H. Koyama

Developmental Competence of Bovine Oocytes Selected by Brilliant Cresyl Blue Staining: Effect of the Presence of Corpus Luteum on Embryo Development

ABSTRACT The local circumstances of the ovary may influenced by the presence of corpus luteum (CL). We investigated the cleavage rate and blastocyst development of bovine oocytes collected from ovaries with or without CL. Oocytes were incubated with brilliant cresyl blue (BCB) for 90 min and separated on the basis of low (BCB+) or high (BCB–) activity of glucose-6-phosphate dehydrogenase. For both types of ovaries, the embryo cleavage rates in the case of BCB+ oocytes were significantly higher than those in the case of BCB– oocytes, but were not higher than the rates in the control group. The percentage of blastocysts developing from BCB+ and BCB– oocytes from the ovaries with CL did not significantly differ. However, in the case of ovaries without CL, a significantly higher (P<0.05) percentage of blastocysts developed from BCB+ oocytes than from BCB– and control group oocytes. The presence or absence of CL did not significantly influence the cleavage rate and blastocyst development. Based on our results, we conclude that BCB staining facilitates the selection of competent oocytes that will develop into blastocysts for IVF better than conventional morphological selection methods. We also conclude that the CL does not significantly influence blastocyst development.

Effect of Meiotic Arrest by Cycloheximide on the In Vitro Maturation of Bovine Oocytes and Their Subsequent Development Following In Vitro Fertilization

ABSTRACT Cycloheximide (CHX) is a reversible inhibitor of bovine meiotic resumption. This study examined the timing of nuclear maturation in bovine oocytes treated with CHX and determined the optimum maturation interval in cultures for their subsequent development. CHX prevented the nuclear maturation of nearly all oocytes for 24 h. In an inhibitor-free medium, the majority of the CHX-treated oocytes underwent germinal vesicle breakdown (GVBD) after 6 h of culture, while the control oocytes did so at 10 h. In the maturation culture, the majority of the CHX-treated oocytes had reached metaphase II by 16 h whereas the control oocytes took 20 h. However, the CHX-treated oocytes that matured for 16 h developed into blastocysts at low rates while those that were matured for 20 h had a development rate similar to that of the control oocytes. These results indicate that the nuclear maturation of CHX-treated oocytes did accelerate, but these oocytes needed the same maturation time as required by the control oocytes for their subsequent development to blastocysts.

Development of cellular polarity of hamster embryos during compaction

Development of cellular polarity is an important event during early mammalian embryo development and differentiation. Blastomeres of hamster embryos at various stages were examined by scanning electron microscopy (SEM) and immunocytochemical staining. SEM observations revealed that 1- to 7-cell-stage embryos showed a uniform distribution of microvilli throughout the cell surface. Microvillous polarization was initially noted in the blastomeres (10-35%) of 8-cell-stage embryos. The polarized microvilli were observed mostly in the basal region of cell-cell contact and occasionally at the apical, outward-facing surface of the blastomere. Fluorescein-isothiocyanate-conjugated concanavalin A failed to reveal any polarity in the blastomeres regardless of the stages of the embryos. Actin staining showed that microfilaments were present beneath the cell surface, and in addition, areas of cell contact were more heavily stained, indicating a thick microfilament domain. Microtubules were located throughout the cytoplasm and were heavily concentrated near the nucleus during interphase, although they became redistributed in the region of the mitotic spindle during karyokinesis. The position of nucleus changed from the cell center to the apical, outward-facing surface of the cell, and it distanced itself from the basal microvillous pole. It is suggested that the changes in the cell surface and nuclear position are the first manifestations of cell polarity in peri-compacted hamster embryos, which appear as early as the 8-cell stage; furthermore, the outward migration of the nuclei may parallel the redistribution of microtubules in the cytoplasm.