5.4 Objective 4: Investigate the potential of vitrified bovine oocytes and cytoplasts for use in CT
5.4.2 Artificial activation of thawed oocytes
After establishing that oocytes can survive the vitrification/thawing process, thawed oocytes underwent ionomycin/DMAP activation to assess whether the nuclear or cytoplasmic components essential for normal embryo development were degraded during the vitrification/thawing process. Parthenogenesis can occur in vitro when a non-enucleated oocyte is artificially activated to produce a diploid embryo with only maternal genetic material (De et al., 2012; Fernandes et al., 2014; Méo et al., 2007).
Artificial activation can be used as a preliminary study before CT to assess whether an oocyte is capable of supporting development.
Lysis was significantly higher in vitrified oocytes after activation than in fresh oocytes.
Fresh oocytes had significantly higher total blastocyst development and significantly higher grade 1-2 blastocyst development than vitrified oocytes. Blastocyst development was taken from the total number of oocytes into IVC and did not exclude oocytes that lysed by day 5. The low blastocyst development of the vitrified oocytes if
a reflection of their low rate of survival to day 5. So, while vitrified oocytes can still support development to the blastocyst stage, the vitrification process lowers this developmental potential. Dinnyés et al. (2000); Hou et al. (2005) also found that artificial activation with vitrified bovine ZI oocytes produced embryos with lower cleavage and blastocyst development than with fresh oocytes. However, when O2
incubation levels were increased from 5%, like in this thesis, to 20% the difference in blastocyst development disappeared. Authors have no included speculation as to the reason for this phenomenon. The exact reason why the vitrification/thawing process negatively affected oocyte developmental competence cannot be confirmed, but it is likely to be because of the effect of vitrification on the temperature-sensitive meiotic spindle. Some studies have observed that vitrification causes cryoinjury in the meiotic spindle which hinders survival and the use of vitrified oocytes (Atabay et al., 2004;
Booth et al., 2001; Dinnyés et al., 2000; Girka et al., 2022; Saragusty & Arav, 2011).
This spindle makes bovine oocytes more difficult to vitrify than bovine embryos. The meiotic spindle is required for the proper separation of chromosomes, completion of meiosis, and normal embryo development (Chen et al., 2003). The heterodimers of the microtubules in the spindle can dissociate in the rapidly decreasing temperatures of vitrification, compromising the spindle. During warming the spindle repolymerizes but it may not be the correct conformation. The alternative reason is that maternal developmental factors, especially those required for early embryonic activation, were damaged during the vitrification/thawing process. Vitrified cytoplasts, which had the meiotic spindle removed, also displayed lowered developmental competence which may suggest that a damaged meiotic spindle was not a detrimental issue in oocytes, only damaged developmental factors. Although if the developmental factors were intact, reprogramming factors may have been a causative problem in cytoplasts while the meiotic spindle was a causative problem in oocytes.
Although it has been suggested that the ZP can provide protection from lysis during the vitrification/thawing process, it does not affect lysis after artificial activation for fresh or vitrified oocytes. There was no significant difference in cleavage or blastocyst development between oocytes with or without a zona, regardless of whether they were vitrified or not (P>0.05). To my knowledge, there are no reports in previous studies
that the ZP impacts development after artificial activation3. Booth et al. (2001) found that the artificial activation of bovine ZI oocytes produced embryos with significantly lower lysis than with ZF oocytes. However, they saw no significant difference in blastocyst development. Even though the difference in blastocyst development in this thesis was not statistically significant, we can conclude that ZF vitrified oocytes can develop to blastocysts while we cannot make that conclusion with ZI vitrified oocytes.
If oocytes are vitrified with an intact zona to increase thawing survival, the zona should then be removed prior to CT. This will not hinder the current CT protocol as CT in this thesis is performed with a zona free system to make enucleation easier and fusion rates higher (Booth et al., 2001; Oback et al., 2003).
The rate of cleavage and blastocyst development in our ZI fresh control PGs was lower than the range in the literature. Both Dinnyés et al. (2000) and Hou et al. (2005) found that ZI fresh PGs had about 1.3-fold higher cleavage and about two-fold higher blastocyst development. After activation, the rate of cleavage in the ZI vitrified oocytes was within the reported range but blastocyst development was nil, much lower than reported. Both Dinnyés et al. (2000) and Hou et al. (2005) found that ZI vitrified PGs had the same cleavage at 50% but approximately 20% higher blastocyst development.
Overall Dinnyés et al. (2000); Hou et al. (2005) observed a reduction in both cleavage and blastocyst development in artificially activated vitrified oocytes compared to fresh oocytes. A reduction was also observed in this thesis but at a lower baseline indicating that the artificial activation system in this thesis was suboptimal. The variation in development between this thesis and the literature is likely to be caused by differences in the composition, concentrations, and the time of exposure of the vitrification media.
The concentration of cryoprotectants and the time of exposure to cryoprotectants is important because they affect the extent of dehydration in the cells (Chian et al., 2004).
Cryoprotectant concentration affects cooling-warming rates during vitrification. Hou et al. (2005) found that increasing the concentrations of cryoprotectants increased oocyte survival by 21%. The type of cryoprotectant used can affect the developmental competence of oocytes. Chian et al. (2004) found that development after IVF with vitrified oocytes was higher when using propylene glycol instead of DMSO. DMSO can cause spindle polymerisation with increased risk for polyploidy. Propylene glycol
3 As of 1st November 2022.
is less toxic and does not cause spindle polymerisation. Dinnyés et al. (2000) opted to exclude DMSO during the vitrification of ZI oocytes and produced blastocysts at higher rate after artificial activation than was displayed in by oocytes in this thesis which were vitrified with DMSO in the media. This could further suggest that the presence of DMSO does damage cells.
Ovary collections alternated between seasons and the ages of the mothers. There are seasonal changes in the membrane phospholipid composition and cholesterol levels in bovine oocytes (Buschiazzo et al., 2017). There is lower cholesterol levels in bovine oocytes in winter and in summer compared to autumn and spring which lowers the membrane fluidity. Lowered fluidity can lower the tolerance of the oocytes to cryopreservation. Murine vitrified oocytes have been observed to have decreasing survival, cleavage rate and blastocyst development rate with maternal age (Yan et al., 2010).
The development of two blastocysts, including a high-quality one, from the artificial activation of ZF oocytes proved that the oocytes are capable of supporting development after the vitrification thawing process.