Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation - Genetic Engineering: Recent Developments in Applications - page 67

concentrations of the reducing agent alpha-monothioglycerol (MTG). This re-
agent was selected because previous work has shown it is necessary for the culture
of ES-cells in sensitive/stressful serum-free systems[22]. The presence of at least
318 µM MTG improved the fertilization proficiency of C57BL/6J sperm (Figure
2). This improvement was not associated with an increase in the proportion of
motile sperm (p = 0.12), providing further evidence that it is essential to evaluate
endpoints other than sperm survival and motility when optimizing cryopreserva-
tion methods.
Figure 2. Dose response of monothioglycerol addition to mouse sperm cryopreservation media. Sperm were
collected from 6 C57BL/6J males. To reduce male-to-male variability, 3 pools of sperm were created by
combining 2 males each. The samples were split 5 ways and the sperm were cryopreserved in the concentrations
of monothioglycerol (MTG) illustrated on the x-axis. The cryopreserved sperm were thawed, placed into MVF
and incubated for 60 min prior to the addition of cumulus oocyte masses from two superovulated C57BL/6J
females (mean±standard deviation; 50±22 oocytes/IVF). Differences among the treatments were determined
using ANOVA and preplanned comparisons to the control of no MTG using Dunnett's method[32].
Excess ROS generation may be linked to the requirement for a sperm pre-
incubation step, as observed in Figure 1a. To test this, C57BL/6J sperm were
cryopreserved in the presence of 477 µM MTG and incubated in MVF media for
30, 60, 90 or 120 minutes prior to adding C57BL/6J oocytes. As shown in Figure
1a, the presence of MTG did not change the incubation time required to reach
maximum fertility. However, the percentage of 2-cell embryos was greater for
sperm frozen in the presence of MTG. Collectively, these observations indicate
that damage by ROS may be partly responsible for the reduction in fertility of
cryopreserved mouse sperm. This effect could be due to the oxidation of lipids[23]
and/or proteins[24] that participate in the fusion and subsequent penetration of
the oocyte by sperm. Reactive oxygen species can also oxidize DNA[25], poten-
tially leading to a reduction in embryo development. Nonetheless, the observa-
tion that cryopreserved C57BL/6J sperm require post-thaw incubation to reach
maximum fertility does not appear linked to ROS generation.