Materials and Methods
Chapter 4 Results
4.2 Objective 2: Investigating the effect of nocodazole cell synchronization on polar body extrusion
Figure 4.1.3 The effect of different blastocyst hatching techniques on embryo-derived outgrowth efficiency. WT blastocysts were non-hatched, pronase hatched, mechanically hatched, or naturally hatched. Outgrowth age refers to number of days since blastocyst plating.
Outgrowth efficiency is the proportion of blastocysts plated that form an outgrowth. Error bars=SEM. Total blastocysts plated; non-hatched N=15, pronase hatched N=11, mechanically hatched N=16, and naturally hatched N=20. Replicates n=7. Significance * = P <0.05, ** = P<0.01.
4.2 Objective 2: Investigating the effect of nocodazole
To analyse the effect of nocodazole and CHX treatment on embryo development, ECT with nocodazole-synchronized donors was performed (n=3). As a control reconstructs were formed with non-synchronized embryonic donors and then were incubated in DMAP after activation. Non-treated donors were likely a mix of cells at different cycle stages. Embryonic donors were pronase-treated, disaggregated, attached to cytoplasts, and fused. Successful fusion was recorded an hour after exposure to DC pulses and was quantified out of couplets stuck, excluding couplets where lysis had occurred in the cytoplast or donor. Fusion between MII cytoplasts and embryonic donors was the same regardless of whether the donor was treated with nocodazole (N=126; 82 ± 2%), or not (N=121; 82 ± 4%, P=1). (Figure 4.2.1).
Figure 4.2.1 Fusion of CT couplets with embryonic donors that were treated with 500 nM nocodazole or non-treated. Fusion was calculated out of number of couplets that successfully fused out of couplets stuck excluding couplets where lysis in the cytoplast or donor occurred. Error bars=SEM. Significance * = P<0.05, ** = P<0.01. Total number of couplets; nocodazole-treated N=121, non-treated N=126. Replicates n=3.
Reconstructs were activated with ionomycin/CHX to allow pseudo-polar body extrusion for the return to a diploid state (N=94, n=3). Four hours after incubation in CHX, embryos were separated into those that formed pseudo-polar bodies (PPB) or no pseudo-polar bodies (NPB) in between the ZP and plasma membrane (Figure 4.2.2).
The proportion of reconstructs that did not form pseudo-polar bodies (78 ± 5%) was
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significantly higher than the amount of reconstructs that did form pseudo-polar bodies (22 ± 4%, P=10-14).
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4.2.2 Development of ECT embryos with synchronized and non-synchronized donors
Reconstructs with non-synchronized donors were incubated in DMAP, which inhibited pseudo-polar body formation. Reconstructs with nocodazole-treated donors that formed PPBs (N=21), reconstructs with nocodazole-treated donors that had NPPB extrusion (N=73) and non-treated reconstructs (N=97) were kept in ESOF culture for 5 days. On day 5, lysis in the embryos reconstructed with the different donor treatments was recorded (figure 4.2.3). Lysis on day 5 occurred in the non-treated group (11 ± 6%) and the nocodazole-treated PPB group (5 ± 4%) but not in the nocodazole-treated NPPB group (0 ± 0%). The difference in lysis between the embryos with non-treated donors and embryos with nocodazole-treated donors is only significant when those embryos did not have polar bodies (P=0.0032). The difference in lysis between the nocodazole-treated NPPB and PPB embryos was not significant.
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Figure 4.2.2 Polar body formation in CT reconstructs with embryonic donors treated with 500 nM nocodazole and activated with CHX. Four hours after CHX activation, embryos were separated into those that formed pseudo-polar bodies or no pseudo-polar bodies. * = P<0.05, ** = P<0.01.
Total number of reconstructs treated with nocodazole N=94. Replicates n=3
Figure 4.2.3 Lysis of ECT embryos with donors treated or not treated with 500nM nocodazole. After CHX activation, treated embryos were separated into those that formed PPB or NPPB. After DMAP activation, non-treated embryos were NPPB. Lysis was calculated on day 5 out of number of reconstructs that went into IVC. Error bars = SEM.
Significance * = P<0.05, ** = P<0.01. Total number of reconstructs into IVC; nocodazole NPPB N=73, nocodazole PPB N=21, non-treated N=97. Replicates n=3.
There was no significant difference in day 5 cleavage or day 7 blastocyst development between embryos with non-treated donors and embryos with nocodazole-treated donors with or without pseudo-polar body formation (P>0.05) (figure 4.2.4). Cleavage was the same after nocodazole treatment with NPPB (68 ± 3%), nocodazole treatment with PPB (67 ± 14) and no treatment (67 ± 4). Total blastocyst development was highest after no treatment (13 ± 3%), and then nocodazole treatment with NPPB (7 ± 1%). No blastocysts developed after nocodazole treatment with PPB (0 ± 0). High grade blastocysts developed from the no treatment group (4 ± 3%), and the NPPB nocodazole treatment group (1 ± 2%).
Data from three repeat experiments was used for the final figures and calculations. A 4th repeat was completed, which showed that it was possible to produce blastocysts from the nocodazole PPB group. However, this run was not included due to an issue with culture medium evaporation that specifically affected the DMAP group. One grade 3 blastocyst (25%) was produced from the nocodazole PPB group in the 4th repeat which was not significantly different from B1-3 development in the NPPB
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group (6%). No embryos from the PPB or NPPB treated group developed into grade 1-2 blastocysts in the 4th repeat.
Figure 4.2.4 Development of ECT embryos with donors treated or not treated with 500nM nocodazole. Cleavage was calculated on day 5 out of number of reconstructs that went into IVC. Development in blastocysts grade 1-3 or grade 1-2 were calculated on day 7 out of number of reconstructs that went into IVC. Error bars=SEM. Significance * = P<0.05, ** = P<0.01. Total number of reconstructs into IVC; nocodazole NPPB N=73, nocodazole PPB N=21, non-treated N=97. Replicates n=3.