Preimplantation genetic diagnosis (PGD) is the technology used to avoid inheritance of genetic disease by selecting unaffected embryos. Furthermore PGD has made genetic screening of embryos possible. The limited number of cells available for genetic tests has been a serious problem with PGD. The first solution was the application of molecular technologies such as PCR, FISH and cell recycling. The next solution was the visualization of metaphase plates and application of advanced FISH technologies. The ideal solution was in vitro culture of blastomeres to increase the number of cells because it would be easy to reexamine them to assure the accuracy of genetic test results and obtain additional genetic information. The culture system for ES cells was applied to culturing mouse isolated blastomeres because it met two requirements: rapid proliferation and maintenance of the normal karyotype during culture. Our trial with mouse embryos successfully demonstrated these two requirements.

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Oocyte activation is a key issue in current animal biotechnology. This study was designed to reveal a suitable condition for activation of the pig oocyte. In-vitro matured oocytes were activated by means of double DC pulses (150 V/mm for 60 μsec, 1 sec apart), the same treatment followed by cytochalasin D (CD, 5 μM for 4 hrs), or two sets of the same electricity. Oocytes were examined for their chromosome configurations and the types of pronuclear formation by fluorescence staining 8 hrs post-activation. High rates of pronuclear formation were obtained after each treatment (93-96%). A greater proportion of oocytes having one diploid nucleus or two haploid pronuclei were found by treatment with electric pulses plus CD (84%) than by treatment with one (33%) or two sets of DC pulses (41%). Two sets of DC pulses caused partial loss of cortical microfilaments and a decrease in density of microfilaments and microtubules in the peripheral ooplasm. Oocytes treated with CD had a wavy layer of cortical microfilaments. The incidence of cleavage 48 hrs post-activation was similar for all treatments (69-78%), but the blastocyst formation 168 hrs post-activation was higher after the combined treatment with electric pulses and CD (24%) than the treatment with one (11%) or two sets of electric stimulation (5%). The results suggest that increased development to blastocysts after a combined treatment with double electric pulses and CD may depend on a higher incidence of diploid parthenogenotes, probably due to altered microfilament organization of the ooplasm caused by the CD treatment rather than the DC pulse itself.

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The purpose of this study was to assess the effect of oxygen condition on the survival and growth in vitro of bovine oocyte-cumulus/granulosa cell complexes isolated from early antral follicles. Complexes comprised of a growing oocyte enclosed in cumulus layers and a piece of mural granulosa cells were isolated from early antral follicles, 0.5-0.7 mm in diameter, and cultured for 14 days in TCM199 supplemented with 5% fetal bovine serum and 4 mM hypoxanthine in an atmosphere of 5% O₂ or 20% O₂. Mean oocyte diameter at the beginning of culture was about 95 μm. Within 4 days formation of the antrum-like structure was evident in virtually all complexes, regardless of high or low oxygen tension, but many of the complexes failed to retain this structure after 8 days. At low oxygen tension, complexes retain the antrum-like structure for slightly longer periods than those cultured at high oxygen tension, yet no significant difference was found. Accordingly, the percentage of oocytes that survived in culture was higher at low oxygen tension. Nevertheless, the oocytes (105.9 ± 8.4 μm) were smaller than those grown at high oxygen tension (112.6 ± 10.5 μm) (P<0.05). Supplementation of estradiol-17 β was effective in increasing the percentage of follicles that retained the cavity, especially at low oxygen tension, and of oocytes recovered as being enclosed with cumulus cells after 14-day cultures, but no promotion of oocyte growth caused by estradiol-17 β was observed. Taken together, a reduced oxygen condition may be beneficial in improving the survival rate of oocytes, but not in promoting growth in the culture system used in this study.

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The state of nuclear maturation and the state of cortical granule (CG) distribution were observed in porcine oocytes cultured for various periods and also in those treated with olomoucine, an inhibitor of cyclin-dependent kinase, and the relationship between nuclear maturation and changes in CG distribution was investigated. CGs were distributed over the cortical cytoplasm (type I) in 98% of porcine oocytes soon after collection and in 90% of those cultured for 8 hrs after collection, and nuclei of these oocytes were all in the germinal vesicle (GV) stage. When cultured for 22 hrs, type I oocytes markedly decreased, while oocytes having CGs in the cortical cytoplasm and also immediately beneath the plasma membrane (type II) increased to 22%, and oocytes having CGs immediately beneath the plasma membrane (type III) appeared at 72%. Nuclei of type II oocytes were in the GV to M I stages, while most nuclei of type III oocytes were in the M I stage. When cultured for 32 and 44 hrs, type III oocyte populations were observed as 70 and 87% of the whole, respectively, and nuclei of these type III oocytes were all in the M I to M II stages. In the oocytes cultured for 22 hrs with olomoucine, nuclei were all in the GV stage, and 83% of the treated oocytes showed the type I distribution of CGs. On the other hand, 91% of control oocytes showed the type II and type III distribution of CGs, and type I oocyte populations were observed only as 10% of the whole. From these results, it was inferred in porcine oocytes that the start and the completion of CG movement into the cytoplasm immediately beneath the plasma membrane are closely related to the time at which GV breaks down and nuclei reach the M I stage, respectively.

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To overcome infertility of male transgenic mice with low-motility spermatozoa, we examined the benefits of the zona pellucida incision using a piezo-micromanipulator (ZIP) on in vitro fertilization (IVF) using low-motility spermatozoa. The incision was about 26 μm long, representing 8% of the perimeter of the zona pellucida of the mature oocyte. Over 95% of cumulus-free oocytes survived after ZIP. The in vitro fertilization rate of ZIP oocytes by low-motility spermatozoa of the transgenic mice was 22-57%, compared with 1-2% of cumulus-intact oocytes. After embryo transfer of ZIP zygotes at 2-cell stage into the oviducts of recipients, 11-20% of the transferred embryos developed to term. Our results indicate that ZIP is a useful technique for IVF of ova by low-motile spermatozoa and subsequent embryo transfer.

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In vitro fertilizability and subsequent developmental competence of inbred RFM/Ms mice, which have high incidence of myeloid leukemia by radiation, was studied. When response of RFM/Ms females to various amount of eCG (1.25-10.0 i.u.) was examined, superovulation (>20 ovulated ova) occurred in animals injected with ≥5.0 i.u. eCG. The kinetics of in vitro fertilization and embryo development were examined and compared among RFM/Ms, C57BL/6J zygotes and their hybrid zygotes. The fertilization kinetics were significantly slower in RFM/Ms zygotes than in C57BL/6J zygotes. Crossbreeding experiments between C57BL/6J and RFM/Ms showed that the speed and incidence of fertilization were affected by paternal and maternal factors, respectively. The in vitro development to the blastocyst stage of RFM/Ms zygotes was significantly slower than that of C57BL/6J zygotes and their hybrid zygotes at 96 h post-insemination (PI), but not at 120 h PI. The nuclear number of RFM/Ms embryos was significantly lower than that of C57BL/6J and their hybrid embryos throughout the culture period (72, 96 and 120 h PI). Conversely, hatching at 120 h PI in C57BL/6J embryos was much slower than that of the other groups of embryos. This study showed that gametes and zygotes of RFM/Ms strain can be fertilized and developed well in vitro and that maternal and paternal factors and their interaction have complicated effects on fertilization and developmental kinetics in vitro.

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Pig early antral follicles (0.5-1.0 mm in diameter) contain oocytes approximately 100 μm in diameter with limited competence for meiotic resumption. Oocyte-cumulus complexes containing parietal granulosa cells (OCGs) were dissected from the ovarian follicles, embedded in collagen gels and cultured in Waymouth's medium containing 5% fetal calf serum. In the first experiment, OCGs were cultured in medium containing 0, 1, 10 or 100 ng/ml of FSH for 8 days, and the viability and growth of oocytes were examined. The mean diameter of the surviving oocytes in each experimental group significantly increased, although the percentage of surviving oocytes was highest in the 10 ng/ml FSH-supplemented group (41% vs. 16-22%). In the second experiment, OCGs were cultured in medium containing 10 ng/ml FSH with or without 2 mM hypoxanthine for 7 days. Regardless of hypoxanthine supplementation, about 45% of the oocytes in each group had normal morphology after the culture and grew to almost the full size of 120 μm. All of these were at the germinal vesicle stage. Recovered oocytes were further cultured for 48 hours so that their meiotic competence could be compared. Of the oocytes cultured in hypoxanthine-free medium, 13% underwent germinal vesicle breakdown, although no oocytes progressed beyond metaphase I. On the other hand, 47% of the oocytes cultured in hypoxanthine-supplemented medium underwent germinal vesicle breakdown, and 9% progressed to metaphase II. These results suggest that hypoxanthine promotes the acquisition of meiotic competence in growing pig oocytes in the medium containing FSH, which supports oocyte viability.

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We examined the effects of butyrolactone I (BL-I), a specific cdc2 kinase inhibitor, on the inhibition of meiotic resumption of porcine oocytes and subsequent developmental competence after somatic cell nuclear transfer (NT). Porcine follicular oocytes were cultured in a medium containing BL-I during maturation culture. BL-I suppressed germinal vesicle (GV) breakdown in a dose-dependent manner and most (98.3%) oocytes were arrested at the GV stage when they were cultured for 28 h with 100 μM BL-I, but after 48 h of culture with 100 μM BL-I half of the oocytes underwent GV breakdown. Twenty hours after release from the BL-I treatment (28-h culture), most (95.9%) oocytes reached the metaphase II stage, whereas most non-treated oocytes reached the metaphase II stage after 40 h of maturation culture. There were no differences between BL-I-treated and non-treated oocytes in meiotic progression. When oocytes were enucleated and fused with serum-starved cumulus cells, the development of NT embryos to the blastocyst stage with the BL-I-treated oocytes (12.3%) was similar to that achieved with non-treated oocytes (12.5%). The results demonstrate that BL-I could reversibly inhibit meiotic resumption of porcine oocytes, and that BL-I-treated porcine oocytes can be used as recipient cytoplasts for NT without compromising developmental competence.

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