The role of contraction in blastocyst hatching was determined by time-lapse videomicrography in mouse blastocysts whose hatching ability had been suppressed by soybean trypsin inhibitor (STI). The hatching rate of blastocysts developed from morulae in a medium containing STI at a concentration of 1.0 mg/ml (STI-treated blastocysts) was 17.2%, which was significantly lower than the 63.9% for blastocysts developed from morulae in a medium without STI (non-treated blastocysts). Over the span of 32 hrs after blastocoel formation, the number of strong contractions was similar in STI-treated and non-treated blastocysts, but the total number of contractions and the number of weak contractions (less than 20% volume reduction) were significantly smaller in STI-treated blastocysts (4.22 and 2.94 times) than in non-treated blastocysts (5.80 and 4.50 times). These STI-treated blastocysts took a significantly longer time for weak contraction and for re-expansion after weak contraction (10.2 and 87.6 min), compared with non-treated blastocysts (7.8 and 58.2 min). It was also confirmed that the activity of trypsin-like proteinase was similar in STI-treated and non-treated blastocysts, and that a small hole was formed in the zona pellucida at the start of hatching in STI-treated blastocysts, as seen in non-treated blastocysts. Nevertheless, the rate of blastocysts with a slit resulting from enlargement of the small hole in zona pellucida was 37.5% in STI-treated blastocysts, which was significantly lower than the 86.7% in non-treated blastocysts. These results suggest that weak contraction and re-expansion after weak contraction of blastocysts play an important role in hatching by slitting the region adjacent to the small hole in the zona pellucida.

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Oocyte maturation is a key issue in current animal biotechnology. This study was designed to examine effects of incubation temperature and cumulus investment on in vitro maturation (IVM) of porcine oocytes. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries. The oocytes, surrounded completely or partially with cumulus cells (completely- and partially-enclosed groups, respectively), were incubated for 44 h at either 37°C or 39°C, and progression of meiosis and changes in the cytoskeleton distribution were evaluated by fluorescence staining. Prior to maturation culture (0 h), 94-100% of the oocytes were at the germinal vesicle (GV) stage. At 24 h IVM, a significantly greater number of the oocytes showed signs of GV breakdown (GVBD) when incubated at 39°C than at 37°C (96% vs. 61% and 92% vs. 40% for completely- and partially-enclosed groups, p<0.01, respectively). Percentages of the oocytes which reached metaphase-II (M-II) at 36 h IVM was higher at 39°C than at 37°C (p<0.01), whereas no significant difference was found in the maturation rate at 44 h IVM. The completely-enclosed oocytes had a significantly higher maturation rate than did the partially-enclosed oocytes (90% vs. 68% for 37°C and 91% vs. 53% for 39°C, p<0.01 respectively). Fluorescence staining showed that transzonal microfilaments were abundant at the GV and M-I stages (0 to 24 h), but decreased in number at the M-II stage (36 to 44 h) and that there was no remarkable difference in the distribution of microtubules and microfilaments within the ooplasm, irrespective of the incubation temperature or the condition of cumulus attachment. These observations suggest that GVBD and meiotic progression may be drastically delayed by a 2°C decrease in incubation temperature and that the cumulus condition may affect porcine oocyte maturation.

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One-cell mouse embryos were cultured in the presence or absence of oviductal tissue in order to investigate the role of oviductal tissue on the developmental competence and morphology of mouse embryos cultured in vitro. To synchronize embryo development, one-cell embryos were treated with nocodazole, an inhibitor of tubulin polymerization. The morphology of 4-cell stage embryos that subsequently developed in the absence of oviduct (phosphate-free medium) differed markedly from that of 4-cell stage embryos developed in an oviductal environment. Four-cell embryos that developed without oviductal tissue had spherical blastomeres whereas embryos developed in co-culture had flattened blastomeres and appeared to have undergone premature compaction. Embryos with flattened blastomeres exhibited E-cadherin localization to regions of cell-to-cell contact.

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We prepared IgM and IgG (PFG-5 and PFG-6, respectively) monoclonal antibodies against granulosa cells prepared from healthy antral follicles of porcine ovaries. PFG-5 antibody specifically recognized two cell-membrane proteins (PFG-5 antigen: 55 kD, pl 5.9, and PFG-6 antigen: 42 kD, pl 5.2), and PFG-6 antibody recognized PFG-6 antigen. Immunochemical reactions of these antibodies were only detected in follicular granulosa cells but not any other ovarian tissues for organs. Both antigens were detected in granulose cells of healthy follicles, but PFG-6 antigen disappeard in granulosa cells of atretic follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing PFG-5 antibody, the cells underwent apoptosis, and co-incubation with PFG-6 antibody inhibited PFG-5 antibody inducible apoptosis. These observations suggested that PFG-5 antigen is a novel cell death receptor, which is different from well-known apoptosis-mediating receptors (Fas or tumor necrosis factor receptor), and that PFG-6 antigen may act as a decoy receptor and inhibit apoptotic signals through PFG-5 antigen.

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Using immunohistochemical techniques, the localization of maturation promoting factor (MPF) and MAP kinase around chromosomes of cumulus nuclei transferred into the porcine mature oocytes was investigated. Nuclear condensation was observed in most of the transferred nuclei within 2 h and was maintained until 6 h after nuclear injection. Cyclin B, a subunit of MPF, and MAP kinase accumulated around the exogenous chromosomes in 47% of the injected oocytes at 6 h after injection. Tubulin assembly around the exogenous chromosomes was detected only in those accumulated by cyclin B and MAP kinase. The pronuclear formation rate after oocyte activation agreed well with the tubulin assembly rate. The present results indicate for the first time that MPF and MAP kinase accumulated around exogenous chromosomes in porcine mature oocytes and suggest that their accumulation correlates with tubulin assembly and pronuclear formation.

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In the present study, to elucidate the cause of different developmental potential in porcine oocytes matured in two media, mTCM199 and mNCSU37, we examined the time course of changes in the nuclear states and p34^cdc2 kinase activity of oocytes during meiotic maturation. The p34^cdc2 kinase activity of oocytes cultured in mTCM199 gradually increased from earlier in the culture time periods than that of oocytes in mNCSU37, and that of oocytes in mNCSU37 rapidly increased from the middle of the culture period, but the oocytes in both media had similar levels of p34^cdc2 kinase activity at the end of the culture period. Oocytes cultured in mTCM199 underwent GVBD and reached the MII stage earlier in the culture period than those in mNCSU37. Moreover, MII oocytes cultured in either mTCM199 or mNCSU37 had a similar level of p34^cdc2 kinase activity at the end of the culture period. It was therefore concluded that the difference in developmental competence of oocytes matured in different media seems to be independent of p34^cdc2 kinase activity.

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The objective of this study was to determine the quality of semen in Japanese men born in the 1970’s. When samples were compared among generations (1970-72, 1973-75 and 1976-78), significant differences were not seen in the volumes and the abnormality rates. But the sperm concentration of the 1970-72 generation (114.8±52.5×10⁶ sperms/mL n=3) was higher than these of other generations (48.8±44.1×10⁶ and 58.0±27.0×10⁶ sperms/mL, respectively 1973-75 n=17 and 1976-78 n=28). There was no correlation between semen quality (sperm concentration and total sperm number) and the date of birth of the subjects. However their parents’ date of birth had an influence on semen quality. Environmental chemicals began to increase around the time the subjects’ parents were born, and it might be possible that parents’ reproductive ability was connected with their children’s semen quality.

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Two pairs of the ovaries were obtained from two giant pandas which accidentally died due to hepatic disease and senility. Of these ovaries, one pair of “twin oocytes” was collected from each pair of ovaries and processed for histological observation with an electron microscope. The results from this study suggest the possibility of the existance of “twin oocytes” in the follicle and of a higher rate of twinning in the giant panda.

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The present study was conducted to examine the effect of Japanese Kampo Medicines (JKMs) on mammalian spermatozoa and in vitro fertilization. Two kinds of experiments were carried out in this study to determine (1) the most effective mix ratios of JKMs which were determined by using PBS supplemented with JKMs, and (2) the most successful in vitro fertilization and subsequent embryonic development. In the first experiment, frozen-thawed bovine and goat spermatozoa were examined. Frozen thawed spermatozoa were kept for 3 h at 37°C in a 5% CO₂ incubator in media with added JKMs at different mix ratios and different concentrations. In the second experiment, in vitro fertilization (IVF) and consequent embryonic celeavage ratios were also determined. The present results show that all of the JKMs were effective at enhancing sperm function and in vitro fertilization, especially Syouhangekabukuryoto (JKMs21) and Hotyuekkito (JKMs41). In addition to this, all of the JKMs were confirmed to enhance spermatozoan function, in vitro fertilization and early embryonic development.

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Judgement of viability is essential for determining the effectiveness of cryopreservation method for ovaries. Freeze-thawed ovaries have been transplanted into the uteri of pseudopregnant rats, but the survival rate of after transplantation has been low. Since it has been reported that the survival rate of transplanted kidneys was improved when blood transfusion was performed before transplantation, the present study was designed to determine the effect of blood transfusion on the survival rate of transplanted mouse ovaries. Before transplantation of the ovaries in the uteri of rats, the recipient rats were given 1∼5 intraperitoneal blood transfusions. The ovaries were frozen using the vitrification method. After dehydrated the ovaries, they were kept in liquid nitogen. Immediatly after thawed the ovaries, they were transplanted into uteri of pseudopregnant rats that had received blood transfusion, and the survival rate of the ovaries was investigated. The viability of the transplanted ovarian follicles was examined using a morphological technique. It was found that the number of healthy follicles increased as the number of blood transfusions was increased, indicating that pre-transplantation blood transfusion into the recipient is an effective method for increasing the survival rate of transplanted ovaries.

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