28 EXPRESSION OF MHC CLASS I IN THE PLACENTA AND THE LYMPHOCYTES OF BOVINE SOMATIC CLONES

Abstract

A major limitation for the development of somatic cloning in cattle is the low efficiency for producing live offspring because only 5 to 25% of the reconstructed embryos reach full-term development, with variation between the genotypes. Hill et al. (2002 Biol. Reprod. 67, 55–63) reported that early fetal losses were due to inappropriate expression of trophoblast major histocompatibility complex Class 1 (MHC1) antigens on the placenta of bovine somatic clones. This abnormal expression was present in all clones, with more cells expressing MHC1 in those with growth retardation. These clones, however, originated from only one genotype, and only one antibody recognizing a monomorphic determinant of bovine MHCI was used. In the present study, MHC1 expression was examined throughout gestation in bovine clone placentas originating from 3 different genotypes. Placentas were collected at Days 32, 62, 180, 260, and 280 (term) in clones (n = 8) and in controls obtained by artificial insemination (n = 6). Four different monoclonal antibodies that recognize different determinants of MHC1 molecules (ILA-A88, IL-A19, H58A, and the β2-microglobulin, i.e. the MHC1 light chain) were used. Results showed MHC1 expression in the maternal tissue but not on the fetal side, regardless of group, gestational age, or fetal growth, although patchy expression of β2-microglobulin could be found in both clones and controls in the fetal mesenchyme in some but not all samples. In conclusion, MHC1 protein expression appears normally down-regulated in the placenta of clones, but the antibodies used do not discriminate between classical and non-classical expression. The mRNA expression of classical and non-classical MHC1 genes is currently being explored. These results cast doubt on the hypothesis that the observed fetal losses would be the result of an immunologic rejection due to inappropriate expression of some placental MHC1 genes. Although early observations suggest that immune functions appear normal (Lanza et al. 2001 Science 294, 1893–1894; Chavatte-Palmer et al. 2006 Reprod. Fert. Dev. 18, 121 abst), immunological deficiencies have been reported in some bovine somatic clones after birth (Renard et al. 1999 The Lancet 353, 1489–1491), and they may be related to an abnormal expression of MHC1 genes. We therefore determined the MHC1 haplotype of cell donors from 4 genotypes and 3 of their healthy clones using the Reference Strand Conformation Analysis method with DNA extracted from lymphocytes. Results showed that the MHC1 type was identical, as could be expected in clones. Animals that died of immune deficiencies, however, may have had abnormal MHC1 expression. Further work is therefore being performed on tissues obtained postmortem from cloned animals having been diagnosed with thymic atrophy.