5 NEUTRAL SEGREGATION OF DONOR CELL MITOCHONDRIA IN FETAL AND ADULT TISSUES OF SOMATIC CELL CLONES IN CATTLE

Abstract

Until now, animal cloning has been extremely inefficient: only 1–2% of nuclear transfer (NT) clones survive to birth. Some of these anomalies may be related to an incompatibility between nuclear and mitochondrial genes (Cummins JM 2001 Hum. Reprod. Update 7, 217–228). Controversy exists as to the levels of donor cell mitochondrial DNA (mtDNA) inheritance in somatic clones (heteroplasmy). Whereas some researchers found very low quantities (0.1–4%) (Steinborn R et al. 2000 Nat. Genet. 25, 255–257), others found levels of heteroplasmy ranging from 6 to 40% (Takeda et al. Mol. Reprod. Dev. 64, 429–437). Since it remains unclear whether mtDNA segregation is neutral or selective, the purpose of this study was to analyze the transmission of the mtDNA from donor somatic cells in fetal and adult clones using a particular mtDNA marker (mtDNA Bos taurus with one mutation in the D-loop of 40 base pairs plus than the wild type). Fibroblasts from a fetus of 60 days were used as donor cells. The fetus was produced by artificial insemination of a Holstein (Bos taurus) heifer carrying an mtDNA mutation with semen from a Zebu (Bos indicus) bull. Oocytes derived from slaughterhouse ovaries of Holstein cows carrying wild-type mtDNA were used as recipient cells. The presence of the mutated mtDNA from the donor cell (heteroplasmy) was analyzed in a male cloned fetus of 60 days and in three adult male clones at 18 months of age. Heteroplasmy was detected in 7 tissues in the foetus: muscle, skin, stomach, testicle, thymus, tongue, and umbilical cord. Three tissues were analyzed from the adult clones: semen, skin, and white blood cells. Heteroplasmy was detected in all the tissues by nested PCR amplification of the D-loop and analyzed by ANOVA and Tukey-Kramer multiple comparison test. The mean (%) of the mutated mtDNA of the donor cell in the seven tissues of the 60-day-old fetus was 1.14 ± 0.34 (SEM). There was no differences in the means of heteroplasmy (%) between the tissues of the fetus (P > 0.05). The mean level of heteroplasmy in the three adult clones analyzed (clones A, B, and C) was 1.41 ± 0.18 (SEM). Analysis of heteroplasmy between the tissues of each clone showed no differences (P > 0.05) with the exception of clone B, where semen was different (P < 0.05) from white blood cells. There were significant differences (P < 0.05) between some clones (taking together all the results of all tissues of each clone). The heteroplasmy in clone B (%) (2.59 ± 0.18 SEM) was different (P < 0.05) from that of both clone A (1.04 ± 0.18) and clone C (1.46 ± 0.18). There was no difference between the heteroplasmy (%) of clone A and that of clone C (P > 0.05). These results show that the tissues of the fetus and the adult clones were heteroplasmic at similar levels, suggesting neutral segregation of the donor cell mtDNA during development and tissue differentiation.