Cryopreservation of porcine oocytes: recent advances†

Abstract

Successful cryopreservation of porcine gametes and embryos has been very challenging due to their sensitivity to cryoinjuries. Although considerable improvements have been achieved in the vitrification of porcine embryos, there has been no offspring born from the vitrified oocytes in this species. Porcine oocytes characteristically contain large amounts of cytoplasmic lipids that are major obstacles limiting efficient cryopreservation. These droplets together with structures such as mitochondria, membranes, cortical granules and basic components of the spindle and cytoskeleton (microtubules and microfilaments) often incur serious damage during cooling and warming. According to recent reports, the proper combinations of permeable and non-permeable cryoprotectants and vitrification with high cooling and warming rates may increase the survival of porcine oocytes. The cryotolerance of porcine oocytes may also be enhanced by removal of the chilling-sensitive lipid droplets, supplementation of cytoskeleton relaxants in vitrification solutions, or high hydrostatic pressure pretreatment of oocytes before cryopreservation. The improvement in cryopreservation methodology for porcine oocytes will no doubt augment other technologies such as pig cloning and the establishment of a gene bank for transgenic pigs.

cryoinjury, cryopreservation, cryoprotectant, taxol, porcine oocytes

Introduction

Cryopreservation of porcine ooctyes has significant agricultural and biomedical importance. If high efficiency of oocyte cryopreservation in the pig can be achieved, then large numbers of viable oocytes would be available for testing the safety of related technologies before embarking on human studies. The pig has many physiological and immunological similarities to the human and, therefore, would be a good model animal to support human studies. In addition, cryopreservation reduces maintenance costs and provides safeguards against loss through infection, disease, genetic drift and against catastrophic loss of rare or endangered animal genetic resources.

Slow freezing and vitrification are currently the only practical methods for oocyte and embryo cryopreservation. Slow freezing is a process where cells are suspended in a relatively low concentration of cryoprotectants (CPAs) and cooled at low rates (Whittingham et al., 1972). Vitrification is the rapid cooling of cells in liquid medium in the absence of ice crystal formation (Rall and Fahy, 1985). The cooling rate, the concentration of the CPAs and their volume all affect the effectiveness of vitrification. Procedurally for the slow-rate freezing, if the cooling rate is too high or too low, the oocyte may have irreparable damage due to the formation of intracellular ice or from so-called solution effects (the abrupt increase in salt concentration that occurs during cellular dehydration). Therefore, slow freezing procedures have to maintain a very delicate balance between adverse factors that may result in damage caused by ice crystallization, osmotic and chilling injuries, and alterations of the cytoskeleton. For vitrification, increasing the cooling rate decreases chilling injury, i.e. damage of the intracellular lipid droplets and the cytoskeleton (Fu et al., 2009), and alleviates the adverse effects that are generally associated with high concentrations of CPAs. This offsets the likelihood of toxic and osmotic effects that are not particularly a concern if oocytes are only exposed to high concentrations of CPA for <60 s (Homburg et al., 2008). Vitrification does not require a programmable freezer, and the technique is easy to use, safe and highly efficient (Hou et al., 2005). More than likely, vitrification will become the method of choice for freezing porcine oocytes in the future.

Using the slow cryopreservation method, Whittingham (1977) successfully cryopreserved mouse oocytes, and much research since then has been conducted on the cryopreservation of mammalian oocytes either by slow freezing or subsequently with the vitrification method, thus resulting in live offspring in human (Chen, 1986), rabbit (Al-Hasani et al., 1989), cattle (Fuku et al., 1992) and horse (Hochi et al., 1994).

To date, however, there is no report on offspring obtained from cryopreserved porcine oocytes. This has led researchers to conclude that the failure to successfully cryopreserve porcine oocytes is due to characteristically larger amounts of cytoplasmic lipids than in other mammals, which influences the survival rate after cooling or vitrification (Fig. 1). Therefore, many protocols have been designed to improve the developmental capacity of cryopreserved porcine oocytes (Shi et al., 2006; Somfai et al., 2006; Shi et al., 2007; Huang et al., 2008; Pribenszky et al., 2008; Fu et al., 2009). The objective of this review is to identify and discuss the types of injuries during cryopreservation, and the methods that have been, and are currently being developed, to resolve the problems and technical difficulties associated with the cryopreservation of porcine oocytes.

Figure 1

Photomicrograph of porcine oocytes showing large amounts of cytoplasmic lipids. Lipid droplets (stained with Sudan red) distribution of porcine in vitro-matured oocytes under different treatment. (A) fresh oocytes; (B) oocytes with toxicity treatment; (C) oocytes vitrified-warmed and (D) oocytes pretreated with taxol and vitrified-warmed. Scale bars = 20 µm.

Cryoinjuries of porcine oocytes

Oocyte cryoinjuries may occur at all phases of the cryopreservation procedure (e.g. addition of CPAs, cooling, freezing and thawing/warming). During cooling, chilling injuries occur at 15 to −5°C, intracellular ice crystal formation occurs at −5 to −80°C and fracture damage occurs at −15 to −50°C to the zona pellucida (ZP) and/or the cytoplasm (Vajta and Nagy, 2006). Chilling injuries predominantly cause damage to the cytoplasmic lipid droplets and microtubules including the meiotic spindle as reviewed by Vajta and Nagy (2006). During the thawing/warming phase, CPAs toxicity and osmotic injury to the oocytes often occur. Cryopreservation also has been shown to induce premature extrusion of the cortical granules that then leads to an abrupt hardening of the ZP and a decrease in sperm penetrability as observed in the mouse (Carroll et al., 1990), ovine (Tian et al., 2007) and bovine (unpublished data).

Porcine oocytes are particularly difficult to cryopreserve, and there are no reports of live offspring production after thawing via in vitro fertilization and embryo transfer. This may be partially due to the large size of the oocyte that has a low surface-to-volume ratio, making it more difficult for water and CPAs to move across the cell plasma membrane. Porcine oocytes also have a greater hypothermic sensitivity resulting from their larger amounts of cytoplasmic lipids when compared with other mammals.

Porcine oocytes are sensitive to cooling. Low temperatures may prohibit subsequent spindle reorganization and nuclear and cytoplasmic maturation of the germinal vesicle (GV) (Liu et al., 2003b). Upon cooling, Liu et al. (2003a) observed that the spindle in most of the in vitro-matured porcine oocytes underwent partial disassembly at 24°C and complete disassembly at 4°C, and the spindle disassembly during cooling was accompanied by dispersion of chromosomes in the cytoplasm.

Wu et al. (2006) reported that oocytes frozen to −196°C had various degrees of cryoinjuries in GV oocytes including the separation of cumulus cells from the cumulus–oocyte complex, fracture of ZP adjacent to cumulus cells, rupture of the gap junctions between cumulus cells and the disruption of microvilli. They concluded that the percentage of oocytes with normal spindle organization and distribution of F-actin was decreased in both vitrified GV oocytes and metaphase II (MII) oocytes. The proportion of spindles with disrupted structure also has been shown to increase when in vitro-matured porcine oocytes were exposed to anisotonic solutions (Mullen et al., 2007), especially to the hyperisotonic sucrose solution during the warming procedure.

The inhibition of F-actin polymerization prevents completion of oocyte meiosis and embryo development during porcine oocyte maturation and embryo development (Wang et al., 2000). Porcine oocytes at different meiotic stages respond differently to cryopreservation. MII porcine oocytes had better resistance to cryodamage than GV stage oocytes (Rojas et al., 2004) in spite of the fact, at least in theory, that immature oocytes present lower microtubular chilling-sensitivities and have no meiotic spindle.

Under normal conditions mitochondria are evenly dispersed throughout the oocyte cytoplasm before fertilization. After vitrification of porcine MII oocytes, however, the normal distribution pattern was altered (Shi et al., 2007) and the distribution rate decreased from 92.9 to 50.8% when compared with in vitro-matured porcine oocytes (Fu et al., 2009). The researchers reported that the lipid droplets in vitrified oocytes were damaged resulting in large numbers of smaller lipid droplets (diameter <5 µm).

Strategies for the improvement of porcine oocyte cryopreservation

The common approaches attempting to deal with the lower success rates of cryopreservation of porcine oocytes are: (i) to modify cryopreservation procedures (CPAs and cooling–warming rates) and (ii) to modify the cells themselves to make them more cryopreservable. For example, possible solutions would be to decrease the intracellular lipid content or pretreat oocytes under high hydrostatic pressure (HHP) before cryopreservation. As the majority of published data strongly purport that vitrification methods are more efficient and reliable than slow freezing, we will focus on strategies that might improve the developmental potential of vitrified porcine oocytes.

Permeating and non-permeating CPAs

The utilization of optimal CPAs to some extent could minimize cryoinjury and facilitate cell regeneration as CPAs might protect intracellular organelles during long-term preservation in liquid nitrogen during cryopreservation (Dobrinsy, 2001).

CPAs are divided into permeating and non-permeating groups. The permeating CPAs, which form hydrogen bonds with water molecules, can prevent ice crystallization and protect cells from solution effects. They act to depolymerize microfilaments and microtubules and may be beneficial for protecting cytoskeletal components during osmotic stresses induced by exposure to, or removal of, CPAs (Dobrinsy, 2001). Ethylene glycol (EG), with high permeability and low toxicity, is one of the most effective permeating CPAs (Wu et al., 2006; Huang et al., 2008) and it is often combined with dimethylsulphoxide (DMSO) (Fujihira et al., 2004; Shi et al., 2007). After intra-cytoplasmic sperm injection (ICSI), no significant differences were observed in cleavage and blastocyst rates of fresh immature porcine oocytes (45.2 and 20.0%, respectively), those treated with cytochalasin B and vitrified in EG (37.8 and 13.5%, respectively) and those vitrified in EG + DMSO (39.3 and 14.3%, respectively), although the rates of matured oocytes in both vitrification groups (23.9–37.1%) were significantly lower than those in the fresh (83.5%) (control) (Fujihira et al., 2004).

Sugars (non-permeating agents) can draw not only the free water from within the cell during freezing but also the intracellular CPAs during thawing/warming. Used by themselves, they have low toxicity to oocytes (Shaw et al., 1997). When they are combined in a CPA solution, they contribute to the overall vitrification properties (Kuleshova et al., 1999), eliminating the possibility of intracellular and extracellular ice formation. Other non-permeating agents, such as polysaccharides [Ficoll and Lycium barbarum polysaccharide (LBP)], disaccharide (sucrose) and monosaccharide (glucose), have been used in vitrification solutions to cryopreserve porcine immature oocytes (Huang et al., 2008). The addition of 0.75 M sucrose or 0.10 g/ml LBP has been shown to be effective with nearly 21% of vitrified oocytes developing to the maturation stage (Huang et al., 2008).

Cryopreservation methods

At the present time, there have been no reports of live piglets born from cryopreserved porcine oocytes utilizing the traditional slow freezing method. Isachenko et al. (1998) used vitrification procedures in an attempt to preserve immature porcine oocytes. In the study, they used a 0.25 ml straw as container, elevated the temperature, pretreated the oocytes with the cytoskeletal inhibitor cytochalasin B and then gradually saturated/removed the CPA. This was the first study to report the in vitro development of vitrified porcine GV oocytes although no blastocysts were obtained.

To facilitate vitrification by using higher cooling rates, it is necessary to minimize the volume of the vitrification solution as much as possible. To minimize the volume of the vitrification solution, special carriers have to be used during the vitrification process. Consequently, cryotop sheet, solid surface vitrification (SSV) and open pulled straw (OPS) methods are currently used in the cryopreservation process (Fujihira et al., 2004; Somfai et al., 2006; Varga et al., 2006; Gupta et al., 2007). Development to the blastocyst stage has been achieved from vitrified porcine GV or MII oocytes fertilized by in vitro methods (Gupta et al., 2007) or ICSI (Fujihira et al., 2004), and from the parthenogenetically activated in vitro-matured porcine oocytes after SSV (Somfai et al., 2006).

Cytoskeleton stabilizers (taxol and cytochalasin B)

Taxol is a cytoskeleton stabilizing agent that alters microtubule dynamics in mammalian oocytes after GV breakdown. It induces the formation of numerous cytoplasmic microtubule asters and causes a change in the shape of the spindle (Sun et al., 2001a). Taxol pretreatment of in vitro-matured porcine oocytes before vitrification significantly improves the parthenogenetic development of oocytes after warming (Shi et al., 2006). The cleavage rate of oocytes vitrified by OPS was shown to increase from 5.6 to 24.3%. The results of this study were in contradiction to an earlier report by Fujihira et al. (2005) who observed that taxol had no positive effect on the developmental capacity of vitrified, in vitro-matured porcine oocytes. The different conclusions from the two studies suggest the need to determine whether or not the addition of taxol to a vitrification solution has beneficial properties.

A potential benefit of pretreating oocytes with taxol is that it could significantly increase the ratio of normal mitochondria distribution in vitrified in vitro-matured porcine oocytes (70.0 versus 50.8%, P < 0.05) (Fu et al., 2009). The extent of microtubule damage during vitrification may effect the transfer of mitochondria within oocytes since the transfer of mitochondria within different areas of mouse oocytes is mediated by the cytoskeletal network of microtubules (Van Blerkom, 1991), and similar results were obtained for porcine oocytes (Sun et al., 2001b). Taxol can decrease the critical tubulin concentration in vitro for assembly and promotes tubulin to reassemble in vitro (Schiff et al., 1979). Fu et al. (2009) also indicated that the taxol pretreatment had a positive effect on vitrified in vitro-matured porcine oocytes in terms of maintaining morphology, distribution and ultrastructure of lipid droplets. It is not clear how taxol reduces the number of larger lipid droplets.

Cytochalasin B is a cytoskeletal relaxant that causes the cytoskeletal elements to be less rigid and is thought to minimize, to some extent, the disruption of the cytoskeleton system induced by the vitrification procedure. Porcine GV oocytes treated with cytochalasin B before vitrification have higher development to the MII stage compared with no cytochalasin B treatment (22.0 versus 5.6%) (Isachenko et al., 1998). Blastocyst development (13.5–14.3%) was observed after ICSI (Fujihira et al., 2004).

Modification of the lipid content

Physical changes of lipids at various freezing temperatures are among the major causes of cellular cryodamage (Pereira and Marques, 2008). To reduce intracellular lipid content and thus cryodamage, different strategies have been developed. Mechanical delipidation has been used in porcine oocytes at the GV stage (Hara et al., 2005; Park et al., 2005). By polarization of the cytoplasmic lipid droplets by centrifugation and then partial removal of lipid by micromanipulation, the in vitro maturation (IVM) of vitrified GV porcine oocytes was for the first time improved (Park et al., 2005). A novel lipid removal method that involves suspension and then centrifugation of oocytes in a hypertonic solution of 0.27 M sugar has been used for the removal of whole lipid droplets without the loss of mitochondria and has improved cryotolerance of porcine GV oocytes (Hara et al., 2005).

IVM rates of delipated GV oocytes are still very low (7–15%) after vitrification (Hara et al., 2005; Park et al., 2005). GV oocytes matured in IVM medium with ruthenium red (an inhibitor of mitochondrial Ca²⁺ uniporter) had improved IVM rates to as high as 39.4% (Nakagawa et al., 2008). They attributed this improvement to ruthenium red blocking the uptake of intracellular Ca²⁺ into mitochondria, which increased with cooling and/or warming disorders after vitrification and subsequent maturation in the IVM medium.

More than 10 years ago, centrifugation of oocytes without the removal of polarized lipids before cryopreservation was found to improve the developmental competence of surviving bovine oocytes (Otoi et al., 1997). For the cryopreservation of the IVM porcine oocyte, this approach is highly detrimental (Somfai et al., 2008a). The development potential of cryopreserved porcine oocytes simply cannot be improved without some removal of intracellular lipids. Even when lipids are removed from the oocyte, there is only a slight improvement in viability and there is not an improvement in the developmental competence of surviving zygotes. Recently, Somfai et al. (2008b) reported the successful development to term of transferred zygotes, from in vitro-matured and -fertilized oocytes, after the zygotes at the pronuclear stage were cryopreserved by SSV.

High hydrostatic pressure

Parthenogenetic development rates of vitrified in vitro-matured porcine oocytes have been shown to improve with a HHP pretreatment (Du et al., 2008; Pribenszky et al., 2008). HHP was first used in food processing to inactivate micro-organisms and enzymes responsible for shortening the storage life of a product (Knorr, 1993). It was then used for pretreatment of mammalian cells to increase the synthesis of heat shock proteins (HSPs) (Kaarniranta et al., 1998). HSPs improve stress tolerance, protein folding and signal transduction in mammalian cells (Csermely et al., 1998), and are necessary for normal development of mouse embryos (Esfandiari et al., 2007). More recently, HHP has been used to pretreat porcine-matured oocytes before cryopreservation (Du et al., 2008; Pribenszky et al., 2008). The ensuing blastocyst rate from parthenogenetic-derived embryos was 13.1%, whereas the control was 0.0% (Du et al., 2008). The improved developmental capacity of vitrified oocytes with HHP pretreatment may partially be due to post-transcriptional stabilization of HSP70 mRNA even though oocytes are generally regarded transcriptionally quiescent (Du et al., 2008).

Conclusions

Due to the biological uniqueness of the pig oocyte, the development of cryopreservation methodologies for porcine oocytes is much more problematic than for many other mammalian oocytes. To date there are no suitable systems for successfully cryopreserving of porcine oocytes with the endpoint being full-term development. However, progress in the cryopreservation of porcine oocytes is encouraging, as summarized in Table I, and offspring derived from cryopreserved oocytes will become a reality in the near future. Once a suitable method is developed it would enhance pig cloning technologies, promote the establishment of a gene bank for transgenic pigs and potentiate the use of the pig model for testing the safety of related technologies in humans.

Table I

Summary of strategies showing improved survival following cryopreservation of porcine oocytes

Strategy	Results	Possible mechanism	Reference
Methods of vitrification
Permeating cryoprotectants ethylene glycol, dimethylsulphoxide	Blastocyst (ICSI) ratesa: 13.5–14.3 versus 20% fresh, although lower maturation rates (23.6–37.1 versus 83.5%)	Cytoskeletal protection	Fujihira et al. (2004)
Non-permeating agents, sugars and polysaccharides	21% maturation rate 0.75 M sucrose/0.10 g per ml polysaccharide	Prevent ice formation	Huang et al. (2008)
Use of solid surface vitrification, cryotop sheetb	Blastocyst ratea (IVF: 3.4–9.5%; PA: 2.6–5.4%; ICSI: 13.5–14.3%)	Minimizes volume of vitrification solution	Fujihira et al. (2004), Somfai et al. (2006) and Gupta et al. (2007)
Cellular modifications
Taxol	Cleavage rate (PA) increased from 5.6 to 24.3%	Mitochondrial distribution, lipid droplets tubulin assembly	Shi et al. (2006) and Fu et al. (2009)
Cytochalasin B	Blastocyst ratea (ICSI: 13.5–14.3%). Improved development to MII 22 versus 5.6%	Cytoskeletal protection	Fujihira et al. (2004)
Delipidation	Maturation rate 7–15%		Hara et al. (2005) and Park et al. (2005)
Ruthenium red	Maturation rate 39.4%	Blocking Ca2+ uptake in mitochondria	Nakagawa et al. (2008)
	Blastocyst ratea (IVF: 28.6%)
High hydrostatic pressure	Blastocyst ratea (PA: 13.1%)	Stabilization of HSP70	Du et al. (2008)
	Cleavage rate (PA: 41.7%)

^aThe blastocyst rate was calculated as the percentage of the number of cleaved embryos after in vitro fertilization (IVF), or of the injected oocytes after intra-cytoplasmic sperm injection (ICSI), or of the activated oocytes after parthenogenetic activation (PA).

^bThese procedures, combined with other strategies (e.g. cytochalasin B and permeating cryoprotectants, etc.), allowed normal, lipid-containing, GV or MII porcine oocytes to be fertilized in vitro, to be parthenogenetically activated or following ICSI, and develop to the blastocyst stage in vitro. HSP70=heatshock protein 70.

Funding

The Major State Basic Research Development Program of China (973 Program) (2006CB102100) and the National Natural Science Foundation of China (30621064).

Acknowledgements

We would like to thank Dr Meng Qinggang and Dr D. Bunch Thomas (Utah State University, USA) for critical reading of the manuscript, Dr Fu Xiang-Wei for providing the figure and Dr Zhu Shien (China Agricultural University, People's Republic of China) for his valuable suggestion.

Author notes

†

This paper was presented at the International symposium of reproductive biology in Beijing October 2008.