Respiration is useful parameter for evaluating embryo quality as it provides important information about metabolic activity. In this paper, we describe a scanning electrochemical microscopy (SECM) technique that is a non-invasive and sensitive method for measuring oxygen consumption by individual embryos. SECM measuring system can non-invasively measure respiration activity by single embryos of several species including human. The bovine embryos with higher oxygen consumption are better candidates to further development into good quality embryos and yielded higher pregnancy rates after embryo transfer. Respiration activity correlates with the embryo quality. SECM technique may be a valuable tool for accurately assessing the quality of embryos and thereby contribute to improving outcomes associated with assisted reproduction, including human in vitro fertilization.

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Microarray technology provides new insights into the field of reproductive biology as well as other fields of medicine and biology. We fabricated a bovine uteroplacental cDNA microarray and investigated the key factors involved in the establishment and maintenance of gestation. Microarray-based global gene expression analyses on bovine placenta and trophoblast cells suggest that the expression profiles of specific genes depend on the cells and tissues in which they are expressed as well as the time of the gestation period. This custom-made microarray revealed that trophoblast-specific genes such as placental lactogen, pregnancy-associated glycoproteins, prolactin-related proteins, and those of the sulfotransferase family were mainly expressed in the trophoblast giant cells and that their expression increased as gestation progressed. The expression of these genes was extremely temporal and spatial. Further, the expression of the transcription factor AP-2 increased in the trophoblast giant cells as gestation progressed. Thus, the AP-2 gene family may play a major role in regulating the functions of bovine trophoblast giant cells. Microarray technology provides information not only on thousands of genes simultaneously but also on their regulatory mechanisms in cells. Bioinformatic tools could greatly aid biological and biomedical research; therefore, active efforts must be undertaken in this field.

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Fully grown mammalian oocytes arrested at prophase I resume meiosis after gonadotropic stimulation. Oocytes undergo a series of changes including chromosome condensation, nucleolus disassembly, germinal vesicle breakdown (GVBD), and spindle formation. The mouse is the best model for studying the molecular mechanisms underlying the maturation of mammalian oocytes. However, some of the maturational events are different from those in other mammalian species. To study these events, we use pig ovaries, which are available as a byproduct from local slaughterhouses. It has long been known that pig oocytes have a dependence on de novo protein synthesis for GVBD, whereas GVBD in mouse oocytes occurs independently of protein synthesis. The reason seems to be the lack of Cyclin B1 molecules in pig GV-oocytes, although the synthesis of other protein(s) may be required for the GVBD. In mouse oocytes, the spindle is formed through the action of cytoplasmic microtubule organizing centers (MTOCs), and the oocytes are able to form the spindle without chromosomes. However, pig oocytes don't have such distinguished cytoplasmic MTOCs and never form the spindle without chromosomes. In this species, the condensing chromosome plays the role of the organizer nucleating spindle microtubules. We should develop some other mammalian models that will help us understand the mechanisms underlying oocyte maturation.

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The method of enucleating recipient oocytes by bisection with a metal-blade has been adapted to somatic cell nuclear transfer (SCNT) using zona-free mammalian oocytes, and the process named handmade cloning (HMC). Besides the simplification of enucleation without using a manipulator, this technique also provides for a reduction in the amount or the elimination of expensive equipment that is usually indispensable for conventional SCNT. In this review, we will highlight some recent studies on HMC and related studies in farm animals, especially cloning using enucleation by the gradient centrifugation of zona-free oocytes to produce a large number of recipient cytoplasts at once.

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The purpose of this study was to evaluate effect of cooling on the cytoskeletal organization affecting the distribution and structure of the meiotic spindle in the porcine metaphase II (MII) oocyte. Fluorescence staining was done for visualization of microtubules, microfilaments and chromosomes. In vitro-matured oocytes were either maintained at 37°C (for controls) or cooled abruptly to 5 or 18°C. Microtubules were preferentially labeled at the meiotic spindle and microfilaments were mainly detected at the cortex of the MII oocyte. The majority of controls (87%, n=97) had a barrel-shaped spindle, whereas the remaining 13% showed a broad, box-shaped spindle. After exposure to 18°C for 30 min, 94% of oocytes (n=48) possessed either a box-shaped spindle (63%) with a shortened interpolar distance (7.4 ± 0.4 μm vs. 9.8 ± 0.2 μm for controls P<0.05), or no spindle (31%). Exposure of oocytes to 5°C for 30 min caused complete disassembly of the spindle in 81% of the oocytes (n=103). Such spindle-disassembled oocytes had increased intensity of microtubule signaling in the cytoplasm and irregular staining of cortical microfilaments. Cortical microfilament staining decreased in intensity in 64% (n=33) and 89% (n=74) of the oocytes cooled to 18 and 5°C, respectively. When oocytes were rewarmed at 37°C after exposure to 5°C for 30 min, the spindles were not restored to normal in any oocyte (n=138) regardless of the rewarming duration (0, 1, 10, 20 or 60 min), although microtubules and microfilaments were reassembled. However, more oocytes contained dispersed chromosomes with no spindle and collapsed microfilament architecture, as rewarming time increased beyond 20 min. These results suggest that in porcine MII oocytes both microtubular spindle and cortical microfilaments are irreversibly affected by a 5°C cold shock, and that the polar microtubule-organizing centers malfunction even after rewarming.

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The purpose of this study was to examine the effect of ovary storage on the development of bovine oocytes after intracytoplasmic sperm injection (ICSI), parthenogenetic activation, or somatic cell nuclear transfer (SCNT). Oocytes were obtained from ovaries stored in PBS for 2 to 6 h (control group) or 26 to 30 h (stored group) at 15°C. The maturation rate of the oocytes was significantly lower in the stored group (67%) than in the control group (78%). The degeneration rate of the oocytes was significantly higher in the stored group (24%) than in the control group (2%). ICSI and parthenogenetic oocytes from stored ovaries had a significantly decreased development to the blastocyst stage compared with the control (ICSI 8% vs. 24%, parthenogenetic activation 15% vs. 31%). However, the development rate to blastocysts of SCNT embryos derived from cumulus cells was not different between the two groups (38% vs. 38%). Also, the storage period of ovaries did not decrease the pregnancy rate of SCNT embryos, and cloned calves were produced in both groups with the same efficiency (21% vs. 21%). In summary, ovary storage at 15°C for 26 to 30 h reduced the maturation rate and in vitro development rate of bovine oocytes after ICSI or parthenogenetic activation, but did not decrease the blastocyst formation rate or survival rate after embryo transfer in SCNT.

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The roles of microtubules and microfilaments on distribution of mitochondria were evaluated by using fluorescent staining in 2-cell embryos of golden hamsters with or without cytoskeletal assembly inhibitors. In 2-cell embryos without treatment (control), most mitochondria were accumulated at the perinuclear region, while some mitochondria were noted at the cell cortex. Microtubules were found around the nuclei, correlating with distribution of the mitochondria. In contrast, microfilaments were stained intensely beneath the cell membrane and especially at the cell-to-cell contact region. In most (82%) of embryos treated with nocodazole (an inhibitor of microtubule polymerization), mitochondria had extended into the subcortical (intermediate) region with varying degree, where they were aggregated in patches. After a treatment of cytochalasin D (an inhibitor of actin polymerization), distributional density of mitochondria decreased at the cell cortex, suggesting that mitochondria moved back around the nucleus. After a treatment of both inhibitors, the distribution pattern of mitochondria was almost similar to that observed after cytochalasin D treatment. Our results suggest that the translocation of mitochondria to the perinuclear region is mediated by microtubules, while the movement of mitochondria to the cell cortex is regulated by microfilaments. Microtubules and microfilaments may function as bidirectional anchors of mitochondria to the perinuclear region and to the peripheral region, respectively.

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The transition from germ cell to embryo is an important event in the creation of new life. During this period, cells change their characteristics through changes in gene expression patterns. Histone methylation is considered to be a stable and important epigenetic marker regulating gene expression. In the present study, we investigated the expression of histone methyltransferases (HMTs) and histone demethylases (HDMs) during preimplantation development. Analysis by reverse transcription-PCR (RT-PCR) revealed that many of these enzymes are abundantly expressed in MII stage oocytes and that their expression levels change dynamically during preimplantation development. These results suggest that HMTs and HDMs are involved in the regulation of gene expression during preimplantation development.

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