The optimum voltage for inducing activation by electrostimulation of porcine oocytes matured in vitro was determined.Oocytes matured for 42 h were exposed to a single squared pulse for 100, μ sec at 100 to 3, 000 V/cm DC and examined 12 h later. More than 90%of eggs pulsed at 250 to 1, 250 V/cm were activated, and the highest rate (98%) was obtained at 750 V/cm. Oocytes matured for 36 to 54 h were subjected to the stimulation at 750 V/cm. The rates of activation in oocytes cultured for 36 and 38 h (47 and 44%, respectively) were significantly lower than in those cultured longer than 42 h (94 to 99%; P<0.01). Succeeding events were examined.Oocytes matured for 42 h were pulsed at 750 V/cm, and anaphase II (32%) and telophase II (32%) were observed 10 min and 30 min after stimulation, respectively. Nuclear formation was completed 4 h after stimulation in 95%of the eggs.Meiotic resumption induced by electrostimulation is thus quite synchronous. Extrusion of the second polar body and formation of a nucleus was observed in 92%of activated eggs. These eggs when further cultured developed beyond the 4-cell stage.

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Effects of oxytocin supplemented into medium on the development of mouse one-cell stage embryos were investigated. When the embryos with and without cumulus cells were cultured in the media containing 10^-14 to 10^-4M oxytocin, the developmental rates of 10^-10M oxytocin-supplemented group in the cumulusfree embryos were significantly increased compared to those of control, but the rates of 10^-4M oxytocin group were decreased. There, however, was almost no effect on the developmental rates in the cumulus-enclosed embryos. In addition, effect of 10^-10M oxytocin on the embryo development was compared to that of coculture with granulosa cells. There were no differences between oxytocinsupplemented and co-culture groups in the developmental rates to each stage from 2-cell to expanded blastocyst. But co-culture system obviously increased the cell number of blastocysts. The additional effect of supplementation with oxytocin into co-culture system on the developmental rate and the cell number of blastocysts was not found.

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We have attempted to sex preimplantation porcine embryos using a polymerase chain reaction (PCR) with the primers made from the SRY (sex-determining region of Y) conserved sequences of mammals.The sense primer was 5'-GTCAAGC GACCCATGAACGC-3'(20 mer) and the antisense primer was 5'-CTGTGCCTCCTG GAAGAATGGC-3'(22 mer).After amplification of male blood samples, the male specific DNA band was detected as a 165-bp fragment.These specific DNA bands were detected in 44-50% of the embryos from 16-cell to blastocyst stage, although the frequencies of positive samples were lower in 2-cell and 4-cell stage embryos. Furthermore, direct sequencing of prepared fragments revealed that there were considerable similarities among the porcine, human, mouse and rabbit sequences.

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In this study the bovine follicular fluid (bFF) from small follicles (2-5mm in diameter) or its fractions were added to maturation medium, and their effects on maturation, fertilization and cleavage of bovine follicular oocytes were examined. Oocytes from slaughtered Holstein cows were cultured in maturation medium supplemented with various concentrations of bFF or its fractions. After 22-24 hr of culture, the oocytes were coincubated with frozen-thawed spermatozoa, then cultured in development medium for 48 hr and at the end of culture evaluated for cleavage, nuclear maturation and fertilization. The bFF at 30% significantly enhanced maturation compared to bFF-free group (control); it also significantly enhanced cleavage beyond the 2-cell stage compared to bFF-free and 60% low molecular fraction (<10KD) groups. The 60% bFF group significantly enhanced fertilization compared to bFF-free group. In addition, the embryos derived from oocytes matured in the 60% bFF significantly proceeded to >2-cell stages. The fractions of bFF were without significant effect on the rates of maturation, fertlization or cleavage, except at 60% where the high molecular fraction (>10KD) significantly enhanced cleavage to 2-cells compared to bFF-free group, but the rates of embryos that cleaved beyond the 2-cell stage was significantly lower (P<0. 01) than in the 60% bFF group. In conclusion, addition of whole bFF (2-5mm follicles) to maturation medium was beneficial for maturation, fertilization and cleavage beyond the 2-cell stage of bovine oocytes at 30% and 60% concentrations. On the other hand, the fractions of bFF did not show significant effect on the rates of maturation and fertlization.

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The purpose of the present study was to investigate the efficacy of Serono Gamete Preparation Medium (GPM), which is used as the medium for human in vitro fertilization, as the fertilization medium for bovine oocytes matured in vitro. Oocytes were inseminated in GPM or GPM supplemented with 5mM caffeine and 10μg/ml heparin (GPM-CH). Brackett and Oliphant's (BO) medium supplemented with 5mM caffeine and 10μg/ml heparin (BO-CH) was used as a control medium. There were no differences in the rates of cleaved embryos and development to blastocysts between GPM-CH and BO-CH (72.0% vs 65.0%, 22.0% vs 25.0%, respectively). However, the rates were significantly low when oocytes were inseminated in GPM alone. It was found that there were no differences in the rates of development to blastocysts by using frozen spermatozoa derived from two different bulls when GPM-CH was used. These results indicated that GPM was successfully used as the fertilization medium for bovine oocytes.

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Our previous studies revealed that porcine oocyte-cumulus complexes that matured in porcine follicular fluid (pFF) and its active fraction resulted in a significant increase in the degree of cumulus expansion as compared with those matured in a modified Krebs-Ringer bicarbonate solution (mKRB) supplemented with bovine serum albumin. In this study, the dif f ereces between an image analyzer and calculation by a formula (LW π/4, where L and W is the length and width of cumulus, respectively) in measuring the degree of cumulus expansion were investigated. Photographs were taken of the oocyte-cumulus complexes matured for 24 h in the different medium and the areas were measured using these two methods. The areas occupied by expanded oocyte-cumulus complexes that matured in pFF-Top fraction were measured accurately only by the formula method. The image analyzer gave significantly lower values due to faint boundary of expanded cumulus. There was no significant difference between the two methods for those unexpanded or very little expanded complexes. Advantages and disadvantages of the two different methods were noted. The advantage in using the image analyzer is that it can measure the natural shape of the oocyte-cumulus complexes. The disadvantages of the image analyzer are: a) the well exp a nded cumulus cell layers can not be measured a ccuret ely, b) it has difficulty in measuring the oocyte-cumulus complexes in the presence of detached cumulus cells and c) only clearly taken photographs can be used for measurement. The advant ages noted for using the formula were: a) oocyte-cumulus complexes printed in the photographs can be measured separately from the detached cumulus cells that are present and b) photographs with either over-or under-exposed prints can be used as a subject for measurement.

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The present experiment was designed to investigate the effect of follicular shell and fluid added to maturation medium on the porcine sperm penetration rate and subsequent male pronucleus formation in oocytes. Pig follicular oocytes were cultured for 47h, with follicular shells, 10% follicular fluid or their combinations added to a medium drop of TCM-199. All treatments had no effect on the proportion of oocytes developing to second metaphase of meiosis. Following insemination, the penetration of oocytes by frozen-thawed sperms was not significantly different among the treated groups. However, the proportion of polyspermic oocytes was significantly lower (p<0.05) when the oocytes were cultured with 10% follicular fluid (25.8%; 16/62) than when they were cultured in other conditions. The percentage of oocytes capable of sustaining male pronucleus formation was higher (p<0.05) when they were cultured with follicular shells combined with 10% follicular fluid (53.8%;43/80) than in all the other maturation conditions. These results indicate that follicular fluid has an effective action on preventing polyspermy in oocytes and that the follicular shells combined with follicular fluid increases male pronucleus formation, inducing the cytoplasmic development of porcine oocytes during their in vitro maturation.

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To investigate the regulation of cell cycle during preimplantationdevelopment, the expression of cyclins was examined in early mouse embryos.Wefirst isolated a cDNA fragment of mouse cyclin A, corresponding to cyclin boxdomain, and determined the sequence. Mouse cyclin A is highly homologous tohuman cyclin A (98.9% in amino acid).The comparison of amino acid sequence ofcyclin A with those of B-and D-type cyclins revealed the sequences specific for A-and B-type cyclins and for A-and D- type cyclins. It is possible that thesesequences are involved in the association with cdc2 and cdk2 kinases, since A- and B- type cyclins are known to be associated with cdc2 kinase and A-and D-typecyclins are with cdk2 kinase. The expression of cyclin mRNAs was exmined by theRT-PCR assay in unfertilized oocytes and the embryos at the 2-cell, 4-cell, morulaand blastocyst stages. Cyclin A mRNA was expressed in unfertilized oocytes and thelevel was gradually decreased to the blastocyst stage. The B-type cyclins whichregulate G2/M phase transition were expressed in a low level at the 2-cell stage.The level of cyclin Bl was almost constant during preimplantation develoment, except for at the 2-cell stage. The level of cyclin B2 was decreased at the 2-cellstage and then increased from the morula to blastocyst stage. The long G2 phase atthe 2-cell stage may be attributable to the decrease in the level of expression of Btypecyclins. The D- type cyclins which regulate Gl/S transition were not constantlyexpressed. The expression of cyclin Dl was detected in only 2-cell embryos andblastocysts. The expression of cyclin D2 was not detected all through the preimplantationdevelopment. Cyclin D3 was detected in unfertilized oocytes. The level wasdecreased to the 4-cell stage and then increased from the morula to blastocyststage.It is possible that the regulation of Gl phase in early embryos is differentfrom that in most somatic cells in which D-type cyclins are required for Gl/Stransition.

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This study was conducted to determine the effect of PMSG and/or hCG (each 15 IU/ml) added to TCM -199 supplemented with 10% FCS and 3mg/m1BSA on the maturation, fertilization and the incorporation of ³H - methionine of pigoocytes. The follicular oocytes were cultured for 48hr with or without supplementationof PMSG, hCG, given each alone or in combination to maturation medium. Theaddition of PMSG with or without hCG to maturation medium was effective ininducting meiotic resumption in pig oocytes. Fertilization rate was significantlyimproved by addition of hCG alone or with PMSG, compared to the control. Fromthese results, it is suggested that the sperm penetration rate of oocyte was increasedby hCG added to the maturation medium. The proportion of polyspermic oocytes, however, was not different among the treatments. Incorporation of methionine intooocytes measured after maturation culture was increased by addition of PMSGand/or hCG as compared to the control. These results indicate that PMSG and/orhCG added to medium during maturation is related to the development of cytoplasmof pig oocytes.

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The present study was designed to investigate the effect of temperatures and duration during removal of ethylene glycol as a cryoprotectant after thawing on the survival of frozen bovine IVF embryos. Bovine blastcysts (7 to 8 days after IVF) were frozen in 1.5M ethylene glycol, and then thawed and diluted by the different dilution procedures as follows. A group: Embryos were expelled from straw within 5 min after thawing and placed directly into Dulbecco' s modified PBS supplemented with 20% calf serum (CS-PBS) at room temperature (22-23°C). B group: Embryos were expelled within 5min and placed directly into warm CS-PBS at 37°C. C group: Embryos were kept in the straw for 40min after thawing and placed into warm CS-PBS for dilution. After washing twice, embryos were co-cultured with granulosa cells in TCM-199 supplemented with 5 % calf serum for 48hrs. Blastcystes developed to the hatching stage were estimated as viable ones. Survival rate in B group was slightly higher than A group, although the difference was not significant. Survival rate in C group was significantly lower than B group. These date suggest that embryos frozen with 1.5M ethylene glycol should be diluted in the warm CS-PBS to obtain better survival rate and the duration in straw after thawing might affect the survival and pregnancy rate in direct transfer.

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The developmental expression of a zona pellucida antigen was examined by use of immunofluorescent stainings and electromicroscopic observations. A monoclonal antibody (MAb-5H4) which recognized an amino acid sequece of Cys-Thr-Val-Leu-Asp-Pro-Glu-Asn-Leu of pZPl was used for detection of the zona pellucida antigen.Electromicroscopic observations revealed that the zona pellucida antigen was deposited around oocytes in the primary follicles in a discontinuous pattern. The amount of the zona pellucida antigen increased according to the development of ovarian follicles in immunofluorescent stainings, suggesting that the zona pellucida antigen is produced continuously through the whole period of follicular development.The antigen was found not only outside but also inside of oocytes in primary follicles with 1-4 layers of granulosa cells. In addition, secretory vesicles containing the zona pellucida antigen were also observed in the cytoplasm of oocytes under the electromicroscope. It is thus concluded that during folliculogenesis the zona pellucida antigen was synthesized in the oocytes, transported to the plasma membrane by secretory vesicles, and was secreted.

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The effects of prostaglandins (PGs) on the development of mouse embryo in vitro was examined. Four cell embryos were obtained from mice after mating following PMS-hCG treatment. The embryos were cultured in the mBWW under 5% CO₂ in air at 37° with indomethacin (IND), PG (E₂, F₂α, I₂)(0-200 μM) and adenosine 3', 5'-cyclicmonophosphathiate sp-isomer (Sp-cAMPS), and the subsequent development of embryos was observed. In the control, 77-87% of 4-cell embryos developed to blastocysts (BL). BL formation was suppressed to 57.1 and 25% by 5 and 10 μM of PGE₂, 52.3% by 100 μM of IND, 25% by 50 μM of PGF₂α, while 200 μM of PGI₂ showed no effect. Sp-cAMPS also suppressed BL formation, but BL formation of these embryos was observed after release from Sp-cAMPS. These results indicate that PGs have a suppressive effect on the develoment of early mouse embryo, and PGE₂ was most effective among the agents used in this stydy. Sp-cAMPS also showed some effect, but its effect was seemed to be different from that of PGs.

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