P-797 Benefit of Replicating Mammalian Gametes

Abstract

Abstract Study question Can haploid parthenogenetic and androgenetic embryos generate haploid embryonic stem cells (haESCs) and function as respective oocytes and spermatozoa? Summary answer Haploid embryos generated blastocysts capable of yielding haESCs. Haploid blastomeres of both sexes served as multiple copies of gamete substitutes. What is known already Although creating diploid digyneic or dispermic embryos through nuclear transfer is possible, the conceptuses rarely achieve normal preimplantation development due to unbalanced epigenomes, demonstrating the requirement of both parental epigenomes in the course of embryogenesis. Haploid parthenogenetic embryos can be achieved by inducing calcium oscillations of a metaphase II oocyte, while the paternal counterpart can be generated through the insemination of enucleated oocytes. These embryos may serve to generate haESCs for heterozygosity identification and can be used for reproductive purposes in animal models. Study design, size, duration To generate parthenogenetic embryos, metaphase II oocytes from B6D2F1 mice were artificially activated. Androgenetic embryos were generated from the insemination of enucleated oocytes. Haploid embryos were cultured to blastocysts and seeded on feeder cells (MEF) to derive haESCs. Haploid blastomeres from 8-cell embryos of either sex were isolated and used as artificial gametes; artificial oocytes were generated by grafting a parthenogenetic blastomere to an enucleated recipient oocyte. Androgenetic blastomeres were used as male gametes. Participants/materials, setting, methods Parthenogenetic embryos were generated by exposing metaphase II oocytes to calcium ionophore. Another cohort of B6D2F1 oocytes were enucleated and underwent piezo-ICSI to generate androgenetic embryos. Sendai-virus–mediated nuclear transfer was performed using parthenogenetic blastomeres as donor nucleuses to generate artificial oocytes and subsequently fertilized. Androgenetic blastomeres were used as sperm substitutes by fusion with an intact oocyte to generate zygotes. Control embryos were generated by piezo-actuated ICSI. Developmental characteristics were recorded by time-lapse microscopy. Main results and the role of chance A total of 129 oocytes were parthenogenetically activated by calcium ionophore, and 119 developed a female pronucleus. Androgenetic embryos were generated from the insemination of 375 ooplasts; 317 developed a male pronucleus. The monopronuclear appearance of haploid embryos of both sexes was comparable to the fertilization of control at 90.7%. Haploid embryo development for both sexes up to the 4-cell stage matched the control. However, from the 8-cell stage onward, parthenogenetic and androgenetic embryos cleaved at a lower rate compared to the control at 76.4%, 59.3% and 92.7% (P < 0.0001), respectively. The haploid genome affected the rate of compaction at 73.6%, 55.3%, and 89.2% (P<0.0001), respectively, and the rate of blastulation at 27.8%, 11.2%, and 80.8% (P<0.00001) respectively. Androgenetic embryos showed delayed cleavage from the 4-cell stage onward, while the parthenogenetic counterparts had similar morphokinetics to the control. A total of 20 parthenogenetic and 18 androgenetic blastocysts were plated on MEF, yielding 5 parthenogenetic and 3 androgenetic haESC lines. Both haESCs yielded beating cardiomyocytes on day 10 of differentiation while maintaining haploidy. The utilization of haploid pseudo blastomeres as gametes yielded 93 reconstructed biparental embryos that resulted in 71 blastocysts (76.3%). Time-lapse morphokinetics were comparable to the control. Limitations, reasons for caution Haploid embryos developed to blastocysts at a remarkably lower rate than the control, particularly the androgenotes; however, they can yield pluripotent haESCs capable of differentiation in the mesoderm and subsequent cardiogenesis prone to self-diploidization. Once this system is replicated in humans, the role of the centrosome needs to be investigated. Wider implications of the findings Our study has demonstrated the feasibility of propagating both male and female genomes from single gametes for reproductive applications. Indeed, once a stable culture of haESCs is achieved, genotyped pluripotent haESCs can be used to help identify heterozygosity for inherited disorders. Trial registration number N/A