Differences in gene expression patterns between somatic cell nuclear transfer embryos constructed with either rabbit granulosa cells or their derivatives.
Successful production of offspring by somatic cell nuclear transfer (SCNT) is affected by the nature of the donor cells used. The purpose of this study was to determine whether characteristic changes induced in donor cells by culture conditions influenced gene expression patterns in the resultant SCNT embryos. Rabbit granulosa cells (rGC) were cultured under different conditions, either with or without hCG, and the two derivative cell types obtained (named respectively cGC+ and cGC-) were used as donor cells for SCNT. There were characteristic differences between fresh rGC and the two derivative cell types: p450scc expression and progesterone secretion were both higher in cGC+ than in cGC-; expression of bmp4 and fgfr2 was decreased in cGC+ and cGC- compared with rGC; and cGC+ and cGC- cell types gained collagenIV expression. Use of fresh rGC, or cGC+ and cGC- derivative cells, did not alter either the developmental potencies of SCNT oocytes or cell numbers at the blastocyst stage. The expression patterns of four genes (bmp4, fgfr2, gata4, oct3/4) in SCNT embryos and in fertilized embryos were analyzed by quantitative RT-PCR. We found that oct3/4 was expressed in all embryos. The expression patterns of the other three genes showed considerable variation between the different types of embryo: bmp4 was found in most fertilized embryos but only some of rGC and none of cGC+ and cGC- derived SCNT embryos; fgfr2 was present in fertilized embryos but was present in some rGC and cGC- NT embryos and in all cGC+ NT embryos; gata4 was not expressed in fertilized embryos but was present in a few rGC and cGC+ NT embryos and in most cGC- NT embryos. Our results suggest that the gene expression patterns in SCNT embryos derived from granulosa donor cells are affected by characteristic changes to the cells during in vitro culture.
Inoue
Fukashi
F
Development and Differentiation Laboratory, Developmental Biology Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. fukashi@affrc.go.jp
Matsuda
Junichi
J
Ohkoshi
Katsuhiro
K
Furusawa
Tadashi
T
Takahashi
Seiya
S
Sasada
Hiroshi
H
Sato
Eimei
E
Tokunaga
Tomoyuki
T
D000818:Animals; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D046149:Embryo Culture Techniques / Q000379:methods / Q000662:veterinary*; D004624:Embryo Transfer / Q000662:veterinary; D047108:Embryonic Development; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006107:Granulosa Cells / Q000378:metabolism* / Q000502:physiology; D053652:Nuclear Transfer Techniques; D011817:Rabbits / Q000196:embryology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary
D006063:Chorionic Gonadotropin