Examination of the nucleus-blastomere relationship indicated that all in vivo embryos had normal morphology, with one nucleus per blastomere. However, because fragmentation and binucleate blastomeres were present in in vitro-produced embryos, the mean number of nuclei varied at each stage examined (Table 2). Moreover, the mean number of nuclei was usually less than the number of blasto-meres. More nuclei were present in in vivo-derived blastocysts than in in vitro-produced blastocysts. Actin filaments were present at the cortex and at the joints of blas-tomeres of all in vivo- and in vitro-produced embryos (Table 2; Figs. 3 and 4). However, considerable differences existed in the perinuclear actin filament distribution. All in vivo-produced 2- to 8-cell-stage embryos and some blas-tomeres of morulae and blastocysts had perinuclear actin filaments (Fig. 3, E and G). However, as shown in Table 2, this type of actin filament distribution was observed only in 31% of 2-cell-, 14% of 3-cell-, and 18% of 4-cell-stage in vitro-produced embryos. Most in vitro-produced embryos had partial perinuclear actin filaments in their blasto-meres (Fig. 4, B, F, and G), and some embryos (5% of 2cell, 9% of 4-cell and 25% of 5-cell) did not have perinuclear actin filaments in their blastomeres (Fig. 4, A, C, and I). As shown in Figure 5, asynchronized cytoplasmic and nuclear division was observed in in vitro-produced 2-cell-and 3-cell-stage embryos. In these embryos, it was found that chromosomes were present in one or two blastomeres or that mitotic division had occurred but cytoplasmic division had not. These situations were not observed in in vivo-derived embryos at any stage. Buy Advair Diskus Online
Effect of Cytochalasin D on the Blastocyst Formation (Experiment 3)
Of 139 2- to 4-cell-stage embryos cultured in medium without cytochalasin D, 81 (58.3%) developed to blastocysts, with a mean nuclear number of 41.4 ± 14.4 per blastocyst. Actin filaments were distributed at the cortex of blastocysts, as shown in Figure 3H. However, no embryos (0/135) cultured in medium with cytochalasin D developed to blastocysts. All of the embryos remained at the 2- to 4cell stages. The nuclei of these embryos divided one or two times, thus forming binucleate (Fig. 6A) or polynucleate (Fig. 6B) embryos. No obvious actin filaments were present in the embryos cultured with cytochalasin D (Fig. 6, A’ and B’).
|Stage of embryos||No. of embryos examined||Mean no. of nuclei||Actin filaments in perinuclear area of: All blastomeres Some blastomeres(%) (%)||None(%)|
|In vivo embryos|
|4-cell||32||4 ± 0||32||0||0|
|Morula||4||24.5 ± 4.2||0||4||0|
|Day 5 blastocyst||24||136.5 ± 60.4||0||24||0|
|Day 6 blastocyst||11||164.5 ± 51.9||0||11||0|
|In vitro embryos|
|3-cell||14||2.6 ± 0.5||2||10||2|
|4-cell||11||3.1 ± 1.2||2||8||1|
|5-cell||4||4.8 ± 0.4||0||3||1|
|6-cell||4||5.3 ± 1.0||0||4||0|
|8-cell||2||7.0 ± 1.4||0||2||0|
|Day 6 blastocyst||31||37.3 ± 11.7*||0||31||0|
a In vivo embryos were collected from the oviducts of gilts on Day 2, Day 4, Day 5, and Day 6 of estrus. In vitro embryos were produced by IVM/IVF, and 2-cell to 5-cell embryos were selected 36 h after IVF, and blastocysts were obtained 144 h after IVF.
* p < 0.001 as compared with the counterpart embryos derived in vivo.
FIG. 5. Confocal photographs of pig embryos produced in vitro showing abnormal nuclear-cytoplasmic relationship during embryo division. A-D show microfilament distribution, and A’-D’ show nuclei in the same embryos. A and A’, and C and C’ show, respectively, 2-cell- and 3-cell-stage embryos with different stages of nuclei. B and B’ show a 2-cell-stage embryo with separated chromosomes but without division of cytoplasm. D and D’ show a 3-cell embryo with only one blastomere containing separated chromosomes and two blastomeres containing normal nuclei.
FIG. 6. Confocal photographs of 2-cell-stage embryos cultured for 4.5 days in medium with cytochalasin D. The embryos still have two blastomeres without obvious microfilaments (A’ and B’), but the nuclei have divided one or two times, resulting in binucleate (arrows in A and B) and poly-nucleate cells (B).