A huge number of small oocytes are contained in the ovaries of the pig and cow. A small number of them grow from the minimal size of 30 μm in diameter to the final size of 120-125 μm, then mature, and are ovulated. A large number of the remaining oocytes do not enter the growth phase or degenerate in the ovary. Oocyte growth takes a long time and is coordinated with surrounding follicle cells. During the growth phase, oocytes are required to be arrested at prophase I (GV stage), and to acquire the meiotic competence to mature to metaphase II. IVG culture systems have been developed for domestic species, although they are still being improved. IVG culture systems for small oocytes are expected to provide a new source of oocytes for livestock production as well as a better understanding of the basic mechanisms of oogenesis/folliculogenesis in the ovary.

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Double stranded RNA (dsRNA) interference is a useful tool for interfering with gene function by promoting the sequence-dependent degradation of targeted mRNA in several organisms. In the present study, in order to confirm and improve the effect of dsRNA, we investigated an inhibitory effect of dsRNA on both transient and stable gene expression of enhanced green fluorescent protein (EGFP) in mouse preimplantation embryos. In the transient expression system, the rates of fluorescent embryos were significantly decreased by co-injection of EGFP dsRNA and EGFP expression vector fragment into the pronucleus of zygotes. In the stable expression system, EGFP expression in transgenic embryos was significantly decreased by injection of EGFP dsRNA into both the pronucleus and cytoplasm of zygotes, but, cytoplasmic injection caused a more significant EGFP inhibition than pronuclear injection. In quantitative PCR analysis, the expression of the EGFP gene was also inhibited by dsRNA injection, whereas the endogenous gene expression was not affected. These data suggest that dsRNA can inhibit the specific gene expression without affecting the development and expression of other genes.

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In the present study, we investigated the role of actin filaments in the blastocoel (BC) of bovine blastocysts produced in vitro by using cytochalasin-D (CD), an inhibitor of actin polymerization. Blastocysts classified as good or poor based on their morphological normality were exposed to 2 μM CD for 1.5-2 hr. After incubation, the presence or absence of BC was observed, and the re-expansion rate was assessed after transferring CD-treated blastocysts with collapsed BC to a non-CD medium. Ultrastructural observation was also undertaken using a transmission electron microscope (TEM). The remaining blastocysts were immersed in a hypotonic solution of sodium citrate so that the cells could be counted to confirm the classification grade of blastocysts in this study. The percent of maintained BC under the presence of CD in the good group was significantly lower (P<0.05) than that in the poor group (3.3% versus 27.7%, respectively). The average cell number of blastocysts in the good group was significantly more (P<0.05) than that in the poor group. In addition, when the blastocysts with shrunken BC in both groups were cultured, re-expansion rates in the good and poor groups were 83.3 and 75.0%, respectively, and no significant difference was observed between groups. Based on observation of the ultrastructure with TEM, the microvilli on the surface of some trophoblast cells of some blastocysts in the poor group in the presence of CD showed a translucent matrix, and their electron density was low compared with that of trophoblast cells of blastocysts in the good and non-treated (control) groups. However, the electron density of microvilli after removal of CD in the poor group increased to a level comparable to those of the good and control groups. These results suggest that polymerizing actin may be required to sustain the blastocoel and microvilli of blastocysts produced in vitro. However, in poor grade blastocysts, the polymerization ability of actin present in the filamentous form in the microvilli in some cells might be lower than that in good grade blastocysts.

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In this study, to investigate the time dependent changes in progesterone receptor (PR) expression in cumulus cells during meiotic resumption of porcine oocytes, each amount of PR-A and PR-B mRNA was analyzed by RT-PCR with primer sets for the PR-B region and the PR-A/B common region. The results showed that the levels of both PR-A/B and PR-B mRNA were very low in cumulus cells immediately recovered from their follicles. The cultivation with FSH and LH significantly increased the level of both PR-A/B and PR-B mRNA in cumulus cell of COCs, whereas the level of PR-B mRNA significantly decreased at 12-hr cultivation. Nevertheless, the higher level of PR-A/B mRNA was maintained up to 20-hr cultivation, suggesting that PR-A was mainly expressed in cumulus cells during cultivation from 12 hr to 20 hr. When COCs were cultured for 10 hr and then further cultured with RU486 for 10 hr, the proportion of oocytes undergoing GVBD significantly decreased in a dose dependent fashion. These results suggest that the high ratios of PR-A to PR-B in cumulus cells of COCs during 12-hr to 20-hr cultivation, are required for meiotic resumption of porcine cumulus-enclosed oocytes in vitro.

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The activities of hydroxysteroid dehydrogenases (HSDs) were histochemically demonstrated in rabbit and hamster oocytes in the process of maturation, and the changes in steroid metabolism during meiotic maturation were examined. In rabbits, the percentages of oocytes showing the activities of Δ⁵-3β-HSD (with DHEA as the substrate), 17β-HSD (testosterone) and 20β-HSD (20β-hydroxyprogesterone) were always high and did not change during maturation, whereas those showing the activities of Δ⁵-3β-HSD (pregnenolone and 17α-hydroxy-pregnenolone), 17 β-HSD (estradiol-17β), 20α-HSD (20 α-hydroxyprogesterone) and 20β-HSD (17α-hydroxyprogesterone) decreased as the time after the hCG injection was prolonged. On the other hand, the activities of Δ⁵-3β-HSD, 17 β-HSD, 20α-HSD and 20β-HSD with eight substrates were almost always observed in hamster oocytes from 0 to 13 hrs after the hCG injection. The present findings suggested that the metabolic abilities of 20β-hydroxyprogesterone and androgen are constantly present in rabbit oocytes in the process of maturation, whereas those of progesterone, 17α-hydroxyprogesterone, 17α, 20β-dihydroxyprogesterone, 20α-hydroxyprogesterone and estrogen decrease during maturation. And it was also confirmed in hamster oocytes that the metabolic abilities of progesterone, 17α-hydroxyprogesterone, 17α, 20β-dihydroxyprogesterone, 20α-hydroxyprogesterone, 20β-hydroxyprogesterone, estrogen and androgen are always present and do not vary with maturation.

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Mouse pronuclear embryos were cryopreserved by a simple and safe vitrification method. In the process, Vitrification Media VT101, Thawing Media VT 102 (KITAZATO. Co. Japan) and the embryos were loaded into a straw; then they were cryopreserved. Different loading methods were examined to determine the safety levels of crystallization for the embryo's survival after thawing. The best condition attained, after thawing, was a 75% embryo survived rate of which 66% developed to the two-cell stage, 71% developed to the morula stage and 27% developed to the blastosyst stage. This development of embryos after vitrification was not significantly different to that of a control group without freezing and thawing. The vitrification method was considered to protect embryos against various infections via liquid nitrogen during cryopreservation. It is expected that the method can be applied to human embryos.

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