protecting those commonly used inbred strains (e.g., C57BL/6,
FVB, and 129/Sv) with single mutations [8–10]. Sperm can be
recovered from unhealthy or dead animals, and a single male may
facilitate regeneration of several thousand of pups.
Although there is a significant interest in effective and consistent spermatozoa cryopreservation protocols for genome banking
for scientifically valuable mouse genetics, there have been reports
on potential variations with regard to post-thaw motility and
in vitro fertilization (IVF) outcome among different laboratories
[11]. These variations may be partially attributed to the extreme
sensitivity of mouse sperm to various stress factors including
mechanical, physical, osmotic, pipetting, centrifugation, oxidative,
and cooling stresses that are collectively imposed throughout cryopreservation procedures [12–15].
Successful mouse sperm cryopreservation protocols were
reported in 1990 by several Japanese research groups [16–
18]. However, among those with various modifications, Nakagata’s
protocol became the most widely used by many research laboratories and repositories around the world [19]. The initial freezing
solution simply contains 18% dehydrated skim milk and 3% raffinose in water, and cooling is achieved in LN2 vapor phase
(~À160
C) for 5 min followed by plunging the samples into
LN2 at À196
C. Since the introduction of this initial protocol,
there have been several changes in an effort to improve post-thaw
fertilization potential of mouse sperm. Ostermeier et al. [20] was
the first to show the beneficial effects of well-known antioxidant
monothioglycerol (447 μM) in the freezing solution for post-thaw
survival of mouse sperm by reducing oxidative stress. Similarly, the
inclusion of L-glutamine (100 mM) has also been shown to increase
post-thaw motility [21].
In addition to modifications in the freezing solution, Takeo
et al. (2008) [22] have made attempts to further improve IVF
potential of frozen-thawed mouse sperm by preincubating them
in capacitation medium containing methyl-β-cyclodextrin
(MBCD), which enhances sperm capacitation via removal of cholesterol from the sperm plasma membrane. The latest refinement
was the addition of reduced glutathione (GSH) to the IVF
medium, which was also suggested to protect spermatozoa against
oxidative stress during IVF [23] as well as weakening the disulfide
bonds within the oocyte zona pellucida glycoprotein filaments,
which collectively increase fertilization rates [23, 24]. This chapter
provides a detailed protocol for mouse sperm cryopreservation as
described by Nakagata and his colleagues with some modifications.
A detailed IVF procedure using frozen-thawed mouse sperm has
recently been described by [19].
402
Yuksel Agca and Cansu Agca
FVB, and 129/Sv) with single mutations [8–10]. Sperm can be
recovered from unhealthy or dead animals, and a single male may
facilitate regeneration of several thousand of pups.
Although there is a significant interest in effective and consistent spermatozoa cryopreservation protocols for genome banking
for scientifically valuable mouse genetics, there have been reports
on potential variations with regard to post-thaw motility and
in vitro fertilization (IVF) outcome among different laboratories
[11]. These variations may be partially attributed to the extreme
sensitivity of mouse sperm to various stress factors including
mechanical, physical, osmotic, pipetting, centrifugation, oxidative,
and cooling stresses that are collectively imposed throughout cryopreservation procedures [12–15].
Successful mouse sperm cryopreservation protocols were
reported in 1990 by several Japanese research groups [16–
18]. However, among those with various modifications, Nakagata’s
protocol became the most widely used by many research laboratories and repositories around the world [19]. The initial freezing
solution simply contains 18% dehydrated skim milk and 3% raffinose in water, and cooling is achieved in LN2 vapor phase
(~À160
C) for 5 min followed by plunging the samples into
LN2 at À196
C. Since the introduction of this initial protocol,
there have been several changes in an effort to improve post-thaw
fertilization potential of mouse sperm. Ostermeier et al. [20] was
the first to show the beneficial effects of well-known antioxidant
monothioglycerol (447 μM) in the freezing solution for post-thaw
survival of mouse sperm by reducing oxidative stress. Similarly, the
inclusion of L-glutamine (100 mM) has also been shown to increase
post-thaw motility [21].
In addition to modifications in the freezing solution, Takeo
et al. (2008) [22] have made attempts to further improve IVF
potential of frozen-thawed mouse sperm by preincubating them
in capacitation medium containing methyl-β-cyclodextrin
(MBCD), which enhances sperm capacitation via removal of cholesterol from the sperm plasma membrane. The latest refinement
was the addition of reduced glutathione (GSH) to the IVF
medium, which was also suggested to protect spermatozoa against
oxidative stress during IVF [23] as well as weakening the disulfide
bonds within the oocyte zona pellucida glycoprotein filaments,
which collectively increase fertilization rates [23, 24]. This chapter
provides a detailed protocol for mouse sperm cryopreservation as
described by Nakagata and his colleagues with some modifications.
A detailed IVF procedure using frozen-thawed mouse sperm has
recently been described by [19].
402
Yuksel Agca and Cansu Agca