The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos.

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

Use of strontium for bovine oocyte activation.

Strontium efficiently activates mouse oocytes, however, there is limited information on its use in cattle. Thus, the objective of this study was to establish a suitable protocol for activating bovine oocyte with strontium. For pronuclear development, the absence of calcium and magnesium in the activation medium (TALP) with 10 and 50 mM strontium (34.4 and 53.1%, respectively) was superior to the complete TALP (6.5 and 19.4%, respectively). In all activation media, better results were observed with 25 and 50 mM strontium (21.9-53.1 and 19.4-53.1%, respectively). Incubation for 4 h promoted similar results in all strontium concentrations. However, strontium at 15, 20, and 25 mM for 6 and 8 h (40.7, 46.7, and 48.3%, and 29.3, 48.3, and 40.7%, respectively) were superior to control (15.5 and 10%, respectively). After in vitro maturation for 26 h, strontium (S; 20 mM in Ca2+- and Mg2+-free TALP for 6 h), ionomycin+strontium (IS), and strontium+ionomycin (SI) (60, 63.3, and 65%, respectively) were similar in pronuclear development and superior to ionomycin (I; 5 microM for 5 min; 36.7%). In treatments S and I, only 1 PN zygotes were observed. In treatment S, most of them had 1 and 2 PB (35.7 and 60.7%, respectively), and in treatment I, 0, 1, and 2 PB (14.3, 57.1, and 28.6%, respectively). Most of the zygotes in treatment IS and SI were 1 PN 2 PB (77.4 and 61.6%, respectively). The number of oocytes with clusters of cortical granules was similar in all treated groups (11-29%). Cortical granule exocytosis in treatment IS (68%) was similar to S (54%) and superior to I, SI, and control (27, 45, and 5.0%, respectively). Cleavage and blastocyst rates were similar for S, I, IS, and SI treatments (61.7-76.7, and 8.3-13.3%, respectively) and the same was observed for ICM, TE, and total cell number, and ICM/total cell ratio (22-25, 64-69, and 86-95, and 0.26-0.27). In conclusion, strontium may be efficiently applied for bovine oocyte activation at 20 mM in Ca2+- and Mg2+-free TALP medium for 6 h.

Karyoplast exchange between strontium- and 6-DMAP-parthenogenetically activated zygotes of cattle.

Ooplasmic factors drive nuclear organization after fertilization and are also important for re-programming in nuclear transfer procedures, in which artificial activation is essential for reconstructed embryos to progress in development. The present research evaluated the effect of pronuclear transfer (PT) between zygotes parthenogenetically activated with ionomycin followed by strontium (S) or 6-DMAP (D) on early embryonic development. PT was performed in the same zygote to obtain embryos in control groups (S-PT and D-PT) and between zygotes activated with S and D to achieve embryos with differentially activated cytoplasm (C) and nucleus (N) (SCDN and DCSN). PT procedure did not affect cleavage and blastocyst rates, respectively, in PT control groups compared to non-manipulated control (S-PT: 73.6% and 7.3% compared with S-Control: 77.9% and 7.8%; and D-PT: 73.3% and 31.7% compared with D-Control: 83.1% and 41.5%). Cleavage, eight-cell, and blastocyst rates, respectively, were similar between SCDN (76.5%, 36.4%, and 6.8%) and DCSN (69.5%, 25.0%, and 4.9%) embryos. Developmental rates in SCDN were similar to S-PT, but inferior to D-PT. Developmental arrest up to eight-cell stage was greater in SCDN and DCSN than in S-PT and D-PT. In conclusion, karyoplast exchange between parthenogenetic zygotes activated with strontium and 6-DMAP can lead to nuclear-cytoplasmic incompatibilities and affect embryonic development to the eight-cell and blastocyst stages.

Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer.

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p < 0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

Mitochondrial genotype segregation and the bottleneck.

Mitochondria can produce a wide range of effects on many physiological systems, and these effects and their severity can vary with the ratio of mutant and wild-type mitochondrial genotype, i.e. heteroplasmy. It is therefore critical to understand the biological mechanisms controlling the segregation of mitochondrial genes, not only in somatic tissue, but also in the germ cell lineage, since the latter is the means of transmission of pathological mutations across generations. The bottleneck hypothesis was proposed to explain the homogeneity of mitochondrial genomes within organisms. This review addresses information available both from in-vitro cellular models and in-vivo animal models that have been designed to investigate mitochondrial DNA segregation in somatic and in germ cells at different stages of development. It appears that segregation occurs in multiple steps during development, and not in a single location or a single time during germ cell transmission. Nonetheless, persistent heteroplasmy of some lineages, replicative advantage of seemingly neutral genotypes and the effect of nuclear background on mitochondrial DNA segregation patterns are only a few of the observations that remain unexplained. Only after further characterization of these mechanisms will we be able to provide proper reproductive counselling to women carrying heteroplasmic mitochondrial DNA.