ICSI-Mediated Gene Transfer Skews Sex Ratio Against Female
Births in Mice
1Moreira
1Pintado
2Montoliu
1Gutiérrez-Adán
P.N.,
B.,
L.,
A.
1Dpto. de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Madrid, Spain
2Dpto. de Biología Molecular y Celular, CNB, Madrid, Spain
• Intracytoplasmic Sperm Injection (ICSI) Mediated
Gene Transfer has been used as an alternative
method to pronuclear microinjection for the genomic
modification of many species.
• With this method, transgenic embryos are produced
by the microinjection of metaphase II oocytes with
spermatozoa previously incubated with foreign DNA.
• Recently it was shown that, the low percentage of
transgenic animals produced from injected oocytes
results from the fact that the expression of foreign
DNA is associated with paternal chromosome
degradation [1], and since it is also known, that sex
chromosomes localize preferentially on the periphery
of the sperm nucleus on sub-acrosomal regions [2],
this suggests that:
• CD1 mice were used as donors of oocytes and sperm for
ICSI-Mediated Gene Transfer experiments.
• Animals were 6-8 weeks old at the time of the experiments.
• Female mice were superovulated with 5 IU of pregnant
mare’s serum (PMS) followed by equivalent dose of human
chorionic gonodotropin (hCG) 48 h later.
• Metaphase II (MII) oocytes with 14 h post-hCG
administration, and
epididymal spermatozoa were collected as previously
described [4].
• M2 medium was used as sperm freezing media.
• 70-100µl aliquots of sperm suspension was transferred into
labeled 1ml cryogenic vials. Each vial was tightly capped
and placed into liquid nitrogen without complete immersion
to avoid internalization of liquid nitrogen. Sperm samples
were subsequently stored for periods ranging from 1 day to
4 weeks at –75°C.
• Sperm samples were thawed at room temperature.
• Mouse ICSI with frozen-thawed spermatozoa was performed
as previously described [1].
• Embryos were cultured in vitro in KSOM (Specialty Media)
until the 2-cell stage and transferred to pseudopregnat CD1
female mice previously mated with vasectomized males of
the same strain.
Hypothesis
Transfer of EGFP and YRT3 alters
Sex Ratio of ICSI Offspring
Objective
To determine if ICSI-Mediated Gene Transfer
skews sex ratio of the offspring
Background
A high level of interaction of sex
chromosomes with foreign DNA molecules
may occur during ICSI-Mediated Gene
Transfer with possible impact on the sex
ratio of the offspring.
Materials and
Methods
• In order to test our hypothesis we have compared
ICSI (no DNA), with ICSI-mediated EGFP (5 Kb
plasmid DNA from Clonetech, Spain) transfer, with
ICSI-mediated YRT3 (a mouse tyrosinase gene
derivative YAC-DNA with 100 Kb; [3]) transfer.
Sex Ratio of the offspring obtained with ICSI, ICSImediated EGFP transfer, and ICSI-mediated YRT3
transfer
Technique
([DNA] in ng/µl)
ICSI (0)
ICSI-EGFP (4-8)
ICSI-YAC (0.2-8)
a,bValues
Embryos Offspring (%) Sex Ratio (? /? )
Transferred
98
223
659
21 (21.4)
22 (9.9)
52 (7.9)
0.43a (9? /12? )
0.64b (14? /8? )
0.65b (34? /18? )
with different superscripts are significantly different (P < 0.05)
Discussion
• Forty three percent of males were obtained with
regular ICSI, while 64% and 65% were the
respective percentages when EGFP or YRT3
DNA was coinjected with spermatozoa.
• This statistically significant (P<0.05, z-test,
Sigma Stat, Jandel Scientific, U.S.A.) sex ratio
deviation, favoring male ICSI offspring when
foreign DNA is coinjected, may result from a
higher female embryo susceptibility to parental
sex chromosome fragmentation induced by the
interaction with foreign DNA molecules.
• Possible impairment of X chromosome
inactivation and dosage compensation resulting
from the fragmentation of the sex chromosome
on X-carrying spermatozoa could explain this
female embryo degeneration.
• Supporting this view, it was recently shown in
mice, that sex ratio can be skewed against
female births by a mutation in a single gene of
the X chromosome (Tsix) involved in such
mechanisms [5].
Conclusion
Mouse ICSI-mediated gene transfer
induces sex ratio deviation favoring male
offspring
References
[1]
Szczygiel, M.A. et al. (2003) Expression of foreign DNA is associated with
paternal chromosome degradation in intracytoplasmic sperm injection-mediated
transgenesis in the mouse.
Biol Reprod 68 (5), 1903-1910
[2]
Sbracia, M. et al. (2002) Preferential location of sex chromosomes, their
aneuploidy in human
sperm, and their role in determining sex chromosome
aneuploidy in embryos after ICSI.
Hum Reprod 17 (2), 320-324
[3]
Montoliu, L. et al. (1996) A locus control region at -12 kb of the tyrosinase gene.
Embo J 15 (22), 6026-6034
[4]
Wakayama, T. et al. (1998) Production of normal offspring from mouse oocytes
injected with
spermatozoa cryopreserved with or without cryoprotection. J Reprod
Fertil 112 (1), 11-17.
[5]
Lee, J.T. (2002) Homozygous Tsix mutant mice reveal a sex-ratio
distortion and revert to random X-inactivation.
Nat Genet 32 (1), 195-200
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