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

Abstract

Somatic cell nuclear transfer (SCNT) is an assisted reproductive technology with potential for its application in agriculture, biomedicine, and biotechnology. However, the SCNT efficiency is low. Failure in embryo production by SCNT could be associated mainly with chemical activation treatments or the donor cell type. In this context, we compare the use of latrunculin A (LatA), instead of cytochalasin B during the activation with roscovitine (Rosco), versus the treatment of donor cells with demecolcine (D-cells) followed by activation just with Rosco to compare cloning efficiency. The aim of this study was to evaluate the invitro 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 ovaries obtained from slaughterhouses and were IVM for 21h. After cumulus-cell removal, enucleation was performed as described by Gambini et al. (2014 PLoS 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 5μM ionomycin for 4min followed by 5-h incubation into different randomly activation groups: D-cells + 50μM Rosco (SCNT-Demec), G0/G1 cells + 50μM Rosco/10μM LatA (SCNT-LatA), and G0/G1 cells + 50μM Rosco/5μgmL−1 cytochalasin B (SCNT-Ctrol). Parthenogenetic controls were also included: Part-Demec, Part-LatA, and Part-Ctrol. Activated oocytes were cultured in synthetic oviductal fluid with amino acids medium until blastocyst stage. Rates of cleavage, morulae, and blastocysts were evaluated at Days 2, 5, and 7 of invitro culture, respectively. Relative abundance of mRNA coding for the four genes was compared between SCNT-Demec, SCNT-LatA, SCNT-Ctrol, and IVF groups by RTqPCR. Data was analysed by Fisher's exact test for invitro culture (P<0.05) or by one-way analysis of variance followed by Tukey post-hoc test (P=0.05). Cleavage rates from SCNT-Demec (n=247, 88%) and SCNT-LatA (n=112, 88%) were significantly higher than those from SCNT-Ctrol (n=123, 76%; Table 1). However, higher blastocyst rates were observed for the SCNT-LatA (n=112, 29%) group than for SCNT-Demec (n=247, 10%) and SCNT-Ctrol (n=123, 14%) (P<0.05). No differences were found for the relative abundance of mRNAs coding for SOX2 and CDX2 between all groups. The NANOG expression was significantly decreased in SCNT-Ctrol and SCNT-LatA compared with IVF embryos (P<0.05). The SCNT-Demec group did not differ from IVF embryos, and OCT4 expression analysis showed no difference among groups. In conclusion, LatA activation improved significantly blastocyst rates, whereas it did not affect gene expression when compared with IVF embryos. Our results suggest that this group could improve full-term developmental efficiency of SCNT embryos. Table 1.Cleavage, morulae, and blastocyst rates among the groups Group1 Treatment n Cleavage (%) Morulae (%) Blastocyst (%) SCNT-Demec Rosco 247 218 (88,26)b 41 (16,60)a 24 (9,72)a SCNT-Ctrol CB/Rosco 123 93 (75,61)a 25 (20,33)a 17 (13,82)a SCNT-LatA LatA 10μM/Rosco 112 99 (88,39)b 42 (37,50)b 33 (29,46)bc Part-Demec Rosco 141 133 (94,33)b 35 (24,82)a 20 (14,18)a Part-Ctrol CB/Rosco 84 76 (90,48)b 22 (26,19)ab 19 (22,62)ab Part-LatA LatA 10μM/Rosco 73 67 (91,78)b 30 (41,10)b 28 (38,36)c a-cStatistical differences between treatments (Fisher's test P<0.05). 1Treatments: donor cells with demecolcine (D-cells) + 50μM roscovitine (Rosco) (SCNT-Demec), G0/G1 cells + 50μM Rosco/10μM latrunculin A (LatA) (SCNT-LatA), and G0/G1 cells + 50μM Rosco/5μgmL−1 cytochalasin B (CB) (SCNT-Ctrol). Parthenogenetic controls were also included: Part-Demec, Part-LatA, and Part-Ctrol.