Itially imprinted from the paternal allele at the 4?-cell stage, but is detectable in bovine, porcine, and human preimplantation embryos from the 8to 16-cell stage onward [27,37,38]. Thus, in other mammals, including pigs, the onset of XCI appears to begin later than in mice. Taken together, these data suggest that in female porcine embryos in which the XIST gene may not be functional, XCI, as a dosage-compensation mechanism, does not occur until the blastocyst stage. Gene expression profiles have been reported to differ both among and within individual clones with Emixustat (hydrochloride) web different types of donorTable 1. In Vitro Development of the Male and Female Tartrazine Cloned Embryos.No. Cleaved ( ){ 328 188 194 164 137 172 183 171 191 (62.464.6) (58.564.9) (61.262.6) (55.664.2) (53.963.8) (55.462.5) (59.163.5) (57.865.1) (58.862.7)Group IVF NT F F1 2 1No. reconstructed 562 328 329 294 260 312 310 296 328 (n = 8) (n = 7) (n = 8) (n = 6) (n = 5) (n = 7) (n = 8) (n = 6) (n = 6)No. blastocyst ( of cleaved) 161 53 46 34 48 71 90 63 85 (32.764.7) (16.161.8) (14.561.8) (11.362.2) (18.161.6) (22.961.7) (30.763.7) (21.465.4) (26.162.5)a c d d b b a b aM M NTSc {F1 F2 M M1{ {a2dthe cleavage rate was counted after 2 days in culture. Sc; scriptaid, which were treated for 14 hr after post-activation. Values with different superscripts within each column are significantly different, P,0.05. doi:10.1371/journal.pone.0051398.tX-Linked Gene Transcripts in Pig BlastocystsTable 2. In Vivo Development of the Cloned Embryos.No. live offspring{ 18 11 ?Cell line F1 M1 MNo. experiments 6 6No. embryos transferred 635 719No.( ) pregnancy* 3 (50) 3 (50) 21 (40)*an initial pregnancy diagnosis was examined via ultrasonography at 27 to 30 days after embryo transfer. the fetuses were aborted from two recipients at Day 55 to 60. { all of the cloned piglets were vaginally delivered. doi:10.1371/journal.pone.0051398.tcells [39]. Also, imprinted gene expression has been shown to be variable in the placentas of deceased and surviving clone piglets [22]. Therefore, a large degree of the variability in expression, particularly in BEX1 and XIST, among individual cloned blastocysts may be attributable to abnormal epigenetic reprogramming that occurs in a random manner. Such incomplete reprogramming reflects the extremely low efficiency of SCNT. Although overall, the IVF embryos had normal ranges of expression, noticeable gene discordance occurred in a small proportion of individuals, especially in females. These differences can be interpreted as being the effects of chromosomal abnormalities that resulted from polyspermy, which usually occurs in the porcine IVF system [40]. This assumption is supported by the observation that increased expression of X-linked genes can occur as a response to both changes in ploidy and to the number of active X chromosomes (Xa) [41]. Nevertheless, we cannot rule out the possibility that female embryos may be more sensitive to environmental conditions than male embryos. Treatment of cloned embryos with histone deacetylase inhibitors (HDACi) was recently shown to improve the success rate of development to term in several species [28,42]. However, this effect may not occur in X-linked gene transcription patterns in cloned embryos with TSA treatment [19]. We found that the blastocyst rate was consistently increased in all Sc treatment groups but relatively little improvement was observed in X-linked gene expression rates, with some exceptions. For BEX1 transcription, reduced.Itially imprinted from the paternal allele at the 4?-cell stage, but is detectable in bovine, porcine, and human preimplantation embryos from the 8to 16-cell stage onward [27,37,38]. Thus, in other mammals, including pigs, the onset of XCI appears to begin later than in mice. Taken together, these data suggest that in female porcine embryos in which the XIST gene may not be functional, XCI, as a dosage-compensation mechanism, does not occur until the blastocyst stage. Gene expression profiles have been reported to differ both among and within individual clones with different types of donorTable 1. In Vitro Development of the Male and Female Cloned Embryos.No. Cleaved ( ){ 328 188 194 164 137 172 183 171 191 (62.464.6) (58.564.9) (61.262.6) (55.664.2) (53.963.8) (55.462.5) (59.163.5) (57.865.1) (58.862.7)Group IVF NT F F1 2 1No. reconstructed 562 328 329 294 260 312 310 296 328 (n = 8) (n = 7) (n = 8) (n = 6) (n = 5) (n = 7) (n = 8) (n = 6) (n = 6)No. blastocyst ( of cleaved) 161 53 46 34 48 71 90 63 85 (32.764.7) (16.161.8) (14.561.8) (11.362.2) (18.161.6) (22.961.7) (30.763.7) (21.465.4) (26.162.5)a c d d b b a b aM M NTSc {F1 F2 M M1{ {a2dthe cleavage rate was counted after 2 days in culture. Sc; scriptaid, which were treated for 14 hr after post-activation. Values with different superscripts within each column are significantly different, P,0.05. doi:10.1371/journal.pone.0051398.tX-Linked Gene Transcripts in Pig BlastocystsTable 2. In Vivo Development of the Cloned Embryos.No. live offspring{ 18 11 ?Cell line F1 M1 MNo. experiments 6 6No. embryos transferred 635 719No.( ) pregnancy* 3 (50) 3 (50) 21 (40)*an initial pregnancy diagnosis was examined via ultrasonography at 27 to 30 days after embryo transfer. the fetuses were aborted from two recipients at Day 55 to 60. { all of the cloned piglets were vaginally delivered. doi:10.1371/journal.pone.0051398.tcells [39]. Also, imprinted gene expression has been shown to be variable in the placentas of deceased and surviving clone piglets [22]. Therefore, a large degree of the variability in expression, particularly in BEX1 and XIST, among individual cloned blastocysts may be attributable to abnormal epigenetic reprogramming that occurs in a random manner. Such incomplete reprogramming reflects the extremely low efficiency of SCNT. Although overall, the IVF embryos had normal ranges of expression, noticeable gene discordance occurred in a small proportion of individuals, especially in females. These differences can be interpreted as being the effects of chromosomal abnormalities that resulted from polyspermy, which usually occurs in the porcine IVF system [40]. This assumption is supported by the observation that increased expression of X-linked genes can occur as a response to both changes in ploidy and to the number of active X chromosomes (Xa) [41]. Nevertheless, we cannot rule out the possibility that female embryos may be more sensitive to environmental conditions than male embryos. Treatment of cloned embryos with histone deacetylase inhibitors (HDACi) was recently shown to improve the success rate of development to term in several species [28,42]. However, this effect may not occur in X-linked gene transcription patterns in cloned embryos with TSA treatment [19]. We found that the blastocyst rate was consistently increased in all Sc treatment groups but relatively little improvement was observed in X-linked gene expression rates, with some exceptions. For BEX1 transcription, reduced.
