Post‐fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs

Abstract

The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P < 0.05) level in SCNT oocytes that were treated post-fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos.

Treatment with the Proteasome Inhibitor MG132 during the End of Oocyte Maturation Improves Oocyte Competence for Development after Fertilization in Cattle

“…Beneficial effects of MG132 on nuclear remodeling, transcript abundance and embryonic development have also been shown for embryos constructed by somatic cell nuclear cloning in mice [22], [23], rats [24], [25], goats [23] and pigs [7], [26], [27]. Unlike for addition from 0–6 h, MG132 added from 16–22 h did not improve oocyte competence by improving nuclear maturation because the percentage of oocytes that were MII at the end of maturation was not affected by MG132 later in maturation.…”

Prolonged Proteasome Inhibition Cyclically Upregulates Oct3/4 and Nanog Gene Expression, but Reduces Induced Pluripotent Stem Cell Colony Formation

“…A role for the proteasome in determining pluripotency is also supported by evidence that gene expression of subunits of the 20S proteasome decreases as human ESCs undergo differentiation (Atkinson et al, 2012). Beneficial effects of MG132 on development of rat (Nakajima et al, 2008), murine (Gao et al, 2005), bovine (Le Bourhis et al, 2010;Tani et al, 2007), and porcine (You et al, 2010) somatic cell nuclear transfer (SCNT) embryos have also been documented.…”

MG132 treatment during oocyte maturation improves embryonic development after somatic cell nuclear transfer and alters oocyte and embryo transcript abundance in pigs

“…Thus, regulation of proteosomal degradation during later stages of maturation may be a useful method for improving the yield of pig embryos produced by PA and SCNT. Here, we used two endpoints to evaluate the ability of MG132 to improve oocyte competence for supporting embryonic development: The first was to measure development to the blastocyst stage, while the second measured transcript abundance for several genes because it has been reported that developmental competence of SCNT embryos is correlated with expression level of genes such as PCNA, OCT4, and DNMT1 (Lee et al, 2006; You et al, 2010a).…”

“…One specific proteasome inhibitor, MG132, can induce germinal vesicle breakdown and arrest oocytes at the metaphase‐I (MI) stage in a dose‐ and time‐dependent manner (Josefsberg et al, 2000; Chmelikova et al, 2004). In other studies on pig SCNT, a beneficial effect of MG132 on development of SCNT embryos has been shown when reconstructed oocytes were treated with MG132 at time points after fusion or activation (Whitworth et al, 2009; You et al, 2010a). It was speculated that MG132 treatment inhibited degradation of maternal proteins that were beneficial for nuclear reprogramming and embryonic development.…”