81 Pronuclear formation and SMARCA4 incorporation after intracytoplasmic sperm injection (ICSI) or assisted ICSI in pig zygotes

Abstract

Pigs are considered an important experimental model for their biological similarities to humans, including their potential as organ donors in xenotransplantation. Unfortunately, in this species conventional invitro fertilization results in high polyspermic rates. ICSI avoids polyspermy and ICSI-mediated gene edition could be a powerful technique to produce genetically modified pigs. However, ICSI is not yet efficient in pigs. Moreover, the ATP-dependent chromatin remodeller, SMARCA4, translocates to the pronuclei soon after fertilization and its mislocalization or reduction leads to poor embryo development. The aim of this study was to assess whether assisted activation or the use of the piezo drill (PD) during ICSI improves pronuclear (PN) formation rates and to analyse SMARCA4 intensity levels in pronuclei. First, cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured invitro for 44h. Matured and denuded oocytes were subjected to (1) ICSI (n=47), (2) ICSI assisted by PD (ICSIp, n=21), (3) ICSI assisted by electrical activation (ICSIe, n=39), and (4) electrical activation as an haploid parthenogenetic control (HAP, n=21). Presumptive zygotes were fixed for 20min in 4% formaldehyde solution 18h after injection or activation and incubated with SMARCA4 antibody (1:100) and Alexa Fluor (1:1000) as a secondary antibody. Then, the zygotes were classified according to the presence of PN in 2 PN (2-PN), 1 PN with the presence of a semi-condensed or condensed sperm (1-PN), and semi-condensed or condensed sperm with no evidence of PN (no activation). Zygotes that exhibited a different pattern were included in the “other” category. A region of interest was drawn around each PN and the average pixel intensity of SMARCA4 was determined with ImageJ image processing software. Data were analysed by Fisher’s exact test and Kruskal–Wallis test using GraphPad software (GraphPad Inc.). Differences were considered significant at P<0.05. We found no significant differences in 2-PN formation rates among groups after ICSI (ICSI n=16, 34.04%; ICSIe n=10, 25.64%; ICSIp n=6, 28.57%). As expected, the majority of the HAP zygotes exhibited 1 PN (n=14, 66.67%). In contrast, in most of the zygotes of all experimental groups, SMARCA4 was found to be localised in both PN, being absent in polar bodies, metaphase plate, or condensed sperm. Interestingly, out of the total 2-PN porcine ICSI zygotes of all experimental groups (n=25), 7 zygotes (28%) showed clear asymmetric intensity levels between PN. The rest of the ICSI zygotes (n=18, 72%) showed a similar SMARCA4 intensity level between PN. In conclusion, our results suggest that neither the use of piezo drill or electrical activation improves PN formation or SMARCA4 pattern. It remains to be determined whether the asymmetric levels of SMARCA4 between PN observed in some zygotes could be associated with a lower embryo developmental competence.