This research was conducted to obtain the basic information on the cell block phenomenon occuring during early development in vitro of mouse embryos. Early embryos were recovered at 3h post-hGG injection(hph). Various chemicals (EDTA, EGTA, DTPA, MA and PRA) were tested to examine the effects of them on the overcoming the 2-cell block phenomenon. One hundreds M of the chelating agents were added to the M16 medium containing embryos. The treated embryos were worked and transferd to fresh M16 medium after 1, 3, 6 and 12h of treatment. Development was examined at 58 and l2Oph injection, respectively. 44.7~68.9% of the treated embryos developed to 4-cell stages at 58hph. Only 17.6~60.3% of the embyos developed upto blastocyst at l20hph. Whereas control embryos showed slightly lower development in M16 medium alone (38.9~42.4%, 4-cell and 3.8~65.5%, blastocyst). Three mitogenic agents were tested. 51.6~63.8% and 43.4~48.1% of embryos developed up to 2-cell and blastocyst stage, respectively when treated in 5 g PHA-M Imi for 5 min, 1, 3 and 6h subsequently cultured in fresh M16 medium. Control embryos only showed 38.8% for 4cell and 5.9% fo blastocyst at 58 and l2Ohph, respectively. 100M PMA was also beneficial for the 2-cell block. Showing better development them that of control (42.4 vs 57.9~59.4% 4cell and 5.9 vs 25.0~55.6% blastocyst, respectively. However 1M butyric acid was toxic to early embryos, thus arresting further development. These result indicate that either chelating or mitogenic agents could be used to overcome the "in vitro 2-cell block" occuring during early development in vitro of ICR embryosCR embryos

The present experirnents on cryopreservation were carried out to investigate effect of solution toxicity, equilibration time and cell stages on the post-thaw survival of mouse morulae and blastocyst embryos cryopreserved by vitrification in EFS solution. The mouse embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen and thawed rapidly. After the mouse morulae embryos were exposed to EFS solution for 2 and 5 ruin. at room temperature and then they were washed in 0.5 M sucrose solution and basal mediurn(D-PBS + 10% FCS), they were cultured to examined cryoprotectant toxicity induced injury during exposure, most of embryos developed to expanded blastocysts(100 and 90.0%). However, when the exposure time was extended to 10 and 20 min, these development rates dropped dramatically in 10 ruin. (75.0%) and 20 ruin. (4.5%), respectively. When the compacted morulae were vitrified in EFS solution after equilibration for 2 and 5 min, the embryos have developed to normal blastocyst following thawing, washing and culture processes was 89.3 and 89.6%. However, when the exposure time was expanded to 10 ruin, this survival rate dropped to 68.8%. When the blastocyst were vitrified in EFS solution after equilibration for 2, 5 and 10 minutes, the survival rate of embryos which developed to normal blastocyst following thawing and culture processing were 58.5, 46.7 and 22.4%, respectively. The optimal time of equilibration of mouse morula and blastocysts in EFS solution seemed o be 2 and 5 ruin.

This study was carried out to investigate the inhibiting or promoting effect of fetal bovine serum fractionated by the molecular weight and to examine the effect of reconstruction of serum fractions on the development of 1- and 2-cell mouse embryos fertilized in vitro (IVEE) . The serum was separated by ultrafiltration or gel filtration methods and added in m-KRB medium for culture of IVFE. The developemental ability(cavitation and hatching) of embryos following culture of day 4 and 6 was compared among fractions. Small molecular weight fraction( <3 kDa) significantly inhibited the development of 1-and 2-cell IVFE to the blastocyst stages, compared with other fractions. One-cell IVFE were more sensitively damaged than 2-cell embryos by that fraction and arrested mainly at 2~4 cell stages. Moreover, small amount(<3%,v /v) of the inhibiting fraction acted even with protein rich fraction(100~30 kDa) and arrested the embryonic development. On the other hand, 100~30 kDa fraction promoted the embryonic development and no inhibiting effect was observed at the level of 50%(v /v) in culture medium In the experiment of gel filtraton, =30 kDa fraction showed the highest promoting effect on the embryonic development, but <4 kDa fraction inhibited significantly the development. These results suggest that serum contains not only small molecular weight inhibitory component(s) but also promoting one rather than albumin on embryonic development. And serum can be more effectively used in the IVF program after removal of inhibitory component(s) by one of above separation methods.

This study was carried out to investigate the aggregation rate of isolated mouse 2-, 4- and 8-cell stage blastomeres in phytohemagglutinin(PHA) solution. The morphologically normal embryos were collected from the oviduct of superovulated female mouse by flushing with M2 and the zona pellucida of embryos were removed with 0.5% pronase. The blastomeres were isolated by pipetting after plunging into Ca++-Mg++free PBS for 20 min. The result showed that aggregation rate in 0.5% (84.9~93.1%) was higher than that in 1.0% PHA(76.0~82.1%). Optimal aggregation time was 60min (83.9~100.0%) when compared with 30min (78.8~87.5%). Developmental to blastocyst in recombinated blastomeres was higher under conditions of 0.5% PHA solution and 60-min aggregation than that under other conditions.

To investigate the effect of extracellular matrix proteins on the in vitro development of ethanol-induced parthenogenetic eggs of ICR strain mice, those were cultured in vitro in fibronectin, gelatin, or collagen precoated culture dishes containing 1.5 ml of NaH-C03-BMOC-3 medium at 37 for 96 hrs. under the atmosphere of 5% and 95% air. Fibronectin, gelatin, or collagen significantly(P1.4, 45.4i1.4, and 44.8O.9, respectively. And the diameter of those eggs ranged 104.61.9, 102.82.3, and 103.4O.8 m, respectively.

To investigate the effect of EDTA on the in vitro development of blastomeres isolated from 2, 4, and 8-cell embryos(termed 1 /2, 1 /4 and 1 /8 blastomeres, respectively) of ICR strain mice, those were cultured in vitro in 35 mm culture dishes containing NaHCO-BMOC-3 medium supplemented with 10, 50, 100, or 500 M of EDTA at 37 for 72hrs. under the atmosphere of 5% and 95% air. EDTA supplementation of 10, 50, or 100 M to medium significantly(P<0.01) increased blastocyst formation rate compared with controls in 1 /2(58.3, 63.7, and 61.3% vs 21.6%), 1 /4(54.7, 57.5 and 62.2% vs. 2L3%), and 1 /8 blastomeres(46.2, 48.7, and 57.7% vs. 19.1%). Whereas, it was significantly(P<0.01) decreased to 4.5, 2.3, and 2.0% for 1 /2, 1 /4 and 1 /8 blastomeres, respectively by the EDTA supplementation of 500 M Both the nuclear number(P<0.05) and diameter of blastocysts(P<0.01) developed from balstomeres were significantly affected by the origin of blastomeres. The nuclear number of blastocysrs developed from 1/2, 1/4, and 1/8 blastomeres ranged 28.3i1.3, 24.18i1.2, and 19.840.9, respectively. And the diameter of those blastocysts was 87.21.1, 56.40.9, and 39.20.8 M, respectively.