Influence of oocyte selection, activation with a zinc chelator and inhibition of histone deacetylases on cloned porcine embryo and chemically activated oocytes development

Abstract

Summary The aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

Strategies to Improve the Efficiency of Somatic Cell Nuclear Transfer

“…Scriptaid supplementation in IVC media promoted a two-fold increase in the blastocyst rate of cloned canine embryos [120]. Scriptaid also significantly increased the total cell number of cloned porcine embryos at the blastocyst stage [121]. The supplementation of Bufexamac, an HDACi in porcine IVM media, had no significant effect on the maturation rate, but it increased the histone H3 lysine 9 (H3K9), histone H3 lysine 14 (H3K14), and histone H4 lysine 8 (H4K8) acetylation levels of porcine oocytes.…”

Monitoring swine virus transmission in embryos derived from commercial abattoir oocytes

“…After 24 h, the oocytes were washed and transferred to 500 μL wells of fresh OMM (OMM-2 for sampling) for 18 h. After use, a 3 mL sample of OMM-1 and OMM-2 was collected for diagnostic testing. After 18 h, 60 oocytes were separated and submitted for diagnostic testing, and the rest of the matured oocytes (n = 200) were chemically activated, as previously described, to produce parthenogenic embryos (17). In brief, oocytes were exposed to 5 μM ionomycin HEPESbuffered medium supplemented with bovine serum albumin and incubated for 4 h, followed by a culture in PZM-3 medium for 7 days (17).…”

“…Parthenogenic embryos can increase the number of embryos for evaluation without completing the complicated SCNT reconstruction process (17,18). Parthenogenic embryos are unfertilized oocytes activated into embryos that cannot progress beyond the early developmental stages and do not produce full-term pregnancies (17,18).…”

“…Parthenogenic embryos can increase the number of embryos for evaluation without completing the complicated SCNT reconstruction process (17,18). Parthenogenic embryos are unfertilized oocytes activated into embryos that cannot progress beyond the early developmental stages and do not produce full-term pregnancies (17,18). Parthenogenic embryos may allow a more accessible alternative to SCNT cloning when screening the potential viral risk of oocytes to embryo reconstruction by increasing the number of embryos available for diagnostic testing.…”

Interleukin 17D Enhances the Developmental Competence of Cloned Pig Embryos by Inhibiting Apoptosis and Promoting Embryonic Genome Activation

“…So far, a broad spectrum of research has characterized the biological, molecular and epigenetic determinants of mammalian SCNT embryo development. The afore-indicated determinants involve: (1) the origin of nuclear donor cells [ 10 , 11 , 12 , 13 , 14 ], (2) the quality of nuclear recipient oocytes determined by the parameters related to meiotic, epigenomic and cytoplasmic maturity [ 15 , 16 ], (3) the methods used to stimulate the embryo-specific developmental program of enucleated oocytes [ 17 , 18 , 19 , 20 ], (4) the incidence of programmed cell apoptosis in in vitro-cultured nuclear donor cells and cloned embryos [ 21 , 22 , 23 ], (5) the intergenomic communication between nuclear DNA and mitochondrial DNA in cloned embryos [ 24 , 25 , 26 , 27 , 28 , 29 ], and (6) the capacity of donor cell nuclei to be reprogrammed in cloned embryos [ 30 , 31 , 32 , 33 ].…”