85 Effects of WNT-inhibition and activin A on in vitro development and pluripotency markers of bovine haploid parthenogenetic embryos

Haploid embryonic stem cell lines (hESCs) from haploid parthenogenetic embryos (HPEs) have been obtained in mice and humans. The hESCs have unique advantages in reproductive development research, genetic screening, and targeted drug therapy that are primarily due to their differentiation potential in vitro and their phenotype being the same as their genotype. However, most bovine HPEs (bHPEs) undergo developmental arrest before reaching the blastocyst stage, which precludes their use for deriving hESCs. Therefore, the present study aimed to investigate whether supplementing the embryo culture medium at the morula stage with a GSK3B inhibitor (CHIR99021) or Activin A (AA) affects the developmental competence and pluripotency of bHPE. To do this, IVM oocytes were incubated with 5 μM ionomycin for 5 min followed by culture in cycloheximide (CHX) for 5 h to induce haploid parthenogenesis. A group of diploid parthenogenetic embryos (DPEs) was included as a control by culturing the oocytes in 6-DMAP instead of CHX for 5 h. Treatments with CHIR99021 (3 μM) and AA (20 ng mL⁻¹) were performed from Day 5 onward (5–6 morulas per group) using a serum-free culture medium (SFCM) system. Morulas cultured in 0.001% of DMSO were used as vehicle control. Embryo development was assessed at 72 h (cleavage), 120 h (morula), and 192 h (blastocyst). Morphological quality, immunostaining analysis of pluripotency markers (CDX2, SOX2, GATA2, and NANOG), and DNA fragmentation were performed at the blastocyst stage. Binomial data sets were analyzed using Fisher’s test. Differences were considered significant at P < 0.05. Figures and statistical analysis were obtained using R software (https://www.R-project.org/). The results showed that the SFCM did not affect the developmental competence or cell allocation into inner cell mass and trophectoderm of DPEs. Although there was a tendency (P = 0.106) for a higher proportion of morula to develop into blastocysts (blastulation rate) of bHPEs with AA (77%) compared with the control DMSO group (55%), all groups of bHPEs showed similar blastulation rates (range, 55%–77%) and similar levels of pluripotency as well as DNA fragmentation (P > 0.05). These data show that GSK3B inhibition and AA supplementation do not affect blastulation and expression of markers associated with embryonic pluripotency of bHPEs. Further studies will be focused on evaluating the effects of these small molecules on the derivation efficiency of hESC lines.

Grant support was provided by ANID–Fondecyt Iniciacion 11230091, Universidad de La Frontera GI24-0039.