50 Ploidy correction during pre-implantation development of bovine triploid embryos

As in other mammals, most cells in bovine tissues are diploid (2N). Yet, polyploid cells are abundant in several tissues such as megakaryocytes, skeletal myocytes, and Purkinje neurons. Triploidy (3N) is a distinct form of polyploidy characterized by the presence of an additional set of chromosomes of one parental origin. Abnormal fertilization, such as polyspermy and the failure of polar body extrusion, can lead to polyploidy, resulting in embryos with 3N or higher complements of chromosomes. Direct cleavage of the first mitotic division, however, can correct ploidy by splitting the zygote into three or more blastomeres (Suzuki et al. 2023 Theriogenology 200, 96–105). Furthermore, it has been reported that pigs can produce offspring from polyspermic zygotes (Somfai et al. 2008 Anim. Reprod. Sci. 107, 131–147). Elucidating ploidy correction in mammals is crucial to enhance our understanding of genetic stability, disease modeling, and developmental biology. Our objective here was to evaluate the extent to which ploidy correction occurs during preimplantation development using bovine 3N embryos as a model. Cytochalasin B (CB) inhibits the extrusion of the second polar body, which results in 3N zygotes. First, we treated bovine oocytes with CB at 0, 7.5, 10, and 15 μg mL⁻¹ for 1 or 2 h before IVF to identify the condition for the generation of the most 3N embryos. Twenty-two hours after fertilization, we stained the pronuclei of the presumptive zygotes with Hoechst 33342 and analyzed the data with logistic regression (Procedure GENMOD). Binomial distribution was considered for live versus dead oocytes, fertilized versus unfertilized, and 2N versus 3N zygotes. Overall, longer CB treatment increased the number of dead oocytes (P < 0.0001). Longer and higher CB levels also decreased fertilization rate (P < 0.05). Within the 1-h treatment groups, 7.5 μg mL⁻¹ CB generated the highest percentage of 3N zygotes (50.7%; P < 0.05) without significantly affecting oocyte viability (97.3% vs. 97.1%) or fertilization rate (94.2% vs. 92%) compared with the control group. Although treatment with 10 μg mL⁻¹ CB for 1 h also generated a high percentage of 3N zygotes (34.0%), it significantly decreased fertilization rate to 79.2% (P < 0.05). Therefore, 7.5 μg mL⁻¹ CB treatment for 1 h was selected for all subsequent experiments, and 77 blastocysts were generated using such a treatment. Karyotype analyses were performed on 10 of these blastocysts, and ploidy correction was observed because a mixture of 2N and 3N cells were present in seven of the blastocysts; the rest of the embryos contained no 3N cells. Furthermore, α-tubulin immunofluorescence staining revealed bipolar and tripolar cell division in blastomeres undergoing mitosis in CB-treated blastocysts, while blastocysts from untreated oocytes did not show any tripolar cell division. Additionally, 15 lines of bovine embryonic stem cells (bESCs) were derived from 36 of the blastocysts. Karyotype analysis revealed that all 15 bESC lines were diploid. Flow cytometry of one bESC line confirmed their 2N status. We also generated 10 lines of bESCs from 15 blastocysts developed from untreated oocytes. All were diploid as revealed by karyotyping. The data clearly demonstrated that a high rate of ploidy correction occurred and 2N was the default ploidy during early bovine development. Further characterization of other embryonic stages by α-tubulin immunofluorescence staining will permit the precise determination of when ploidy reduction is initiated. Fluorescence in situ hybridization is another method for confirmation of ploidy correction.