Pronuclear formation and SMARCA4 incorporation after ICSI or assisted ICSI in pig zygotes.

O BRISKI; A GAMBINI; LD RATNER; DF SALAMONE

Congreso; International Embryo Technology Society?s 47th Annual Meeting; 2021

International Embryo Technology Society

Pigs are considered an important experimental model for their biological similarities tohumans including their potential as organ donors in xenotransplantation. Unfortunately, inthis species conventional in vitro fertilization results in high polyspermic rates. ICSI avoidspolyspermy and ICSI-mediated gene edition could be a powerful technique to producegenetically modified pigs. However, ICSI is not yet efficient in pigs. Moreover, the ATP-dependent chromatin remodeler, SMARCA4, translocates to the pronuclei soon afterfertilization and its mislocalization or reduction leads to poor embryo development. The aimof 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 intensitylevels in pronuclei . First, cumulus–oocyte complexes were collected from slaughterhouseovaries and matured in vitro for 44 h. Matured and denuded oocytes were subjected to: a)ICSI (ICSI, n= 47), b) ICSI assisted by PD (ICSIp, n=21), c) ICSI assisted by electricalactivation (ICSIe, n=39) and d) electrical activation as an haploid parthenogenetic control(HAP, n=21). Presumptive zygotes were fixed for 20 min in 4% formaldehyde solution 18 hafter 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 thepresence of PN in: two PN (2-PN), one PN with the presence of a semi-condensed orcondensed sperm (1-PN) and semi-condensed or condensed sperm with no evidences ofPN (no activation). Zygotes that exhibited a different pattern were included into the “other”category. A region of interest was drawn around each PN and the average pixel intensity ofSMARCA4 was determined with ImageJ image processing software. Data was analyzed byFisher’s exact test and Kruskal-Wallis test using GraphPad software. Differences wereconsidered significant at P < 0.05. We found no significant differences in 2-PN formationrates 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 one PN (n=14, 66.67%).On the other hand, in most of the zygotes of all experimental groups, SMARCA4 was foundto be localized in both PN, being absent in polar bodies, metaphase plate or condensedsperm. 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 ofthe ICSI zygotes (n=18, 72%) showed a similar SMARCA4 intensity level between PN. Inconclusion, our results suggest that neither the use of piezo drill or electrical activationimproves PN formation or SMARCA4 pattern. It remains to be determined if the asymmetriclevels of SMARCA4 between PN, observed in some zygotes, could be associated with alower embryo developmental competence.