Vitrification of immature oocytes in pigs

3.1 The vitrification method

To date, several vitrification methods have been applied for the preservation of immature porcine oocytes with very variable results (Table 1). Among these reports, there is little or no consistency in the exact vitrification protocol used. The phrase “vitrification method” may refer to either the device only or the device combined with the CPA treatment procedure. Therefore, the following sub-chapters address these points separately. Furthermore, it is important to point out, that some studies reported different results using the same vitrification protocol (Table 1) which indicates that factors other than the vitrification protocol (such as the skills of the technician) may also influence the results. It is worth to note that large numbers of porcine oocytes used for IVEP are usually obtained from slaughterhouse-sourced ovaries. Female pigs are commonly slaughtered at a market weight that is attained prior to reaching sexual maturity. It is known that the quality of oocytes from prepubertal gilts are inferior to those from mature sows (Grupen, 2014) This raises the question about the intrinsic quality of the oocytes used in vitrification studies which may explain some differences between studies. Other factors which may affect results (hence differences between studies) are season, follicle size and the IVM system (reviewed by Grupen, 2014).

3.1.1 Device

It is generally accepted that the vitrification device determines the cooling/warming rate during the process; hence it has been considered as major factor for the success for vitrification (Kuwayama, 2007). Among many devices, the Cryotop employing a minimum volume (less than 0.1 μl) of the vitrification solution was proven to provide a relatively high cooling rate (approximately 37,500 C/min) (Kuwayama, 2007; Sansinena et al., 2011). A study on matured porcine oocytes demonstrated the superiority of the Cryotop device compared with the Open Pulled Straw (OPS) method (Liu et al., 2008). However, using a different CPA treatment protocol, immature porcine oocytes could be effectively vitrified by the direct plunging of the oocytes into liquid nitrogen in 1- to 2-μl microdrops (MDs) of the vitrification solution with success rates similar to those achieved by the Cryotop device with minimum volume cooling (Appeltant et al., 2018). This suggests that when optimum CPA treatment is used, vitrification devices employing 2 μl or less of vitrification solution may be similarly effective. This suggestion is in line with the earlier results of Fernández-Reyez et al. (2012) and a similar conclusion was made on in vitro produced porcine blastocysts (Bartolac et al., 2015).

In respect of the above mentioned, the capacity and biosafety, availability and the price should determine which type of vitrification device is optimum for oocyte cryopreservation for each case (discussed in the last chapter). Many vitrification devices such as Cryotip, Cryoloop and Cryotop are available in closed version to prevent cross-contamination during storage (Castelló et al., 2018; Larman et al., 2014). However, they can accommodate only a limited number (3–10) of oocytes, hence they are not optimum when large numbers (over 100) of oocytes are to be vitrified at once. Vitrification of oocytes in MDs either in liquid nitrogen (LN) or on a cold metal surface (solid surface vitrification = SSV) is a deviceless approach which allows the fast cryopreservation of oocytes in groups of up to 100 in multiple MDs (Somfai & Kikuchi, 2021). Vitrified MDs can be stored in closed cryotubes (Figure 3). Such high-capacity vitrification method is advantageous in pigs which are multiparous (thus, several embryos must be transferred to one recipient to achieve pregnancy), and their IVEP is often characterized by limited embryo production rates (Grupen, 2014) (hence, high numbers of oocytes are required for each experiment). Therefore, the MD vitrification is clearly advantageous when oocytes are to be cryopreserved to conduct large scale research experiments. Nevertheless, this approach has a disadvantage that approximately 1%–5% of the oocytes are lost during preservation. Therefore, when only limited number of oocytes are available (e.g., gene banking for individual animals with low reproductive performance) other vitrification devices may offer a better choice.

3.2 The presence of cumulus cells

In previous studies, it has been suggested that the presence of cumulus cells on the zona pellucida might reduce the success of immature oocyte vitrification due to blocking/slowing CPA permeation (Casillas et al., 2014, 2015). Therefore, these authors denuded the oocytes before vitrification. This approach requires a co-culture with granulosa cells for efficient embryo production (Casillas et al., 2014, 2015). On the other hand, cumulus cells are known to be important during IVM for the cytoplasmic maturation of porcine oocytes by maintaining intracellular glutathione at a level necessary to support embryo development (Maedomari et al., 2007). Also, cumulus cells regulate the meiotic resumption of oocytes (reviewed by Appeltant et al., 2016). For this reason, our group fundamentally refrains from oocyte denudation before vitrification. In a recent study, we compared the survival after vitrification/warming of cumulus-intact and denuded immature porcine oocytes in our system and revealed that denudation before vitrification significantly reduced the percentage of membrane intact oocytes after warming (Nguyen et al., 2021; Figure 4). Therefore, we concluded that the presence of an intact cumulus compartment is in fact beneficial for oocyte survival during vitrification/warming. A similar conclusion was made in an earlier report using horse oocytes (Tharasanit et al., 2009). An interesting approach was reported by Jia et al. (2019) who co-cultured vitrified COCs with non-vitrified ones which improved the developmental competence of vitrified oocytes, suggesting the possibility that paracrine factors excreted by healthy COCs might be beneficial for the recovery of vitrified oocytes from non-lethal damages.

3.3 The lipid content of oocytes

In the early studies by Nagashima et al. (1994, 1995), it was demonstrated that the high intracellular lipid content of porcine oocytes and resultant embryos is responsible for their sensitivity to low temperatures which hinders traditional slow freezing. Mechanical lipid removal indeed dramatically improved the freezing tolerance of early porcine embryos (Nagashima et al., 1995). However, adoption of this technique for the vitrification of immature oocytes resulted in moderate success (Park et al., 2005). The negative aspects of this technique are that (1) it requires the removal for a certain amount of cumulus cells (which itself may hinder development potential) and also that (2) it comprises the integrity of zona pellucida which does not meet the international standards of sanitary handling (Stringfellow, 1998). Interestingly, our research revealed a significant difference in the lipid content of the oocytes of native Vietnamese Ban pigs and those of a western cross breed (Landrace x Large White) (Somfai, Nguyen, et al., 2019). However, when we applied our vitrification protocol on oocytes of these breeds, the survival and development rates were not significantly different (Somfai, Nguyen, et al., 2019). This suggests that, when optimum vitrification protocol is used, the lipid content of the immature oocyte may not be a decisive factor for the success of vitrification.

3.4 Post-warming culture (IVM)

Previous studies revealed that vitrification at the GV stage can induce several different types of intracellular damages and alterations in porcine oocytes which can potentially reduce subsequent embryo development. Some examples are the fragmentation of the nucleolus, the damage of microfilaments, premature nuclear progression, and the damage of mitochondria (discussed in the following chapters). Interestingly, oocytes seem to be able to recover from some of these alterations at least to some extent during their post-vitrification IVM culture, such as in case of nucleolus, microfilament and mitochondrial damage (Appeltant et al., 2017; Ito et al., 2020). Although the exact mechanisms of the nucleolus and microfilament recovery in oocytes are not clear, it can be assumed that the composition of the medium (i.e., the presence or lack of necessary factors) may affect these processes. For instance, the recovery from vitrification-related mitochondrial damage and thus subsequent embryo development in immature porcine oocytes could be enhanced by medium supplementation with resveratrol during post-warming IVM culture (Ito et al., 2020). Furthermore, the reagents used for meiosis synchronization during IVM have a major effect on the efficacy of subsequent in vitro embryo production from vitrified oocytes (Nguyen et al., 2022). These examples demonstrate that the medium composition during post-warming culture can indeed affect the developmental competence of vitrified oocytes and that the enhancement of recovery processes is a possible way to improve embryo production rates.

4.1 The fragmentation of the nucleolus

Phase contrast microscopy revealed that the CPA treatment during the vitrification process causes the fragmentation of the nucleolus precursor body (NBP) in the GV in approximately 50% of the surviving immature oocytes whereas none of the 125 control oocytes showed such phenomenon (Appeltant et al., 2017). On the other hand, the ratio of the oocytes having multiple NPBs instead of a single NPB in the GV was significantly reduced after subsequent IVM for 20 h in both the vitrified and CPA groups, suggesting the recovery from NPB fragmentation. Neither the mechanisms of recovery, not the impact of NPB fragmentation, on subsequent development has been clarified to date. Nevertheless, it is noteworthy that the CPA treatment of our vitrification protocol does not reduce the competence of vitrified GV stage oocytes to develop to the blastocyst stage (Somfai et al., 2013), which suggests that NPB fragmentation may not be a major factor responsible for reducing the competence of vitrified oocytes.

4.2 The damage of the cytoskeleton and transzonal projections

The alteration of actin microfilaments in vitrified immature porcine oocytes was reported firstly by Rojas et al. (2004). In their report, both CPA treatment (EG) and OPS vitrification dramatically reduced the percentage of oocytes with normal actin microfilaments which was associated with the complete failure of cleavage after IVF (Rojas et al., 2004). Nevertheless, it is not clear if the investigated oocytes were alive or dead (i.e., membrane damaged) since selection of live oocytes for assay is not described in the study. It is noteworthy that, in our studies, vitrification of immature pig oocytes in solely EG-based media was associated with low survival rates (Somfai et al., 2010, 2013). Later, using a similar vitrification protocol, Wu et al. (2006) confirmed the alterations of cortical microfilaments in surviving oocytes. Since we use a completely different vitrification protocol employing a CPA treatment which itself does not reduce subsequent development, our group also investigated the effects of vitrification and CPA treatment on microfilaments in immature COCs. We have confirmed that vitrification but not CPA treatment caused the deformation of cortical microfilaments of the surviving oocytes which was manifested by the formation of clusters of actin under the oolemma immediately after warming (Appeltant et al., 2017). Interestingly, these clusters completely disappeared within 20 h of subsequent IVM culture suggesting a recovery mechanism for vitrification-related malformations of filamentous actin (F-actin). As a result, at 44 h IVM, the morphology of actin microfilaments did not differ between the vitrified and non-vitrified oocytes (Appeltant et al., 2017) which corroborates our previous results (Egerszegi et al., 2013). On the other hand, another study reported abnormal actin microfilaments in early embryos obtained from porcine oocytes vitrified at the GV stage (López et al., 2021), suggesting that repair mechanisms can be compromised. However, this study applied a different vitrification protocol and the effect of CPA treatment on the results remained unclear. Further research is necessary to clarify the contribution of F-actin alterations to the compromised developmental competence of vitrified oocytes and the impact of the CPA protocol.

In our previous study (Appeltant et al., 2017), visualization of filamentous actin by confocal microscopy allowed the study of transzonal projections (TZPs) within the zona pellucida which are known to play a pivotal role in gap junctional communication between the oocyte and the cumulus cells (Clarke, 2022). We found that vitrification itself but not CPA treatment of COCs significantly reduced the number of TZPs by approximately 30% and the difference between the vitrified and non-vitrified oocytes could be detected even after 20 h of subsequent IVM culture (Appeltant et al., 2017). Gap junctional communication through TZPs between the oocyte and the cumulus cells is important for the control of meiotic resumption and the cytoplasmic maturation of porcine oocytes (Appeltant et al., 2016; Mori et al., 2000) and cumulus expansion during IVM (Appeltant et al., 2015). Nevertheless, in vitrified COCs, the cytoplasmic maturity of oocytes (measured by glutathione and ATP contents) and the extent of cumulus expansion was similar to those of the non-vitrified control (Appeltant et al., 2017). This indicates that the reduction of TZP abundance by 20%–30% was not extensive enough to affect these features. On the other hand, vitrification but not CPA treatment was found to trigger premature nuclear resumption of oocytes (Appeltant et al., 2017) (discussed in the next chapter) which could theoretically be related to TZP damage.

4.3 Premature meiotic resumption

In porcine oocytes, cAMP-dependent protein kinase (PKA) maintains meiotic arrest at the GV stage during the first 10–20 h of IVM and cumulus cells play an important role in this process through jap junctional communication (GJC) (Appeltant et al., 2016). However, when immature COCs were vitrified by our method, approximately 20% of the oocytes resumed meiosis prematurely even in the presence of 1-mM dibutyryl-cAMP (dbcAMP), which is a membrane permeable cAMP analogue used to block premature meiosis during the first half of IVM in order to synchronize subsequent maturation (Appeltant et al., 2017). Since vitrification also compromised the integrity of TZPs, it seemed plausible that a reduction in GJC might be responsible for the vitrification-related premature meiosis. To clarify if gap junctional damage can lead to premature meiosis, we mechanically removed all cumulus cells from some freshly collected COCs with a fine glass capillary eliminating GJC, whereas other oocytes were processed as intact COCs. Then, in both groups, some oocytes were vitrified/warmed, whereas others were processed without vitrification. Then all oocytes were subjected to IVM culture in the presence of 1-mM dbcAMP for 22 h. The meiotic status of denuded oocytes (DOs) without or after vitrification were compared by orcein staining with those of intact COCs without or after vitrification. Interestingly, vitrification triggered premature meiosis resumption in approximately 20% of oocytes in both vitrified COCs and DOs in a similar manner whereas in both non-vitrified COCs and DOs dbcAMP could maintain the GV stage with similar efficiency (Figure 4). This demonstrates that in the presence of dbcAMP, the total damage of gap junctions itself in the group without vitrification cannot trigger premature meiotic resumption of oocytes and that vitrification-related premature meiosis occurs irrespective of the presence/absence of cumulus cells and the damage of gap junctions. Nevertheless, the mechanism of vitrification-related premature meiosis remains to be clarified.

The impact of vitrification-related premature nuclear progression on embryo development is also a matter of question. During IVM, premature meiosis of some oocytes is known to cause heterogeneity in cytoplasmic maturity and aging status in the population of cultured oocytes which reduces the efficacy of in vitro embryo production. For this reason, synchronization of oocyte maturation by the temporal inhibition of meiosis at the prophase (i.e. the GV stage) during the first 20–24 h of IVM has become a common practice in pigs (Grupen, 2014). In porcine oocytes, it can be achieved either by maintaining high intracellular cAMP levels (Appeltant et al., 2016) or by the inhibition of the metaphase promoting factor (MPF) (Motlik et al., 1998). The former approach can be achieved using 1-mM dbcAMP (Funahashi et al., 1997), whereas the latter way is achieved effectively using reversible inhibitors of p34^cdc2 protein kinase (the catalytic subunit of MPF) such as roscovitine (Marchal et al., 2001) which applied at 25 μM was reportedly beneficial for fresh porcine oocytes (Zhu et al., 2020). We compared these two approaches for the nuclear synchronization of vitrified immature oocytes and found that dbcAMP could impede but not completely prevent premature meiosis in vitrified oocytes whereas roscovitine was more efficient. However, for subsequent embryo production, the use of roscovitine was adverse whereas dbcAMP was beneficial (Nguyen et al., 2022). Since the problem of vitrification-related premature meiosis could not be resolved yet, further research will be needed to address this point. Also, it remains to be elucidated whether the vitrification -related premature meiosis is the cause of reduced development or only an indicator of another problem in surviving oocytes. For instance, meiotic resumption is known to require intracellular Ca²⁺ ([Ca²⁺]i) oscillations in oocytes (He et al., 1997). [Ca²⁺]i release from intracellular stores might initiate meiotic resumption. The Ca²⁺ homeostasis in oocytes is controlled by cytoplasmic organelles such as the endoplasmic reticulum (ER) and mitochondria which store Ca²⁺ (Dumollard et al., 2007; Wakai et al., 2019). Although the effects of vitrification on the ER of porcine immature oocytes remain to be elucidated, it reportedly exerts stress on the ER in matured mouse oocytes (Zhao et al., 2015). Mitochondria are also known to be damaged by vitrification (discussed in the next chapter). It was demonstrated in bovine matured oocytes that vitrification induces the release of Ca²⁺ from intracellular stores into the cytoplasm (Wang et al., 2017). In this respect, premature nuclear progression may be an indicator of damage of Ca²⁺ regulation system. Further research will be necessary to verify this theory.

4.4 Mitochondrial damage

In mammalian oocytes, mitochondria play pivotal roles in energy production, Ca²⁺ homeostasis, signaling during fertilization, ROS regulation, and apoptosis (Dumollard et al., 2007). Previous studies demonstrated that, in matured porcine oocytes, vitrification may alter the mitochondrial properties which contributed to reduced developmental competence via the activation of the apoptotic cascade (Dai et al., 2015, 2016). However, in immature oocytes, this may not be the case since vitrification by our method at the GV stage did not trigger apoptosis in porcine oocytes and resultant embryos (Somfai et al., 2020). Furthermore, similar levels of cytoplasmic glutathione, ATP, and hydrogen peroxide in vitrified and non-vitrified immature porcine oocytes during subsequent IVM (Appeltant et al., 2017; Santos et al., 2018; Somfai et al., 2010) suggested that mitochondrial activities may not be dramatically altered by vitrification. Nevertheless, there is evidence that mitochondrial damage occurs to some extent in the vitrified immature oocytes as well. Nguyen et al. (2023) demonstrated reduced mitochondrial activity in vitrified immature porcine oocytes. Although it appeared to be normalized during subsequent IVM, mitochondrial damage contributed to the reduced developmental competence since it could be mitigated by pretreatment with cyclosporine A and docetaxel resulting in improved embryo development (Nguyen et al., 2023). In an earlier study, supplementation of the IVM medium with resveratrol, known as an antioxidant, improved the developmental competence of oocytes irrespective of cytoplasmic ROS levels and apoptosis (Santos et al., 2018). Later, Ito et al. (2020) demonstrated that resveratrol acts through the activation of sirtuin 1, which induces mitochondrial synthesis facilitating the recovery of vitrified-warmed oocytes. These examples demonstrate that, in immature porcine oocytes, the recovery from certain vitrification-related non-lethal damages can be assisted during post-warming IVM culture.

4.5 DNA damage

Although several previous studies verified the detrimental effect of cryopreservation on the integrity of DNA in matured mammalian oocytes (for a review see Kopeika et al., 2015), this issue was not explored in porcine oocytes until recently. Also, studies on DNA damage that occurs during oocyte vitrification at the GV stage in any species are lacking. In early porcine embryos, increased levels of DNA double-strand breaks (DSBs) are associated with delayed early development and a compromised ability to reach the blastocyst stage (Bohrer et al., 2015). Recently we demonstrated that vitrification but not CPA treatment generates DSBs in the DNA of porcine oocytes at the GV stage which are transmitted to the subsequently developing cleavage stage embryos during IVM, IVF and embryo culture (Somfai et al., 2023). This is associated with a delay of early development and compromised capacity to reach the blastocyst stage. However, it does not seem to affect the quality of resultant blastocysts in terms of cell numbers and the frequency of apoptosis. These results suggest that vitrification-induced DNA damage is a major factor that contributes to the compromised development of vitrified GV-stage oocytes. Furthermore, vitrification of oocytes at the GV stage was found to activate the expression of the RAD51 gene, which is responsible for DNA damage-repair by homologous recombination (Somfai et al., 2023). This suggests the possible importance of DNA damage-repair by homologous recombination during post-warming culture in the vitrified oocytes for their development to the blastocyst stage. Further research will be necessary to clarify mechanisms and regulation of DNA damage-repair in vitrified oocytes and subsequently developing embryos.

4.6 Epigenetic alterations

During early development of mammalian embryos, epigenetic regulation of gene expression is crucial for the normality of subsequent fetal and even postnatal development (reviewed by Sciorio et al., 2023). Cryopreservation was found to induce various types of epigenetic modifications in mammalian cells including oocytes (Chatterjee et al., 2017). Also, oocyte vitrification was found to cause abnormal expression of certain genes in the organs of mice at the adult age (Zhang et al., 2022) and alter physiological indexes such as blood pressure and triglyceride level at the first filial generation (Huo et al., 2020). These raise concerns for the health of children conceived at human fertility clinics where vitrification has been accepted as a standard procedure for oocyte cryopreservation (Sciorio et al., 2023). In humans, ethical restrictions underline the importance of animal models for the research of this matter. In mice and cattle, vitrification of matured oocytes was reported to reduce the levels of global DNA methylation and to increase histone acetylation even in subsequent embryos which were associated with reduced blastocyst development (Chen et al., 2016; Liang et al., 2012; Yodrug et al., 2021). This suggests the contribution of altered epigenetic features to developmental failure. When porcine immature oocytes were vitrified in our system, reduced levels of global DNA methylation were detected in the oocytes after IVM which was apparently caused by the CPA treatment (Somfai et al., 2021). In another report, however, vitrification of mouse oocytes at the GV stage did not induce alterations of global DNA methylation measured by the intensity of 5-methylcytosine immunostaining after subsequent IVM (Yan et al., 2014). The differences in results between the two studies may be species-specific since the CPA used for vitrification and the method of DNA methylation analysis were similar. Interestingly, after porcine oocyte vitrification, the DNA methylation levels were restored during early embryo development (Somfai et al., 2021). The DNA methylation levels were similarly affected in the vitrified and CPA-treated groups; however, the latter showed no reduction in the percentage of blastocyst development whereas the former showed reduced blastocyst development. This suggests that, in the current system, CPA-related reduction in DNA methylation levels may not be responsible for the compromised ability of vitrified oocytes to develop to blastocysts. Nevertheless, its effect on fetal and postnatal development remains to be explored. Furthermore, vitrification was reported to alter histone acetylation and methylation in matured porcine oocytes (Spinaci et al., 2012). However, in immature oocytes the effects of vitrification on histone acetylation and methylation status and its impact on subsequent development is yet to be investigated.