51 PROTEOMICS ANALYSIS OF PLACENTOMEGALY IN CLONED MICE

Abstract

A variety of mammalian species have been cloned during the past few years. However, the success rate of somatic cell nuclear transfer in animals has been extremely low with many problems. Particularly, placentomegaly is a frequent finding in cloned mice and cattle (Wakayma et al. 1999 PNAS USA 96, 14 984–14 989; Niemann et al. 2000 Theriogenology 53, 21–34). To assess protein expression profile in the placentomagaly of cloned mice produced by nuclear transfer of embryonic stem cells, we have used global proteomics approach by 2-D gel electrophoresis and mass-spectrometry with the differential protein patterns using the 3 placentae of cloned mice and 4 normal mouse placentae. Proteins within isoelectric point range pH 4.0~9.0 and molecular weight range of 20–100 kDa separately were analyzed by 2-D gel electrophoresis with 3 replications of each sample. A total of approximately 3500 spots were detected 2-D gel. In the comparison of normal and cloned placenta samples, a total of 49 protein spots were expressed differentially, of which 28 spots were up-regulated proteins including alpha-fetoprotein, aspartyl aminopeptidase, placental lactogen 2, tissue inhibitor of metalloproteinase 2 (TIMP-2), etc., and 21 spots were down-regulated proteins including peroxiredoxin 6, creatine kinase, pre-B-cell colony-enhancing factor 1 (PBEF), etc. Eight spots could not be identified. One of differentially up-regulated proteins in cloned mouse placenta was identified as TIMP-2 protein that is related to extracellular matrix degradation and tissue remodeling processes. Western blot was performed with placental sample used in the 2-D gel electrophoresis analysis and normal mouse placenta samples on Days 11.5 to 18.5. Indeed, Western blot analysis confirmed a significant increase of TIMP-2 protein level in cloned mouse placenta compared with normal. The expression levels of TIMP-2 in normal mouse placenta were highest at the normal mid gestation (Day 13.5) and exhibited prominent decrease at late gestation period during normal pregnancy. However, the expression levels of TIMP-2 in placenta of cloned mice appeared to be similar to levels of mid gestation normal mouse placenta. And one of down-regulated protein in NT placenta was identified as PBEF protein that is known to be related to induction of spontaneous labor. PBEF protein level in cloned mice placenta was revealed to decrease remarkably compared with normal. The expression levels of PBEF in normal mice placenta exhibited a gradual decrease between Day 11.5 and Day 18.5 without complete depletion. However, the expression levels of PBEF in placenta of cloned mice appeared to be markedly lower than those of the same day normal placenta. In conclusion, abnormal expression of placental proteins associated with tissue remodeling and labor induction may be the cases of placentomegaly in cloned mice.