Effects of combined treatment of MG132 and scriptaid on early and term development of porcine somatic cell nuclear transfer embryos.
Although improving, the efficiency of producing offspring by somatic cell nuclear transfer (SCNT) is still low (<1.5%). Our laboratory has demonstrated that histone deacetylase inhibitor (Scriptaid) treatment of reconstructed embryos enhances blastocyst formation and cloning efficiency in pigs. It has also been shown that proteasomal inhibitor MG132 treatment for 2 h after activation of oocytes increases blastocyst rate and pregnancy rate. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early embryo development in vitro and on term development in vivo. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10 μM MG132 for 2 h in the presence of 500 nM Scriptaid, washed and treated with Scriptaid for an additional 14 to 15 h, then cultured in porcine zygote medium 3 (PZM3) until day 6. There was no difference in percent cleavage (58.1 ± 7.2%, 62.7 ± 7.2%, and 62.5 ± 7.2%) on day 2, or total cell number (23.1 ± 2.2, 24.0 ± 2.0, and 24.5 ± 2.3 for the 0, 1, and 10 μM MG132 groups, respectively) on day 6 among the three groups. Interestingly, there was no difference in percentage of blastocysts between the 0 (18.5±4.7%) and 1 (25.1 ± 4.7%) μM MG132 treatment groups; however, compared with the 10 μM MG132 group (14.0 ± 4.7%), more embryos from the 1 μM MG132 group developed into blastocysts (p<0.05). To determine the effects on term development in vivo, two MG132 groups were included (0 and 1 μM MG132), and embryos were treated as above and transferred into synchronized surrogates after treatment. There was no difference in the oocyte-donor cell fusion rate, number of embryos transferred, pregnancy rate at days 28, 60, and at term, pigs delivered per embryo transfer, litter size, body weight at birth, nor cloning efficiency between the Scriptaid-alone control and MG132+Scriptaid combined groups. In summary, the combined treatment of MG132 and Scriptaid did not improve term development compared to Scriptaid treatment alone.
National Swine Resource and Research Center, University of Missouri, Columbia, MO 65211, USA.
Jiude
J
Mao
Kimberly
K
Tessanne
Kristin M
KM
Whitworth
Lee D
LD
Spate
Eric M
EM
Walters
Melissa S
MS
Samuel
Clifton N
CN
Murphy
Lisa
L
Tracy
Jianguo
J
Zhao
Randall S
RS
Prather
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004624:Embryo Transfer; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006898:Hydroxylamines / Q000494:pharmacology*; D007976:Leupeptins / Q000494:pharmacology*; D053652:Nuclear Transfer Techniques; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D011804:Quinolines / Q000494:pharmacology*; D013552:Swine
D056572:Histone Deacetylase Inhibitors; D006898:Hydroxylamines; D007976:Leupeptins; D061988:Proteasome Inhibitors; D011804:Quinolines; C410733:scriptaid; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde