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Hum. Reprod. Advance Access originally published online on June 12, 2009
Human Reproduction 2009 24(9):2255-2263; doi:10.1093/humrep/dep213
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© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

DNA methylation patterns of spermatozoa and two generations of offspring obtained after murine spermatogonial stem cell transplantation

E. Goossens1,5, M. De Rycke1,2, P. Haentjens3 and H. Tournaye1,4

1 Research Laboratory for Embryology and Genetics, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium 2 Centre for Medical Genetics, UZ Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium 3 Centre for Outcomes Research and Laboratory for Experimental Surgery, UZ Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium 4 Centre for Reproductive Medecine, UZ Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium

5 Correspondence address. Tel: +32-2-477-46-44; Fax: +32-2-477-66-92; E-mail: ellen.goossens{at}uzbrussel.be

BACKGROUND: Apart from its use in research, spermatogonial stem cell transplantation (SSCT) may have important clinical applications. This controlled study aimed at evaluating the safety of SSCT by analyzing the DNA methylation pattern of Igf2, Peg1 and {alpha}-Actin both in spermatozoa and live born offspring obtained after SSCT in mice.

METHODS: Testicular cell suspensions were transplanted to the testes of genetically sterile WW recipients. Transplanted males were mated with fertile females and their first and second generation offspring were examined and compared with controls with respect to weight, length and DNA methylation patterns. Sodium-bisulfite treated genomic DNA extracted from post-transplantation spermatozoa, liver, kidney and placenta of first and second generation offspring was PCR-amplified to obtain Igf2, Peg1 and {alpha}-Actin gene fragments. Pyrosequencing was used to individually quantify the resulting artificial C/T sequence variation at CpG sites.

RESULTS: First and second generation offspring developed normally with their length and weight not being different from controls. Also the DNA methylation patterns of Igf2, Peg1 and {alpha}-Actin were not different among controls and first and second generation offspring after SSCT.

CONCLUSIONS: SSCT between syngenic individuals was not associated with changes in fetal development nor with differences in the DNA methylation patterns of Igf2, Peg1 and {alpha}-Actin in spermatozoa or other tissues from two subsequent generations of offspring obtained after SSCT.

Key words: DNA methylation/offspring/safety/spermatozoa/spermatogonial stem cell transplantation

Submitted on February 3, 2009; resubmitted on May 12, 2009; accepted on May 20, 2009.


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