Improvement of the developmental competence of bovine somatic cell nuclear transfer embryos using latrunculin A during activation

G VANS LANDSCHOOT; V SAVY; LD RATNER; V ALBERIO; DF SALAMONE

Nueva York

Congreso; International Embryo Technology Society?s 46th Annual Meeting; 2020

International Embryo Technology Society

Somatic cell nuclear transfer (SCNT) is an assisted reproductive technology with potential for its application in agriculture, biomedicine, andbiotechnology. However, the SCNT efficiency is low. Failure in embryo production by SCNT could be associated mainly with chemical activationtreatments or the donor cell type. In this context, we compare the use of latrunculin A (LatA), instead of cytochalasin B during the activation withroscovitine (Rosco), versus the treatment of donor cells with demecolcine (D-cells) followed by activation just with Rosco to compare cloningefficiency. The aim of this study was to evaluate the in vitro developmental competence as well as the gene expression pattern of key genes (CDX2,OCT4, SOX2, and NANOG) in blastocysts obtained from the two treatments. To do this, cumulus-oocyte complexes were collected from cow ovariesobtained from slaughterhouses and were IVM for 21 h. After cumulus-cell removal, enucleation was performed as described by Gambini et al. (2014PLoS ONE 9, e110998; https://doi.org/10.1371/journal.pone.0110998). The G0/G1 cells or D-cells were fused to the oocytes. For activation,reconstructed zygotes were treated with 5 mMionomycin for 4 min followed by 5-h incubation into different randomly activation groups: D-cells þ50 mMRosco (SCNT-Demec), G0/G1 cells þ 50 mMRosco/10 mMLatA (SCNT-LatA), and G0/G1 cells þ 50 mMRosco/5 mgmL1 cytochalasin B(SCNT-Ctrol). Parthenogenetic controls were also included: Part-Demec, Part-LatA, and Part-Ctrol. Activated oocytes were cultured in syntheticoviductal fluid with amino acids medium until blastocyst stage. Rates of cleavage, morulae, and blastocysts were evaluated at Days 2, 5, and 7 ofin vitro culture, respectively. Relative abundance of mRNA coding for the four genes was compared between SCNT-Demec, SCNT-LatA, SCNTCtrol,and IVF groups by RTqPCR. Data was analysed by Fisher?s exact test for in vitro culture (P,0.05) or by one-way analysis of variancefollowed by Tukey post-hoc test (P¼0.05). Cleavage rates from SCNT-Demec (n¼247, 88%) and SCNT-LatA (n¼112, 88%) were significantlyhigher than those from SCNT-Ctrol (n¼123, 76%; Table 1). However, higher blastocyst rates were observed for the SCNT-LatA (n¼112, 29%)group than for SCNT-Demec (n¼247, 10%) and SCNT-Ctrol (n¼123, 14%) (P,0.05). No differences were found for the relative abundance ofmRNAs coding for SOX2 and CDX2 between all groups. The NANOG expression was significantly decreased in SCNT-Ctrol and SCNT-LatAcompared with IVF embryos (P,0.05). The SCNT-Demec group did not differ from IVF embryos, and OCT4 expression analysis showed nodifference among groups. In conclusion, LatA activation improved significantly blastocyst rates, whereas it did not affect gene expression whencompared with IVF embryos. Our results suggest that this group could improve full-term developmental efficiency of SCNT embryos