CSIRO PUBLISHING

www.publish.csiro.au/journals/rfd Reproduction, Fertility and Development, 2007, 19, 13–23

Oocyte cryopreservation: oocyte assessment and strategiesfor improving survival

Sergio LeddaA,B,D, Luisa BoglioloB, Sara SuccuA, Federica AriuB, Daniela BebbereA,

Giovanni Giuseppe LeoniC and Salvatore NaitanaA

ADepartment of Animal Biology, Veterinary Faculty, University of Sassari, 07100 Sassari, Italy.BInstitute of Animal Pathology, Pathological Anatomy and Obstetric and Surgery Clinics, Veterinary Faculty,

University of Sassari, 07100 Sassari, Italy.CDepartment of Physiological, Biochemical and Cellular Sciences, Veterinary Faculty,

University of Sassari, 07100 Sassari, Italy.DCorresponding author. Email: giodi@uniss.it

Abstract. Despite significant progress in cryopreservation of mammalian oocytes and embryos, many of themolecular and biochemical events that underlie this technology are poorly understood. In recent years, researchershave focused on obtaining viable oocytes that are developmentally competent. Even under the most favourableconditions, experimental approaches have achieved only limited success compared with fresh oocytes used inroutine in vitro embryo production. Chilling injuries and toxic effects of the cryoprotectants are the major adverseconsequences following cryoprocedures. To overcome these problems, different strategies have been developed forimproving cryopreservation results. These strategies include reducing container volumes, increasing the thermalgradient, changing the cell surface/volume ratio, enhancing cryotolerance by supplementation with various additivesor modifying the lipid composition of the oocyte membrane. In order to develop new strategies for reducing thevarious forms of stress associated with oocyte cryopreservation, it is fundamental to gain a better understanding ofthe major changes responsible for poor post-thaw survival. With this knowledge, we hope that oocyte cryostoragewill become a fully reliable reproductive technique in the near future.

Introduction

Oocyte cryopreservation certainly represents one of the mostattractive developments in the field of reproductive technolo-gies and will become an important tool for the creation ofgenetic resource banks in domestic animals (Ledda et al.2001;Arav et al. 2002). Despite some successes with differentspecies and the publication in the past years of several fun-damental cryobiology studies, oocyte cryopreservation as anestablished procedure, able to compete with the efficiency ofsperm and embryo freezing, remains largely unaccomplished(Shaw et al. 2000; Woods et al. 2004).

Many of the problems associated with the cryopreser-vation of mature and immature oocytes are related to lowtemperature sensitivity (chilling injury) and exposure to cryo-protectants (CPA; toxic effects), which exert considerablemorphological and functional damage during cryopreserva-tion (Zeron et al. 1999; Paynter 2005). Currently, chillinginjury is one of the main obstacles to successful short- andlong-term oocyte cryopreservation and several reports havesuggested that the exposure of mammalian oocytes to sub-physiological temperatures affects the cell structure at severallevels. Conversely, CPA themselves, usually added at highconcentrations to the cryopreservation solutions to avoid the

formation of ice crystals, may alter cell activities depend-ing on the duration and temperature of exposure (Chen et al.2000). Extensive research in past years has resulted in thedevelopment of new approaches to create an acceptable bal-ance between the positive and negative effects of CPA bydecreasing CPA toxicity using combinations of CPA andmodifying the cooling rate. New devices, such as automatedcooling rate machines, or new CPA cocktails endowed withlow toxicity and a high vitrification capability, together withchanges in their concentration, exposure time and temper-atures, have also been used (for a review, see Vajta andKuwayama 2006).

In recent years, alternative strategies have been proposedto increase viability and developmental competence. Two ofthese approaches include the modification of cytoplasmicmembrane lipid content by the addition of lipids such ascholesterol or egg-phosphatidylcholine during the refriger-ation and freezing steps (Zeron et al. 2002a, 2002b; Horvathand Seidel 2006) and removal of excess cytoplasmic lipidsby micromanipulation to avoid their negative effect duringthe freezing procedures (Nagashima et al. 1994; Hara et al.2005). Other approaches address the question of which mei-otic stage is the most suitable for oocyte cryopreservation.

© IETS 2007 10.1071/RD06126 1031-3613/07/010013

14 Reproduction, Fertility and Development S. Ledda et al.

The cell cycle stage during meiosis seems to influence thesurvival of mammalian oocytes and affects the results ofcryopreservation due to variable sensitivity to cooling proce-dures. Metaphase (M) II oocytes remain the preferred stagefor cryostorage owing to better membrane stability duringchilling procedures. Conversely, immature oocytes wouldnot be directly affected by the problem posed by the mei-otic spindle at the MII stage, because their chromosomesremain confined within the nucleus. However, the numberof experiments using immature oocytes remains low becausedata obtained, thus far, indicate that immature oocytes aremore susceptible to cryoinjures. Several alternatives havebeen proposed to improve the viability of immature germinalvesicle (GV) oocytes: vitrification of isolated GV using theopen pulled straws (OPS) method (Kren et al. 2005); partialremoval of cytoplasmic lipid before vitrification (Park et al.2005); pre-treatment with cytoskeletal inhibitors (Fujihiraet al. 2004); or the addition of taxol (Fuchinoue et al. 2004).However, the purpose of the present paper is not to reporton the state of the art of oocyte cryopreservation in domes-tic animals, but to underline the crucial points that affectthe successful application of this reproductive technique andto suggest strategies that could be adopted to overcome thesubsequent poor development.

Criteria to assess the quality of cryopreserved oocytes

Non-invasive assessments

It is quite clear that successful oocyte cryopreservation isclosely related to oocyte quality assessment before cool-ing and after warming. These criteria, called ‘non-invasive’or ‘invasive’, are not sufficiently defined at the momentand we cannot objectively evaluate the post-warming oocytestate to provide clues for setting the appropriate standardsof quality (Coticchio et al. 2004; Coticchio 2005). Usu-ally, the first criterion used to assess post-thawing viabilityof domestic animal oocytes is the presence or absence ofobvious degeneration or gross cytoplasmic abnormalities,such as extensive vacuolisation or zona pellucida fractures.Although these indicators are relatively accurate with regardto post-thaw development, they cannot provide any pre-cise correlation between morphological intact oocytes beforecryopreservation and developmental post-fertilisation rates.Recently, studies on human oocyte cryopreservation havecontributed greatly to this area. Visualisation of the meioticspindle using a polarised microscope apparatus (PolScope;Cambridge Medical Instrumentation, Boston, MA, USA) hasallowed observation of the polymerisation of the meiotic spin-dle after warming. The correct reversible polymerisation ofthe spindle after warming could be used as an indicator ofthe developmental potential of cryopreserved oocytes (Rienziet al. 2005). Unfortunately, this approach cannot be used withoocytes of domestic animals owing to their high cytoplas-mic lipid content, which prevents direct spindle examination.

Damage to oocyte cytoskeletal structure in these species canbe observed only through invasive methods, such as fluo-rescence microscopy and biochemical or molecular biologyanalyses.

The need for non-invasive methods to assess predictivefactors of oocyte quality should stimulate research towardsthe development of new evaluation markers that could guidethe establishment of effective cryopreservation systems.If the gene expression of the cumulus cells surroundingoocytes during maturation could be correlated with develop-mental rates, then gene expression could be used as a markerof oocyte quality before freezing and help in the selectionof those oocytes most suitable for cryostorage (Kumamotoet al. 2005). Another non-invasive criterion could be thedetermination of the volumetric response of matured oocytesto changes in osmolarity during preparation for freezing.Dynamic measurements of volumetric responses to increas-ing concentrations of CPA should allow precise calculationsof the ideal timing and concentration of CPA exposure (Agcaet al. 1999, 2000).This may help to decrease the unfavourableosmotic and toxic effects caused by excessive concentrationor exposure timing (Newton et al. 1999).

Invasive assessments

As reported previously, invasive assessments of oocyte qual-ity are the most frequently used criteria to define thedevelopmental competence of cryopreserved oocytes fromdomestic animals. These evaluations focus on morphologicaland functional alterations induced by cryoprocedures.

Morphological criteria

It has been observed that cryopreservation of oocytes ofdomestic animal results in ultrastructural damage that affectsdevelopmental competence and in low offspring generationrates (Fuku et al. 1995; Hyttel et al. 2000). Damage dueto cooling and warming procedures was observed at dif-ferent ultrastructural levels (Diez et al. 2005). In humanoocytes, changes in the zonae pellucida or zona harden-ing by premature release of cortical granules, which couldbe responsible for the decrease in fertilisation rates, havebeen reported (Ghetler et al. 2006). Other reported damageincluded diminished plasma membrane selective permeabil-ity, microvilli loss, extensive ooplasm disorganisation andchanges in microtubules and microfilaments in the spindleapparatus (Diez et al. 2005). Ultrastructural changes, such asmitochondrial swelling, together with reduced matrix density,disorganisation of junctions between oocytes and cumuluscells and the presence of vacuoles in the ooplasm periphery,have also been described in cryopreserved human and mouseeggs (Sathananthan et al. 1987;Valojerdi and Salehnia 2005).

Coupling changes between oocyte and cumulus cells

Different lines of evidence clearly indicate that surround-ing cumulus cells play a fundamental role in the maturation

Oocyte assessment and cryopreservation Reproduction, Fertility and Development 15

process (Gilchrist et al. 2004; Li et al. 2006). These cellsand the oocyte are functionally and physically connected,establishing a sophisticated network of mutual interactions,which ultimately confers to the oocyte full development com-petence. The possibility of using cryopreserved immatureoocytes will depend on the ability to preserve not only theviability of the female gamete, but also the structural andfunctional integrity of the entire cumulus–oocyte complex.Little information is available on the effects of cryopreserva-tion on cumulus cells, with the exception of a few studiesin which exposure to CPA resulted in disorganised actinfilaments within the transzonal processes through whichcumulus cells establish physical contacts with the oocyte(Younis et al. 1996; Diez et al. 2005). It is still debated asto whether it is necessary to maintain cumulus cells dur-ing cryopreservation of immature oocytes, because this needmay be species specific (Modina et al. 2004; Fujihira et al.2005; Ruppert-Lingham et al. 2006). However, these stud-ies focused mainly on viability and described ultrastructuralchanges of the cumulus–oocyte complexes without infor-mation on their functional coupling. We have performedseveral experiments analysing the gap junction communica-tion between the oocyte and surrounding cumulus cells inovine oocytes before and after vitrification by injection ofLucifer yellow fluorescent dye, following the fluorocrome asit spread into the cumulus cells. We found that cooling andwarming significantly reduced oocyte–cumulus cell commu-nications compared with control immature oocytes (Fig. 1).In order to better evaluate how the presence or absence ofcumulus cells could benefit post-viability and maturation ofimmature oocytes, we removed cumulus cells at differenttimes (0, 2 and 6 h) during incubation for in vitro maturation(IVM) before vitrification. We evaluated the post-warmingviability rate and meiotic progression compared with vitri-fied cumulus-enclosed oocytes. Removal of cumulus cells at0 h or after 2 h of incubation significantly increased the via-bility of vitrified oocytes compared with cumulus-enclosedoocytes. The IVM rate of warmed denuded oocytes was sig-nificantly higher than that of vitrified oocytes with cumuluscells, both at 0 and 2 h of incubation. When cumulus cellswere removed after 6 h of incubation, a marked reduction inviability and meiotic competence was observed (Fig. 2).

Effects on spindle and cytoskeletal organisation

Many reports point out that physical–chemical conditionsduring cryopreservation may influence spindle response andcould cause the irreversible loss of spindle microtubules (Rhoet al. 2002; Albarracin et al. 2005; Tharasanit et al. 2006).It is well known that the MII oocyte spindle in domesticanimals is extremely sensitive to low temperatures. In fact,after conventional slow freezing or vitrification, the propor-tion of MII oocytes with a morphologically normal spindleis reduced compared with control oocytes. These alterationsmay vary depending on the species involved and on the

cryopreservation procedures (for a review, see Chen et al.2003).

Exposure of bovine and pig oocytes to room temperatureleads to depolymerisation of microtubules and disrupts thenetworks of the meiotic spindles (Aman and Parks 1994;Liu et al. 2003). Similarly, spindle alterations have beendescribed after vitrification of MII horse oocytes (Tharasanitet al. 2006). It has been observed that highly concentratedCPA may lead to injury of the meiotic spindle (Chen et al.2000). Spindle alterations are also observed even if oocytesare cryopreserved at the GV stage. In fact, we observed invitrified immature sheep oocytes a high percentage of spin-dle abnormalities after IVM (Fig. 3). This may result froma defective spindle assembly checkpoint induced by cryop-reservation and, thereby, reduce the accuracy of chromosomesegregation in meiosis I during the IVM of the thawed GVoocytes.

Functional and molecular criteria

Oxidative metabolism

In studies on cryo-injury mechanisms, damage caused to awide range of cellular structures has been investigated exten-sively, but surprisingly little information is available on theeffects of cryopreservation on biochemical processes, such asenzyme inactivation, ionic disturbance or free radical attack.Conversely, this kind of information is coming from studieson somatic and sperm cells, where freezing and cooling havebeen observed to alter the activity and stereospecificity ofenzymes acting as anti-oxidants, such as catalase, glutathioneperoxidase (GPx), superoxide dismutase (SOD) or otherscavengers, subsequently reducing their beneficial effects(Baumber et al. 2005; Gadea et al. 2005; Lai et al. 2005).Irreversible loss of oocyte mitochondrial polarity was foundin cryopreserved human oocytes (Jones et al. 2004). Thisloss could influence the developmental competence of theoocytes by altering ATP levels or cytoplasmic ability to regu-late intracellular Ca2+. These defects could have downstreamconsequences for normal embryogenesis. Cryopreservationprocedures also result in the generation of reactive oxygenspecies (ROS) that could affect viability and developmentalcompetence. It has been recently reported that the additionof cysteamine in high concentrations during IVM reducedthe negative effects of the ROS and improved the develop-ment of embryos derived from cryopreserved bovine oocytes(Kelly et al. 2006). Clearly, it appears that metabolic func-tion in the cryopreserved oocytes remains largely unknownand there is a great need for a more systematic approach thatcould elucidate what happens during the cryopreservationprocedures.

Gene expression

Information is also lacking on cryopreservation-inducedchanges at the molecular level. Knowledge of these changes

16 Reproduction, Fertility and Development S. Ledda et al.

(a) (b)

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Fig. 1. Evaluation of gap junction functional status analysed by microinjection of Lucifer yellow (LY) fluorescentdye in cumulus-enclosed ovine oocytes (a,b). Ten minutes after injection, LY spread from the oocyte cytoplasm tothe surrounding cells, indicating a complete functional coupling, in fresh oocytes (d ), whereas a marked reduction ofcommunication was observed after injection of LY into vitrified oocytes ( f ). (c,e) Controls. Magnification ×40.

could improve cryoprocedures and suggest optimal systemsto preserve viability and developmental capacity. Extensiveanalyses of gene expression in response to low temperaturehave been reported only in mammalian somatic and sperm

cells (Fuller 2003; Meng 2003). In these studies, prelimi-nary information suggests that changes in stress-related geneexpression and genes related to important cell function couldbe used to assess post-thaw oocyte quality. The few studies

Oocyte assessment and cryopreservation Reproduction, Fertility and Development 17

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Fig. 2. (a) Viability and (b) meiotic maturation of immature ovine oocytes vitrified with (COCs) or without (DOs) cumuluscells after 0, 2 and 6 h of culture. P < 0.01 for a v. b and a v. d; P < 0.05 for a v. c.

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Fig. 3. Immature vitrified ovine oocytes after in vitro maturation stained immunocytochemically with anti-α-tubulin monoclonal antibody tovisualise microtubules and Hoechst 33342 to visualise chromosomes. (a) Normal barrel-shaped metaphase II spindle with compact chromosomesarranged at the equator of the structure. (b,c) Abnormal spindle structure associated with disorganised chromosomes. Magnification ×100.

that have been conducted in the mouse reported a correla-tion between the level of expression of stress-related genes,embryonic stage and cryopreservation systems used. It wasshown that an up-regulation of stress-related genes is evident3 h post thawing in pronuclear-stage zygotes compared withcontrols, whereas no differences are observed at the eight-cell and blastocyst stages (Boonkusol et al. 2006). It hasalso been reported that altering gene expression in maturedmouse oocytes by injection of aquaporin-3 mRNA, whichresults in increased expression of channels for water and CPA,improved post-thawing viability (Edashige et al. 2003).

Owing to the scarce information available on gene expres-sion in cryopreserved oocytes of domestic animals, weperformed a series of analyses in vitrified MII ovine oocytesto evaluate the pattern of gene expression after warming. Wechose genes that have been reported previously to be corre-lated with oocyte quality in sheep (Leoni et al. 2006). Ourpreliminary data showed that cryopreservation does affectthe normal pattern of gene expression of vitrified IVM ovineoocytes. Levels of cyclin B, p34cdc2, Na+/K+-ATPase andE-cadherin mRNA drop in vitrified oocytes after thawing

(2 h post thawing) compared with non-cryopreserved coun-terparts, whereas non-significant differences were detectedin the levels of Z variant of the H2A histone family (H2A.Z)and α-actin mRNA (Fig. 4).

Protein content and localisation

Biochemical and molecular analyses could be used toobjectively assess the post-warming oocyte state. It is reason-able to suppose that the low performance of cryopreservedoocytes could be due to an expression deficiency of cru-cial proteins involved during fertilisation and early cleavage.In reference to this, we analysed the levels of maturation-promoting factor (MPF) and mitogen-activated protein kinase(MAPK), known to play a pivotal role in meiotic and mitoticcell cycles regulation, in vitrified MII sheep oocytes usingdifferent devices (OPS, cryoloops and cryotops). We foundthat, after warming, MAPK activity did not differ from thatin the control group, whereas MPF activity showed a tran-sitory decrease (Succu et al. 2006). These lower levels ofMPF after vitrification could be related to the low devel-opmental competence and should be taken into account if

18 Reproduction, Fertility and Development S. Ledda et al.

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Fig. 4. Gene expression values (mean ± s.e.m.) in in vitro-matured metaphase II oocytes subjected (vitrified) or not (control) tovitrification procedures, measured by real time reverse transcription–polymerase chain reaction. (a) H2AZ; (b) cyclin b; (c) p34cdc2;(d ) E-cadherin; (e) Na+/K+-ATPase; ( f ) α-actin. Bars with different letters (a,b) differ significantly (P < 0.05). Scales of expressionvalues vary with genes.

cryopreserved oocytes are to be used as cytoplasts for thenuclear transplantation technique (Hou et al. 2005).

Cryopreservation could also change the polarisation ofmRNA and proteins within the oocyte. It was observed inmouse oocytes that expression of Cdx2 mRNA was concen-trated in one-half of the cytoplasm, usually on the oppositeside to the first polar body (Deb et al. 2006). After fer-tilisation, there was a reorientation of the Cdx2 mRNArelative to the position of the polar body, from the vegeta-tive pole towards the animal pole, so that Cdx2 transcriptsbecame concentrated only on one side of the axis thatbisects the animal and vegetative poles. Presumably, thisshift reflected the reorganisation of cytoskeletal componentsafter fertilisation. The asymmetry in Cdx2 mRNA distribu-tion observed in oocytes persisted in the early embryonicstages. Consequently, Cdx2 mRNA, and presumably othertranscripts associated with cytoskeletal components, couldbe distributed unequally between the two blastomeres whenthe zygote divides. We cannot exclude that this reorientationcould be strongly affected by cryopreservation due to deepalterations shown in the cytoskeleton.

Genetic stability

There are few investigations on cell genetic stabilityduring cryopreservation. Nevertheless, it is necessary to

determine the impact of cryopreservation on genetics. Someindirect data in the literature provide information on thissubject. These reports suggest that damage was caused tonuclear DNA, mitochondrial (mt) DNA and other genome-related structures after cryopreservation, even if the effect onoffspring remains unknown.

Some investigators have focused their attention on theeffects of cryopreservation on the nuclear genome (Kola et al.1988; Cobo et al. 2001; Men et al. 2003). Alterations inploidy were found in animal and human embryos obtainedfrom cryopreserved oocytes (Chia et al. 2000). Conversely,the issue of potential induction of mutations in mtDNAduring oocyte cryopreservation of domestic animals has sofar been largely ignored and the effects of such treatmentsneed to be determined. Studies of zebrafish blastomeres sim-ply exposed to CPA or vitrified indicated a significantlyincreased frequency of mtDNA mutations compared withcontrols (Kopeika et al. 2005). Several factors can potentiallycause lesions in DNA: excessive production of free radicalsafter cryopreservation could be one of the causes for mtDNApoint mutations. Mitochondrial DNA is generally more sensi-tive to ROS than nuclear DNA. It is estimated that nucleotidesubstitution (point mutation) is at a 10-fold higher frequencyin mtDNA than in nuclear DNA (McConnell and Petrie 2004).The higher mutation rate of mtDNA can be attributed to

Oocyte assessment and cryopreservation Reproduction, Fertility and Development 19

at least two general factors: (1) lack of chromatin structureand histone protection; and (2) a more limited repertoire ofenzymatic DNA repair systems.

Improving strategies

Technical improvements: slow freezing v. vitrification

Cryopreservation strategies to minimise damage and helpcells regenerate are based on two main factors: utilisation ofoptimal CPA and cooling–warming rates. Oocytes are verysensitive to chilling and this can be related to several fac-tors, such as the size and shape of the cell, cell cycle, speciesand origin (in vitro and in vivo). So far, two major meth-ods have been used for oocyte cryopreservation: conventionalslow-rate freezing and vitrification.

Conventional slow freezing represents the first systemused for embryo cryopreservation and this method is highlystandardised with considerable industrial and commercialbackground. In this system, controlled cooling rates allowextracellular and intracellular fluid exchange without seri-ous osmotic effects and changes in cell shape. Furthermore,toxic CPA solutions are usually added when the temperatureis rather low, with a consequential decrease in toxicity. Thistechnique has been used successfully to freeze oocytes ofspecies (e.g. mouse, cat) that are less sensitive to chilling(Luvoni et al. 1997; Ruppert-Lingham et al. 2006) and hasbeen used extensively to freeze human oocytes and embryos(Fabbri et al. 2001; Byrd 2002). Conversely, poor resultshave been reported for species more sensitive to chilling (e.g.pig, cattle, sheep and horse). Furthermore, an important con-straint to the use of conventional slow freezing is the criticalperiod of oocyte exposure to CPA and the necessity to performthis technique only in a laboratory equipped with automatedfreezing systems.

An alternative method is vitrification, which uses veryhigh concentrations of CPA and rapid cooling rates, result-ing in solidification of solutions. This technique avoids theformation of ice crystals and consequent cellular damage(Liebermann et al. 2002a). In the past few years, consid-erable effort has gone into finding the ‘ideal CPA’: it shouldhave high permeability and low toxicity, even at high con-centrations. Ethylene glycol (EG) is one of the most effectiveCPA and it is usually combined with dimethyl sulfoxide(DMSO). However, the optimal CPA formulation is notyet available and these molecules, in any case, exert toxiceffects on oocytes during cryopreservation. Recently, vitrifi-cation procedures were improved by tools that substantiallyincrease cooling rates, such as OPS (Vajta et al. 1998), theflexipet-denuding pipette (Liebermann et al. 2002b), micro-drops (Papis et al. 2000), electron microscopic cooper grids(Martino et al. 1996), nylon mesh (Matsumoto et al. 2001),cryoloops (Lane et al. 1999; Liebermann et al. 2002b), cryo-tops (Kuwayama et al. 2005) and solid surfaces (solid statevitrification; Dinnyes et al. 2000; Bagis et al. 2005). Details

on these cryostorage methods have been covered extensivelyby Vajta and Kuwayama (2006).

Following the recognition that little was known aboutthe physiological modifications caused by oocyte cyopreser-vation and that new approaches were needed to diminishthe ensuing detrimental effects, new strategies were inves-tigated to improve viability and the developmental capacityof cryopreserved oocytes. These include studies on the useof protein stabilisers, cytoskeletal inhibitors and on changesin the membrane lipid composition.

Cytoskeletal inhibitors, membrane and protein stabilisers

One possible way to improve the cryotolerance of matureand immature oocyte is cytoskeleton stabilisation usingcytochalasin B (CB). In MII oocytes, CB reduces damage tomicrotubules and may enhance spindle microtubule stabili-sation during vitrification (Rho et al. 2002). In GV oocytes,no organised meiotic spindle is present and the relaxant effectof CB may preserve the function of gap junctions betweenoocyte and granulosa cells and allow a faster and more uni-form penetration of CPA (Vieira et al. 2002). There areother factors that reduce structural damage: the addition oftaxol greatly improved post-thawing development of vitrifiedmouse and bovine oocytes (Park et al. 2001; Fuchinoue et al.2004), although it did not seem to improve the developmen-tal competence of IVM, vitrified porcine oocytes (Fujihiraet al. 2005). In addition, trehalose protein-protective andmembrane-protective properties were proposed to be benefi-cial to the cell and to improve cryotolerance: microinjectionsof trehalose into human oocytes significantly improved cryo-preservation (Eroglu et al. 2002, 2003). As noted above,kinases such as MPF and MAPK involved in ovine oocytescell cycle regulation are affected by vitrification, with a sig-nificant reduction in MPF. This can cause an increase inparthenogenetic activation and fragmentation rates (S. Succu,personal communication). Theoretically, the stabilisation ofthese molecules could avoid these adverse effects. It hasbeen reported that molecules such as caffeine could restoreMPF activity in porcine and ovine oocytes by reducing p34(cdc2) phosphorylation (Kikuchi et al. 2002; Lee and Camp-bell 2006). On this basis, we analysed the level of MPF andspindle morphology of vitrified ovine oocytes that had beenincubated in presence of caffeine for the last 2 h of IVMand during the cryopreservation procedure. After warming,ovine oocytes showed a significant increase in MPF activityand displayed a normal spindle configuration compared withcontrols (S. Ledda, unpublished data).

Modification of membrane lipid constituents

Several groups have attempted to change plasma membranecomposition during in vitro procedures to improve cryo-preservation of embryos and gametes (Hochi et al. 1999;Arav et al. 2000; Zeron et al. 2002a; Seidel 2006). It was

20 Reproduction, Fertility and Development S. Ledda et al.

observed that spontaneous association of cells with lipo-somes containing cholesterol, lecithin or sphingomyelin didnot improve cryosurvival (Pugh et al. 1998). A reduction inchilling sensitivity was obtained through the modificationof the lipid phase transition temperature following phos-phatidylcholine or dipalmitoylphosphatidylcholine transferto matured eggs (Zeron et al. 2002a). In other studies, theaddition of linoleic acid–albumin to culture media improvedcryosurvival of morulae and enucleated oocytes (Hochi et al.1999, 2000). In addition, oocyte cryosurvival and early cleav-age rates could be improved by incubation before vitrificationwith cholesterol-loaded cyclodextrin (Horvath and Seidel2006). However, all these studies showed that, although sig-nificant improvements in post-thaw oocyte viability and earlycleavage were observed after modification of lipid membranecomposition, blastocyst rates remained comparable to thosederived from non-modified cryopreserved oocytes.

Differences between species

Surveying all the principal studies on the cryopreservation ofdomestic animals performed in recent years, it is quite evi-dent that different cryostorage systems have been used fordifferent species. In fact, meiotic stage, devices, technicalprocedures and CPA vary among all laboratories (Arav et al.2002; Abe et al. 2005; Vajta and Kuwayama 2006). We reportseveral examples that show how difficult it is to comparedata obtained by different groups. For example, the beneficialeffects of CB during cryopreservation are not very clear andthe results are controversial, probably depending on the ani-mal species used. Some authors have reported that treatmentof immature porcine oocytes with CB decreases cryoinjuriesduring vitrification and results in enhanced post-warmingIVM rates (Fujihira et al. 2004; Somfai et al. 2006). How-ever, in other studies, CB pretreatment before vitrification ofGV bovine oocyte (Albarracin et al. 2005) and sheep imma-ture and mature oocytes had no detectable effect (Silvestreet al. 2006; S. Ledda, personal observations). It has beenalso reported that vitrification/warming procedures are moreeffective in cumulus-enclosed porcine oocytes, whereas theirpresence does not seem to give particular beneficial effectsduring vitrification of ovine and bovine immature oocyte(Modina et al. 2004; Bogliolo et al. 2006b). Differences incryopreservation successes and failures could also be relatedto the cryopreservation technique used, because some pro-cedures work well in one species but may not be suitable inanother. For example, we compared the behaviour of vitrifiedimmature horse and ovine oocytes using the same vitrificationprocedures (cryotop device and EG and DMSO as the CPA)and found that, after rewarming and IVM, the rate of meioticprogression and normal spindle configuration was higher inhorse than ovine oocytes (Bogliolo et al. 2006a, 2006b).

According to relevant publications, the most encourag-ing results following oocyte cryopreservation in domestic

animals have been obtained in cattle. Vitrification of imma-ture (Vieira et al. 2002) and IVM oocytes (Papis et al. 2000)has resulted in healthy offspring after in in vitro fertilisationand culture. Promising results have been also achieved inpigs, mainly after vitrification of immature oocytes (Fujihiraet al. 2004; Varga et al. 2006) and after activation of vitrifiedIVM oocytes (Somfai et al. 2006). Few data are availablefor other domestic species, such as horses and cats, whereimmature and IVM oocytes have been cryopreserved byslow conventional freezing and vitrification (Murakami et al.2004; Bogliolo et al. 2006a; Luvoni 2006; Tharasanit et al.2006) and from less conventional species, such as buffaloand rabbit (Cai et al. 2005; Gasparrini et al. 2006). A lim-ited number of oocyte cryopreservation experiments hasbeen performed in small ruminants, where poor developmen-tal rates have been obtained after vitrification of immature(Al-aghbari and Menino 2002) and matured sheep (Kellyet al. 2006; Succu et al. 2006) and goat (Begin et al. 2003)oocytes.

Conclusions

Despite the considerable interest surrounding the applicationof oocyte cryopreservation as a new tool for the preservationof genetic material from valuable animals, this technique can-not yet be considered as a well-established procedure. Lowsurvival rates, the absence of comprehensive information onthe status of essential biological attributes of oocytes afterthawing and insufficient developmental rates all contributeto the need for further improvement. More realistic advancescould come from the optimisation of freezing protocols forimmature and matured oocytes using vitrification techniques,which result in better preservation of viability and develop-mental capacity than slow conventional freezing. Knowledgeof the major changes in gene expression and enzyme activ-ities after hypothermic treatment could suggest directionsfor the development of new strategies that could help thesecells to re-establish normal metabolic and biochemical func-tions after the stress posed by the cryoprocedures. Strategiesthat involve changes in membrane lipid composition andthe introduction of intracytoplasmic cryotolerance-helpingmolecules may provide improvements in oocyte viability anddevelopmental performance.

Acknowledgments

Work from the authors’ laboratories was supported by FIRB2001 Project by the Italian Minister of Research and Univer-sity (RBNE01HPMX_003). The authors thank Paola Melisfor assistance during the preparation of the manuscript.

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