagues examined the possibility of in vivo growth of cryopreserved follicles isolated from the mouse ovary by enzymatic dissociation. They transplanted fresh or frozen-thawed follicles into the ovarian bursa of individual ovariectomized mice after suspending them in a plasma clot. They subsequently obtained live offspring (Carroll and Gosden 1993; Gosden 1990). A significant achievement in the field of ovarian tissue cryopreservation and transplantation was the birth of a lamb after autografting frozen-thawed ovarian tissue slices dissected from the cortical region of a sheep ovary (Gosden et al. 1994b). Full-term development from cryopreserved fetal mouse (Cox et al. 1996) and adult mouse and rat ovaries (Aubard et al. 1998; Gunasena et al. 1997b) after orthotopic transplantation were also important steps forward. Besides auto- and allografting studies in mice and rats, a substantial research effort has been directed toward vivo follicle growth of other species. Immunodeficient mice have been successfully used as animal models for in vivo assessment of cryopreserved ovarian tissue xenografts of the sheep and cat (Gosden et al. 1994a), marmoset (Candy et al. 1995), elephant (Gunasena et al. 1998), human (Oktay et al. 1998b; Weissman et al. 1999), and bovine (Semple et al. 2000) (Table 1).

Review of Oocyte Cryopreservation

Although Sherman and Lin (1958) investigated the survival of unfertilized mouse oocytes during cooling and warming, it was not until 1977 (Whittingham 1977) that live births from frozen-thawed MII mouse oocytes were obtained. Mouse oocytes have been more broadly investigated compared with other mammalian species' oocytes, and these findings have actually illuminated many questions regarding problems associated with oocyte cryopreservation. Several groups began investigating the fundamental cryobiology of mouse oocytes in regard to oolemma permeability to water and CPAs, intracellular freezing temperatures, and effects of various low temperatures, CPAs, and cooling rates on survival (Leibo 1980; Leibo et al. 1975; Parkening et al. 1976). Adverse effects of cryopreservation procedures on oocytes have been well studied in terms of loss of one or more requisite structural and/or functional integrities of cellular components (Glenister et al. 1987; Hotamisligil et al. 1996; Kola et al. 1988; Parks 1997; Van Blerkom 1989). These studies have indicated that oocytes are particularly sensitive to non-physiological conditions.

Investigators have had varied success (in 10-40% of studies) in attempts to cryopreserve both GV and metaphase II mouse oocytes, assessed by blastocyst formation after in vitro fertilization using either slow cooling (Candy et al. 1994; Carroll et al. 1993; Karlsson et al. 1996; Schroeder et al. 1990; Stachecki and Willadsen 2000) or ultrarapid cooling protocols (Kono et al. 1991; Nakagata 1989; Wood et al. 1993). Permeating CPAs such as DMSO, PG, and EG were sometimes supplemented with nonpermeating solutes (e.g., sugars) (Rayos et al. 1994) and macromolecules (e.g., fetal calf serum) (Carroll et al. 1993). Van Blerkom (1989) first attempted to cryopreserve mouse GV oocytes, and the first live birth was obtained by Candy et al. (1994) using a slow cooling procedure in the presence of 1.5 M DMSO. Compared with the mouse, very few attempts have been made to cryopreserve rat oocytes. A slow freezing protocol with 1.5 M DMSO was used to cryopreserve rat ovarian oocytes, and 33% of frozen-thawed oocytes were penetrated by sperm (Kasai and Iritani 1979). In another investigation, morphological and functional properties of rat GV oocyte cumulus complexes were examined after freezing in 1.5 M DMSO. It was found that oocytes with more cumulus cell layers have better survival rates than those with few cumulus cell layers (Pellicer et al. 1988). Although live births have been reported from MII oocytes after vitrification in 2 M DMSO + 2 M acetamide + 4 MPG, survival rates in terms of development into fetuses have been very low (3%) (Nakagata 1992).

Factors Affecting the Viability of Cryopreserved Oocytes and Ovarian Tissue

Oocytes

The critical issues associated with oocyte cryopreservation are at the organelle/subcellular level and therefore have been found to be more complex than in developing preimplantation embryos. Oocytes have a relatively complex subcellular structure within which many of the subcellular components are particularly temperature sensitive (Magistrini and Szollosi 1980) and osmotically and ionically sensitive (McWilliams et al. 1991, 1995; Stachecki et al. 1998a). Designing successful cryopreservation protocols for mammalian oocytes requires recognition of the contribution of cytoskeletal elements to events associated with both cell cycle progression (organelle movement, spindle morphogenesis) and somatic cell interaction at the level of the plasma membrane (Albertini 1992, 1995). To date, several factors have been determined to be important for developing optimal cryopreservation protocols for oocytes (Critser et al. 1997).

Ovarian Tissue

Although there have been substantial numbers of studies on factors affecting cryosurvival of oocytes, studies investigating these effects on ovarian tissues have been limited (Paynter et al. 1997). Compared with a suspended single cell, tissue cryopreservation presents serious physical constraints related to heat and mass transfer (Karlsson et al. 1994). Furthermore, because it is a multicellular structure for which cell-to-cell interactions are known to exist within the ovary, the dynamics of CPA permeation into and out of the tissue during cryopreservation must be considered. When tissue is placed in hyperosmotic CPA solutions, water will be osmotically drawn from the individual cells and will accumulate in the extracellular spaces and in the blood vessels. The fate of this water is the formation of large ice structures, which can injure the tissue during freezing and subsequent warming (Mazur 1977). In this regard, better survival may be expected from primordial follicles because of their smaller size and lack of follicular fluid.

Although whole ovaries from mice and rats survive freezing because of their smaller sizes, successful cryopreservation of whole ovaries from other mammalian species such as human and nonhuman primates as well as livestock species can be difficult due to heat and mass transfer limitations as well as posttransplantation limitations. Cryopreserving small pieces (1-2 mm³) of ovarian cortex is necessary from a cryobiology point of view because the rate of CPA/cellular water exchange is affected by the amount of tissue through which the CPA must diffuse. During the cooling stage of cryopreservation, the relative distance of cells in the interior of the ovary from the exterior affects the rate at which these cells undergo cooling. Because current transplantation procedures do not utilize vascular anastomosis, grafts are solely dependent on posttransplantation vascularization. The ability of cells in the graft to obtain nutrients from their surroundings before permanent revascularization depends on the diffusion of those nutrients through the surrounding tissue. Previous studies with fresh transplants suggest that 50% of the follicular population are damaged after transplantation in mice (Felicio et al. 1983) and 26% in human studies (Newton et al. 1996). This result confirms that substantial numbers of follicles are lost during graft establishment.

Oocyte Developmental Stages

Gook and colleagues (1993) and Van Blerkom and Davis (1994) have examined the effects of cryopreservation on cellular organization in GV and Mil mouse oocytes. Their studies demonstrated a normal nucleus and cytoplasm in frozen-thawed GV oocytes that were capable of maturation and implantation. However, MII mouse oocytes have been associated with an increase in the frequency of aneuploidy. Thus, there are both advantages and disadvantages of cryopreserving oocytes at GV and MII stages. The cryopreservation of GV oocytes might circumvent some of the potential problems associated with MII oocyte cryopreservation such as spindle disorganization, chromosomal scattering, and associated low fertilization (Candy et al. 1994; Van Blerkom and Davis 1994). Eroglu et al. (1998a) assessed the effect of cryopreservation on the cytoskeleton of GV mouse oocytes and concluded that cryopreservation at the GV stage is particularly advantageous to prevent the spindle damage and increased likelihood of aneuploidy, which occurs at the MII stage. In addition, in the course of maturation, chemical and physical changes occur in the oolemma (Ashwood-Smith et al. 1988) that may alter its permeability characteristics to water and to CPAs, which are important determinant factors for the cell survival during cryopreservation (Agca et al. 2000).

Effects of Low Temperature on Oocytes

Cooling affects spindle fiber integrity (Pickering and Johnson 1987) and cortical granule vesicles (Vincent and Johnson 1992). Depolymerization of the spindle fiber is likely to lead to aneuploidy (Bouquet et al. 1995; Kola et al. 1988), and premature release of the cortical granule vesicles is likely to lead to zona hardening (George and Johnson 1993; Vincent and Johnson 1992). However, the mouse oocyte has the capacity to reverse the disruption of the meiotic spindle fibers and "repolymerize" in an appropriate manner (Bouquet et al. 1993). Similarly, Eroglu et al. (1998b) demonstrated that a short period of incubation of cryopreserved mouse MII oocytes after thawing allows restoration of cytoskeletal elements and thus increases fertilization rate. It is also known that transient cytoplasmic calcium concentration changes play an important role in mammalian fertilization (Miyazaki et al. 1993). Ben-Yosef et al. (1995) studied the changes in Ca²⁺ during oocyte activation by cooling and demonstrated that chilling is associated with Ca²⁺ oscillation in MII rat oocytes. This finding suggests that chilling itself may induce parthenogenetic activation and may prevent normal postthaw fertilization in the rat.

Effects of CPAs and Duration of Exposure

Oocytes

Despite the protective effect of CPAs during freezing, they may impose concentration-, time-, and temperature-dependent toxicity (Fahy et al. 1990). Therefore, investigating the choice of CPA and exposure time and temperature before freezing is necessary to optimize a cryopreservation procedure. It has been shown that CPAs have adverse affects on the organization of the microtubule system in mouse oocytes (Johnson and Pickering 1987; Vincent et al. 1990a). Furthermore, aneuploid embryos have been obtained from oocytes exposed to 1.5 M DMSO without cooling, which suggests CPA induced DNA damage (Bouquet et al. 1993). Alterations of zona pellucida glycoproteins, especially ZP2, are reported to be responsible for zona hardening in mouse oocytes (Moller and Wassarman 1989). A reduced in vitro fertilization rate due to changes in zona pellucida and premature exocytosis of cortical granules has also been reported in mouse oocytes exposed to 1.5 M DMSO and PG (Carroll et al. 1990; Schalkoff et al. 1989; Vincent et al. 1990b). However, when Hotamisligil and coworkers (1996) evaluated the effects of EG on membrane integrity, microfilament organization, and developmental potential of mouse MII oocytes after treatment with 6 MEG +0.5 M sucrose, they found no significant differences in development during the two-cell and blastocyst stages between CPA-treated and control oocytes. Similar to the effects of cooling on the Ca²⁺ transient in MII rat oocytes are changes in Ca²⁺ in MII mouse oocytes after exposure to 1.5 MPG, DMSO, or glycerol at 21°C, which Litkouhi and colleagues (1999) investigated. In their study, whereas exposure to PG significantly increased intracellular Ca²⁺, DMSO, or glycerol did not contribute any detectable changes.

To develop quantitative strategies for minimizing injury associated with cell volume excursions and CPA toxicity, it is necessary to characterize the fundamental cryobiological properties. These properties include (1) the hydraulic conductivity, (2) the cryoprotectant permeability coefficients of the cell membrane, and (3) how the latter two values change with temperature. A theoretical optimization approach has been proposed to develop better cryopreservation protocols for mammalian oocytes (Karlsson et al. 1996; Newton et al. 1999; Paynter et al. 1999). For example, calculations were used to predict intracellular DMSO concentration after a one-step addition of either a 1.5 M or 6 M DMSO solution. The way intracellular DMSO concentrations change with time, depending on the temperature at which the exposure is performed, can be seen in Figure 4 (Agca et al. 1998).

Ovarian Tissue

Early studies have demonstrated the deleterious effects of prolonged exposure (-1 hr at room temperature) of mouse and rat ovaries to glycerol (Parkes and Smith 1953; Parrott 1960). Newton et al. (1996) demonstrated the difference in survival using various CPAs. In their study, whereas human ovarian tissue frozen in the presence of glycerol lost 90% of the primordial follicle population, tissues cryopreserved in solution containing DMSO, EG, and PG lost 25, 15, and 55% of the primordial follicles after transplantation in SCID mice, respectively. These findings were also supported by another report suggesting that DMSO caused minimal damage compared with 2,3-butanediol and glycerol (Paynter et al. 1997). Studies conducted on mammalian oocytes also revealed that the permeability coefficient of glycerol is much lower than DMSO and EG or PG (Jackowski et al. 1980; Le Gal et al. 1995), which may explain the low survival rate of mouse and rat ovarian tissue cryopreserved in the presence of glycerol in the past.

Candy et al. (1997) have compared the effects of four different CPAs on the survival of mouse ovaries after exposure to 1.5 M DMSO, PG, EG, or glycerol for 5 to 60 min at room temperature before freezing. More primordial follicles survived when ovaries were frozen in DMSO, PG, and EG (81-94%) than in glycerol (4-28%). Furthermore, it was found that prolonged exposure (60 min) to EG decreased the survival rate, whereas increasing the exposure to glycerol (12 min) increased the survival rate. Overall, the total number of follicles remaining in grafts of ovaries frozen in DMSO and PG represented 42 to 46% of follicles present in non-grafted ovaries, which was not significantly different from grafts of fresh ovaries (63%). To achieve optimal cryoprotection, it is essential that freezing protocols allow uniform penetration of CPAs throughout the ovarian tissue. Thus, the rate of CPA permeation is an important determining factor in developing better cryopreservation protocols for ovarian tissues. Newton et al. (1998) have investigated the DMSO, PG, EG, and glycerol diffusion into human ovarian tissue at both 4°C and 37°C and have observed that at 4°C, PG and glycerol penetrate the tissue significantly more slowly than either EG or DMSO. At the higher temperature (37°C), however, all four CPAs penetrate at a faster rate. Some studies used the more sophisticated technique of ¹H nuclear magnetic resonance spectroscopy to measure DMSO permeation in human and porcine ovarian tissue (3-5 mm³) during slow freezing. Thomas and colleagues (1997) reported that by the end of the 20-min exposure at 0 to 2°C, the mean tissue concentration reaches 0.68 M and 0.76 M in the porcine and human, respectively, indicating that CPA permeation into both tissues is incomplete in currently used protocols.

Effects of Osmotic and Ionic Stress on Oocytes

During the cryopreservation procedure, cells and tissues undergo volume changes due to different osmotic pressures between the intracellular and extracellular solutions (Bernard et al. 1988; Oda et al. 1992). These changes in cell volume affect several parameters that play a role in the cryosurvival of oocytes, including integrity of the plasma membrane (Hotamisligil et al. 1996) and subcellular organelles (Carroll et al. 1989; Magistrini and Szollosi, 1980; McWilliams et al. 1991; Schalkoff et al. 1989). Cells generally demonstrate an ideal osmotic response. This response has been characterized by the Boyle Van't Hoff equation in which cell volume is a linear function of 1/osmolality for MII mouse oocytes (Hunter et al. 1992). Osmotic tolerance limits must be known to avoid excessive shrinkage and swelling and to predict optimal addition and removal of cryoprotectants (Hotamisligil et al. 1996; Mazur and Schneider 1986; McWilliams et al. 1991; Pedro et al. 1997).

Biggers and colleagues (1993), Mazur et al. (1984), and Stachecki and coworkers (1998a) have suggested that high intracellular and extracellular solute concentrations during slow cooling may be responsible for cell damage due to solution effects. To avoid damage due to these high concentrations, CPAs are added to the cell suspension to maintain the solutes at a concentration tolerated by the cells and a given cooling rate (colligative effect) (Fahy 1986). Although the exact mechanism underlying this damage is not fully explained, Stachecki et al. (1998b) have recently demonstrated that reducing or replacing NaCl with choline significantly improved the survival of mouse oocytes after slow cryopreservation.

Animal Studies of Ovarian Tissue Transplantation

Although it is important to evaluate whether ovarian tissues and follicles are viable and competent after the cryopreservation procedure, the current in vitro models to study preantral follicle growth (particularly in human tissue) is not well established. One way to evaluate the function of frozen-thawed tissues is to transplant the grafts and then monitor the developmental competence in the host animal. However, to avoid immune rejection of the graft in hetero- or xeno-transplantation, allogeneic transplantation into a recipient with suitable histocompatibility complex or into an immunodeficient recipient is necessary. Immunodeficient mice have been used effectively as an in vivo research animal model for better understanding the nature of tumor cell growth derived from both humans and animals. Numerous cancer cell lines have been xenografted into these mice, and significant knowledge has been gained as a result of these studies (Strieter et al. 1999). Similarly, ovarian xenografts in immunodeficient mice also serve as research models for studying follicle development in other mammalian species. Currently two of these animal models--athymic nude (nu/nu) mice, which are lacking mature T cells, and severe combined immunodeficient (SCID¹) mice, which are lacking both mature B and T cells--are being used for testing steroidogenic activity. This animal model is also useful for the assessment of in vivo follicular development competence of ovarian tissues from various mammalian species.

Use of nonhuman primate ovaries in combination with immunodeficient mice would be the best model to extrapolate tissue viability for humans because of the similarities in ovarian morphology and follicular development between primates and humans. Candy and colleagues (1995) reported successful transplantation of frozen-thawed marmoset ovarian tissue (-1 mm³) under the kidney capsule of athymic nude mice. In the majority (83%) of the recipients, endocrine function resumed and large antral follicles developed. Their studies have shown that despite a drastic reduction in the total number of primordial follicles, it is still possible to recover an acceptable proportion of the potentially usable follicles from cryopreserved ovarian tissues. Moreover, ovarian tissue xenografts in immunodeficient mice can serve as experimental models for investigating follicle development in species in which in vitro follicle growth and studies of the parent animal are not possible. However, it must be noted that these studies are still in an experimental stage and thus require further investigation to evaluate the apparently normal follicles for complete nuclear and cytoplasmic maturation to permit successful fertilization and embryo development.

Human Reproductive Medicine

Extremely low postthaw survival rates of human oocytes necessitate banking ovarian tissue for patients who will soon receive radiotherapy or chemotherapy (Oktay et al. 1998a). As in other reproductive technologies, the research carded out in cryopreservation and transplantation animal studies has provided useful information in transferring methods to treat human infertility (Aubard 1999). When frozen-thawed ovarian tissue slices were transplanted into sheep, follicular survival and endocrine function, as well as restoration of fertility, were achieved in sterilized hosts (Baird et al. 1999). The success achieved in orthotopic transplantation of cryopreserved ovarian tissue from both mouse and sheep resulted in the subsequent use of this technique in human reproductive medicine.

Although recent studies have demonstrated that it is possible to have follicular survival in frozen-thawed human ovarian tissue, the success has not been sufficient for extensive clinical use. However, experimenting with tissue survival after thawing in human subjects may not be an option because of ethical issues. Transplantation studies performed in immunodeficient mice have provided extremely important information that is useful in understanding some of the serious issues in human reproductive medicine. For example, one of the most serious concerns in regard to autologous transplantation of ovarian tissue collected from a cancer patient is the transmission of malignant cells (Gosden et al. 1997; Meirow et al. 1998). Shaw et al. (1996) used a mouse lymphoma model to test cancer cell transmission via ovary transplantation in immunodeficient mice. Their study revealed that both fresh and frozen ovarian tissue from a donor with lymphoma transmitted the disease and caused death of healthy recipient mice that received a small piece of ovarian tissue. This study clearly revealed the need for continued basic research on laboratory animals before this technology can be applied to human infertility programs.

Another significant finding was the demonstration of improved follicle growth upon administration of exogenous follicle stimulating hormone (FSH¹) to the recipients. Oktay et al. (1998b) xenografted human ovarian tissue under the kidney capsules of SCID mice to study the early stages of ovarian follicular growth in vivo. In the absence of FSH, after 17 weeks of grafting, the most advanced follicles contained two layers of granulosa cells. Follicular growth up to the antral stage was significantly improved by employing a daily injection of 1 IU of FSH. Similarly, Weissman et al. (1999) attempted to evaluate the development of follicles in human ovarian cortex grafted under the skin of nonobese diabetic SCID mice. Exogenous gonadotropin administration 12 weeks after transplantation resulted in follicle growth in 51% of the grafts.

Rodent Ovarian Primordial Follicle Cryopreservation

The ability to bank primordial follicles is advantageous for two reasons. First, of the thousands of potentially usable oocytes present in the ovaries of prepubertal animals, most degenerate and never become antral follicles. Second, cryopreserving primordial follicles may be a good alternative to isolate oocyte cryopreservation because they are smaller in size (30-50 gm), are arrested in prophase I of meiosis, and do not have a clear zona pellucida, cortical granules, and spindle fibers, which are reported to be sensitive to cryopreservation procedures (Shaw et al. 2000).

Carroll and Gosden (1993) and Gosden (1990) have shown that it is possible to restore fertility by transplanting enzymatically isolated, fresh and cryopreserved primordial follicles into the ovarian bursa of ovariectomized mice. Significant efforts have been directed toward improvement in follicular recruitment and their in vitro maturation and fertilization (e.g., Leibfried-Rutledge et al. 1997). Roy and Greenwald (1996) described the methods for the enzymatic isolation and in vitro culture of preantral follicles from ovaries of several mammalian species including mouse, rat, hamster, pig, and human. Significant progress has been made in the complete development of mouse oocytes in vitro from primordial stage follicles as well as preantral follicles (Cortvrindt et al. 1996; Eppig and O'Brien 1996; Eppig and Schroeder 1989). In these studies, live pups were obtained after in vitro culture of both primordial and preantral follicles derived from mouse ovaries. In addition, in an in vitro ovulation model study in mice, Rose and colleagues (1999) recently demonstrated that isolated secondary follicles can develop and spontaneously rupture in vitro with hormonal supplementation; furthermore, blastocyst-stage embryos can be developed after in vitro fertilization of the resulting oocytes.

Although the current success in obtaining live births from these procedures has not been perfected to the point of an established repeatable method, it has certainly provided an important step toward the ability to grow oocytes to the MII stage, which can then be successfully fertilized. There is no doubt that an in vitro system would provide an excellent means of studying follicular growth on both cellular and molecular levels. Therefore, similar studies are acutely needed to improve methods for in vitro follicular maturation in other species, including human. For example, autografting ovarian tissues from a cancer patient is risky due to the possible reintroduction of diseases in treated individuals. If these improvements on in vitro maturation systems can be incorporated within ovarian tissue cryopreservation schemes, it will be possible to create various options by which fertility can be successfully restored. Although several imperfections are associated with these technologies, the preliminary studies conducted in mice have clearly established a framework for future improvements in the fields of human reproductive medicine and wildlife conservation programs.

Rodent Ovarian Tissue Cryopreservation

Many genetically engineered mouse and rat models have been created and have been extremely helpful for better understanding genes that are related to human diseases (McBride and Li 1998; Sharp and Mobraaten 1997). However, maintaining the availability of these live animal stocks with current animal breeding programs has increased the financial burden to the scientific community. To date, embryo cryopreservation and storage has provided a cost-effective alternative to the maintenance of live animal colonies with unique genotypes, and thousands of offspring have been born as a result of cryopreserved mouse embryos (Glenister et al. 1990). However, in the case of poor response to super-ovulation, embryo cryopreservation is often not an option. Furthermore, the reproductive capacity of genetically engineered laboratory animals is often somewhat impaired. Therefore, the ability to store genomes in the form of ovaries may permit greater overall flexibility and assurance in management of this process.

Advances in this area commenced with the pioneering effort by Parrott (1960), who slowly froze mouse ovaries to -79°C in the presence of 12% glycerol but obtained low survival. Harp and colleagues (1994) successfully cryopreserved whole mouse ovaries in the presence of 1.4 M DMSO by cooling them to -55°C at 0.5°C/min and plunging into liquid nitrogen. Later, using 1.5 M DMSO, Cox et al. (1996) froze fetal mouse ovaries (16 days) to -80°C from -7°C using a cooling rate of 0.3°C/min. After orthotopic transplantation, 33% of the recipients became pregnant, which resulted in an average litter size of 2.7. In later studies, using the Harp et al. protocol, Gunasena and coworkers (1997b) were able to produce live offspring from 73% of the recipient mice with an average litter size of 4.7 after autologous orthotopic transplantation. However, transplantation into athymic nude mice resulted in a lower delivery rate (25%) and average litter size of 2.0 (Gunasena et al. 1997a).

Overall these results demonstrate that both adult and fetal cryopreserved ovaries can be used to restore fertility in mice. Although frozen-thawed transplantation studies produce relatively smaller litter size, it allows propagation of the lines in one step compared with embryo transfer, or in vitro fertilization, which requires multiple steps to propagate certain strains. While efficiencies are currently sufficient for ovarian tissue cryopreservation to be considered as an adjunct method for genome banking, further improvements are required for it to be used exclusively for genome banking. To improve the efficiency of the frozen-thawed ovarian tissue transplantation, Sztein and coworkers (1998) performed orthotopic transplantation of one half of the frozen-thawed ovaries using recipients with one or both ovaries excised, demonstrating that fertility can be reestablished (pregnancy rate of 57%, average litter size of 3.2 pups). Sztein et al. (1999) also accomplished successful rederivation by transplanting frozen-thawed ovaries derived from a terminally ill transgenic mouse strain that could not reproduce through other means. Those reports clearly show that cryopreservation of ovaries would be a very useful procedure for banking mouse genomes, particularly mouse strains with a low fertility rate (Figure 5).

Similar approaches can also be used effectively to store genetically important rat strains. Deanesly (1954) conducted studies to evaluate cryopreserved rat ovarian tissue and investigate immature rat ovaries grafted after freezing and thawing; however, survival was poor. A recent study by Sugimoto et al. (1996) revealed that after directly plunging neonatal rat ovaries into liquid nitrogen after exposure to a vitrification solution (12.5% DMSO, 25.0% acetamide, and 5% PG), 25% of the follicles survived alter thawing. von Eye Corleta and colleagues (1998) later demonstrated that the steroidogenic activity of the ovary can be restored with a very high rate (75%) after autologous subcutaneous transplantation of fresh ovarian tissue slices. Although fertility was restored for a small proportion of animals, orthotopic autografts of fresh ovarian tissue in ovariectomized rats resulted in normal offspring (Aubard et al. 1996). A subsequent study by Aubard et al. (1998) aimed to cryopreserve the ovarian cortical slices in rats using either 1.5 M DMSO or EG with slow freezing and rapid thawing. After allogeneic orthotopic transplantation into the irradiated rats, pregnancies were obtained from both fresh and frozen-thawed groups, although the percentage of growing follicles was greater in the fresh group than in the cryopreserved group. The number of follicles surviving cryopreservation in the presence of DMSO and EG was not different. Development of effective cryopreservation protocols in combination with utilizing immunodeficient rats (Festing 1981) (e.g., Hsd:RH-rnu, athymic nude rats, New Zealand nude rats) as an ovarian recipient would have significant benefit in rederivation of rat strains. Using these techniques, it would be theoretically possible to rederive rat strains if ovarian tissue can be effectively cryopreserved and transplanted under the bursa of the ovariectomized immunodeficient rat, which would then be bred to produce heterozygous offspring. The pups could then be bred to reestablish the strains and or back-crossed to homozygosity if required.

Summary

From the foregoing review of the structural and functional consequences of cryopreservation processes on oocytes and ovarian tissues, we can conclude that banking female germ plasm in the form of oocytes and/or ovarian tissue constitutes a powerful tool for potential application in the fields of biomedical research and human infertility. The current challenge is to develop optimal cryopreservation protocols that will protect oocyte and ovarian structures throughout the cryopreservation procedure and subsequently allow oocytes to develop fully under either in vivo or in vitro conditions. Thus, the improvement of the other assisted reproductive technologies, such as developing optimal in vivo or in vitro conditions for growing follicles, will be important determining factors for the success of this technology.

Acknowledgments

The author thanks Steve Mullen for his editorial help and the anonymous reviewers for their helpful comments.

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¹Abbreviations used in this article: CPA, cryoprotective agent; DMSO, dimethylsulfoxide; EG, ethylene glycol; FSH, follicle-stimulating hormone; GV, germinal vesicle; MII, metaphase II; PG, propylene glycol; SCID, severe combined immunodeficient.

Figure 1 Potential strategies by which cryopreservation of oocyte and ovarian tissue can be used to restore fertility. IVFC, in vitro fertilization and culture; IVMFC, in vitro maturation, fertilization and culture; ET, embryo transfer.

Figure 2 Schematic representation of mammalian ovary. Reprinted with permission from Donnell TC. 1948. General Endocrinology. Philadelphia: W.B. Saunders Co.

Figure 3 Schematic representation of the cytological features of Germinal vesicle (A) and Metaphase II (B) mammalian oocytes. Reprinted with permission from Parks JE. 1997. Hypothermia and mammalian gametes. In: Karow AM, Critser JK, eds. Reproductive Tissue Banking. Scientific Principles. San Diego: Academic Press. p 229-261.

Figure 4 Simulation of MII mouse oocytes after exposure to either (A) 1.5 M DMSO or (B) 6 M DMSO at various temperatures and the corresponding intracellular DMSO concentrations. Reprinted with permission from Agca Y, Liu J, McGrath J J, Peter AT, Critser ES, Critser JK. 1998. Membrane permeability characteristics of metaphase II mouse oocytes at various temperatures in the presence of DMSO. Cryobiology 36:287-300.

Figure 5 Offspring delivered after allogeneic transplantation of frozen-thawed 101-R1 mouse ovaries into athymic nude mice (Y. Agca, unpublished study).

Table 1 Some of the selected studies in the area of ovarian tissue cryopreservation and transplantation

Species	Fresh/frozen	CPA	Graft site - type of graft	Results	Reference (see text)
Mouse	Fresh	N/A^a	Orthotopic-autograft	Offspring	Robertson 1940
Hamster	Frozen	12% glycerol	Orthotopic-autograft	Offspring	Parrott 1959
Mouse	Fresh	12% glycerol	Orthotopic-autograft	Offspring	Parrott 1960
Mouse	Frozen	1.4 M DMSO^a	Orthotopic-autograft	Steroidogenic activity	Harp et al. 1994
Mouse	Frozen	1.4 M DMSO	Orthotopic-autograft into athymic nude mice	Offspring	Gunasena et al. 1997b
Rat	Frozen	15% glycerol	Orthotopic-autograft	Steroidogenic activity	Deansley 1954, 1957
Rat	Fresh	N/A	Orthotopic-autograft	Offspring	Aubard et al. 1996
Rat	Fresh	N/A	Heterotopic-autograft (s.c.^a)	Steroidogenic activity	von Eye Corleta et al. 1998
Rat	Frozen	1.5 M DMSO or EG^a	Orthotopic-autograft	Offspring	Aubard et al. 1998
Human	Fresh	N/A	Heterotopic-xenograft into SCID^a mice (r.c.^a)	Antral follicle formation	Oktay et al. 1998b
Human	Fresh	N/A	Heterotopic-xenograft into NOD-SCID^a mice (s.c.)	Antral follicle formation	Weissman et al. 1999
Human	Frozen	1.5 M DMSO, EG, PG,a Glycerol	Heterotopic-xenograft (s.c.)	Antral follicle formation	Newton et al. 1996
Monkey	Frozen	1.5 M DMSO	Heterotopic-xenograft into athymic nude mice (s.c.)	Antral follicle formation	Candy et al. 1995
Cat	Frozen	1.5 M DMSO	Heterotopic-xenograft into SCID mice (s.c.)	Antral follicle formation	Gosden et al. 1994b
Bovine	Frozen	1.5 MEG	Heterotopic-xenograft into NOD-SCID mice (s.c.)	Antral follicle formation	Semple et al. 2000
Elephant	Frozen	1.4 M DMSO	Heterotopic-xenograft into athymic nude mice (o.b.^a)	Antral follicle formation	Gunasena et al. 1997a
Sheep	Frozen	1.5 M DMSO	Heterotopic-xenograft into SCID mice (r.c.)	Antral follicle formation	Gosden et al. 1994b
Sheep	Frozen	1.5 M DMSO	Orthotopic-autograft	Offspring	Gosden et al. 1994a

^aDMSO, dimethylsulfoxide; EG, ethylene glycol; N/A, not applicable; NOD-SCID, nonobese diabetic severe combined immunodeficient; o.b., ovarian bursa; PG, propylene glycol; r.c., renal capsule; s.c., subcutaneous.