1 REPLICATION OF SOMATIC MICRONUCLEI IN BOVINE OOCYTES

Microcell-mediated chromosome transfer was developed to introduce a low number of chromosomes into a host cell (Fournier and Ruddle 1977 PNAS 74, 319–323). As a first approach to individual chromosome manipulation, we designed a new technique that consists of injecting a micronucleus into an enucleated oocyte to replicate a low number of chromosomes. Additionally, we studied the capability of such micronuclei to be marked with a transgene. To this aim, micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg mL–1 of demecolcine for 48 h followed by 2 mg mL–1 of mitomycin for 2 h. Cells were finally treated with 10 μg mL–1 of cytochalasin B for 1 h. The cumulus-oocyte complexes aspirated from slaughtered cow ovaries were in vitro-matured under standard conditions for 21 h. MII oocytes were mechanically enucleated and injected with somatic micronucleus, which were previously exposed (Mi*) or not (Mi) to 50 ng μL–1 of pCX-EGFP in 10% PVP. Sham and parthenogenetic (PA*) controls were injected with 50 ng μL–1 of pCX-EGFP in 10% PVP. A PA control was also included. After 2 h, oocytes and reconstructed embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Embryos were cultured in SOF. Cleavage stage and egfp expression were evaluated at Day 2 and 4 of IVC, respectively. At Day 2, some Mi and PA embryos were fixed and stained with DAPI. Nuclei were visualised under blue light (488 nm). Cleaved embryos with more than one nucleus were considered to have replicated their DNA. At Day 2, Mi and PA embryos were karyotyped. An IVF group was also included (Brackett and Oliphant protocol, 1975). Briefly, cleaved embryos were treated with 1.25 μg mL–1 of colchicine for 6 h. After Carnoy fixation, they were stained with Giemsa to determine the chromosomal complement of each blastomere. Embryos were classified as follows: less than 15 chromosomes, euploid (1n and 2n) and others (4n, mixoploid and aneuploid). Differences among treatments were determined by Fisher's exact test (P ≤ 0.05). The Mi*, PA* and PA groups showed higher cleavage rates than the Mi treatment [93/108 (86.1%), 111/136 (81.6%) and 160/186 (86%), respectively vs 89/131 (67.9%); P ≤ 0.05]. Cleavage rates of the Sham* group [78/105 (74.3%)] did not differ from Mi and PA* treatments (P ≤ 0.05). Interestingly, a low number of Mi* embryos showed egfp expression (2.2%). Expression levels were significantly lower than those of the PA* group (38.7%) and did not differ from the Sham control (0%; P ≤ 0.05). Although rates of Mi embryos with more than 1 nucleus (63.6%, n = 22) were lower than those for the PA group (100%, n = 28), DAPI staining confirmed replication of micronuclei. Karyotype analysis revealed that 100% of Mi evaluated embryos (n = 11) had less than 15 chromosomes per blastomere (varying from 1 to 13), whereas none of IVF and PA controls showed such results (P ≤ 0.05). Rates of euploid embryos were 75 (n = 20) and 45% (n = 20) for IVF and PA groups, respectively. In conclusion, we have developed a new method for somatic micronuclei, which could be a useful tool to transfer a small number of specific chromosomes and to target transgenesis to a reduced area of the genome.