5.3 Objective 3: Optimise cytoplast treatment for bovine ECT
5.3.1 Cytoplast age
The proportion of donors in S-phase used in the aging experiments is unknown but is likely to be half as shown by staining of outgrowths cultured in the same conditions as in this thesis (Wilson, 2021). This means approximately half of the reconstructs made with MII cytoplasts were likely to be unviable. To be compatible with the large population of donors in S-phase, recipient cytoplasts with a low MPF environment can be used (Dinnyes et al., 2006; Du et al., 2002). A cytoplast with this environment exists in an interphase-like stage and will not perform nuclear breakdown and chromatin remodelling on the donor while in this state. Replication can be completed in the S-phase donors before the nuclear membrane breakdown and chromosome condensation.
Aging followed by cooling is one technique used to lower MPF in cytoplasts (Bordignon & Smith, 1998; Fulka Jr et al., 1998; Gall et al., 1996). Advancing cytoplast age causes a decline in MPF activity (Fulka Jr et al., 1998). MPF activity plateaus at 20-24 hours after maturation, holding the bovine oocyte in MII (Wu et al., 1997). MPF remains high until 30 hours of maturation where it decreases until basal levels at 44-48 hours. To ensure MPF lowers to a point where the cytoplast no longer induces nuclear envelope breakdown in donors, aging for at least 44 hours is required.
The aging must be accompanied by cooling as high levels of MPF were detected in cytoplasts that were aged at room temperature, but low levels were detected in cytoplasts aged at 10°C (Gall et al., 1996). H1 kinase activity, which is indicative of the oocyte being meiotic, was lower in cytoplasts that were aged and cooled to 12°C compared to in cytoplasts that were aged but not cooled (Bordignon & Smith, 1998).
Temperature reduction may be crucial as it has been shown to stimulate repeated calcium oscillations which could induce activation and the release from MII in some mammals. Enucleation was also required before cooling and aging to create an S-phase compatible environment. Enucleation alone did not affect MPF, as MPF remained high
in enucleated cytoplasts with aging and cooling. Lee and Campbell (2006) also established that the enucleation of ovine oocytes did not affect MPF activity. Li et al.
(2014) observed that a large fraction of cyclin B, a subunit of MPF, accumulated around the spindle which gets removed during enucleation. They found that MPF activity in oocytes was decreased following porcine enucleation, but this decrease was not statistically significant as a fraction of MPF was cytoplasmic and not removed.
However, in combination with aging and cooling, enucleation can lower MPF (Gall et al., 1996). High MPF levels were detected in aged, cooled, non-enucleated oocytes but basal MPF levels were detected in aged, cooled, enucleated oocytes.
The cytoplast environment after aging and cooling was also compatible with donor cells in G2/M-phase as these cells had completed replication and could degrade their nuclear membrane for chromatin remodeling without a high MPF environment (Dinnyes et al., 2006; Du et al., 2002). Donor cells in the G1/G0-phase were not compatible as they needed a high MPF environment to degrade their nuclear membrane for chromatin remodeling. The proportion of donors in G1/G0 was likely to be ≈23% so the total proportion of cells predicted to be compatible with aged and cooled cytoplasts was up to ≈77% (Wilson, 2021).
Fusion was higher for aged cytoplasts than non-aged cytoplasts, although this was not statistically significant. Muggleton-Harris and Hayflick (1976) discovered that aged cytoplasts had higher fusion rates with somatic donors and Sendai viruses than the non-aged cytoplasts and speculated that this could be due to a change in the membrane with age that facilitates easier fusion with other cellular membranes. This varied from a study by Liu et al. (2000) that noticed that the fusion rates of donor cells with aged cytoplasts enucleated at the TII stage were lower than those with non-aged cytoplasts enucleated at the MII stage, potentially due to the membrane becoming more fragile and vulnerable to electrofusion with age.
Lysis on day 5 was significantly higher after aged treatment. This suggests the aging process may have compromised cell integrity which could contribute to lower blastocyst development.
Both cleavage and blastocyst generation rates were higher in the non-aged group than in the aged group. This difference was significant for total blastocyst development but
not significant for high-quality blastocyst development. This result could suggest that the aging process did not sufficiently reduce the levels of MPF or that the proportion of donors in G1/G0 was higher than expected. The results in this thesis did not correlate with literature which found that aging cytoplasts followed by cooling in bovine CT appears beneficial (Heyman et al., 1994; Misica-Turner et al., 2007) or was not different to controls (Shiga et al., 1999). The lowered blastocyst development of aged cytoplasts in this thesis was supported by other literature, which observed that oocyte aging could decrease viability after CT as prolonged culture may have affected some crucial cytoplasmic constituents required for normal development (Bordignon &
Smith, 1998).
Gall et al. (1996) established that enucleation must accompany aging and cooling to cause a drop in MPF levels. However, using non-enucleated oocytes for aging is more common and has been reported to have an advantage for blastocyst development (Bordignon & Smith, 1998; Liu et al., 2000). Yang (1991), on the other hand, noted that using aged oocytes instead of cytoplasts may be an issue because aging can lead to alterations in the cytoskeleton that cause inward migration of the metaphase plate which may reduce the rate of enucleation of the aged oocytes and affect CT efficiency.
An alternative method to create a low MPF environment in recipient cytoplasts is chemical pre-activation. Chemical pre-activation involves activating cytoplasts with ionomycin before fusion and putting them into a presumed G1/S-phase (Du et al., 2002). Then the cytoplast is kept in an inhibitor of protein phosphorylation or synthesis until fusion is completed. Stice et al. (1994) discovered that using preactivated cytoplasts in bovine CT is beneficial when using blastomeres as donors. Du et al.
(2002) observed that pre-activating bovine cytoplasts improved blastocyst development by 25% when using blastomere donors. Kurosaka et al. (2002) discovered that pre-activating bovine cytoplasts improved blastocyst development by 16% when using somatic donors in S-phase. Bordignon and Smith (1998) observed that blastocysts constructed bovine blastomeres had higher development when using preactivated-aged oocytes compared to using aged-cooled cytoplasts.