OBJECTIVE: This study was to determine the effect of different concentration of calcium in medium on the preimplantational development of zygotes and early 2-cell embryos. METHODS: Female mice of ICR strain (5~8 weeks old) were superovulated and mated with fertile males. Zygotes or early 2-cell embryos were collected by flushing the oviducts 31~32 hours after hCG injection. The embryos were cultured in various concentrations of Ca2+ in medium or with EDTA, EGTA and Ni2+. RESULT AND CONCLUSION: Treatment of high concentration of Ca2+ (3.42 mM (2X)~17.1 mM (10X) in medium didn't develop well compared to the control Low concentrations of Ca2+ (0.214 mM (1/8X)~0.855 mM (1/2X)) were deterimental to development beyond 2-cell stage. EDTA, Ca2+ chelating agent was treated with ranged concentrations of eDTA (0.014 mM~0.107 mM) to medium contaning 1.71 mM Ca2+ showed beneficial effect to development to blastocyst compared to the control. EGTA, extracellular Ca2+ chelator, was treated with ranged concentrations of EGTA (0.014~0.107 mM) to the medium contaning 1.71 mM Ca2+. There is no significant difference with the control. Ni2+ (50 micrometer), T-type Ca2+-channel blocker was treated to medium contaning low concentration of Ca2+. It overcame 2-cell block significantly. Rate of degenerated embryos decreased and developmental rate to morula and blastocyst increased more than low Ca2+ concentration alone. Further studies are needed for the overcoming effect of 2-cell block by Ni2+.
Animals ; Blastocyst ; Calcium ; Edetic Acid ; Egtazic Acid ; Embryonic Structures* ; Female ; Flushing ; Humans ; Male ; Mice* ; Morula ; Oviducts ; Zygote

OBJECTIVE: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. MATERIAL AND METHOD: Immature mouse oocytes were obtained from the ovarian follicles of 3~4 weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at 37degrees C, 5% CO2 and 21% O2 (95% air) or 5% CO2, 5% O2 and 90% N2. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of 0.0001 micrometer, 0.01 micrometer or 1.0 micrometer. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. RESULTS: Under 21% oxygen condition, oocytes cultured in the presence of 0.01 micrometer melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with 0.0001 micrometer or 1.0 micrometer melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of 0.01 micrometer melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. CONCLUSION: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.
Animals ; Bucladesine ; Female ; Free Radicals ; Hypoxanthine ; Mammals ; Melatonin* ; Metabolism ; Mice* ; Oocytes* ; Ovarian Follicle ; Oxygen ; Pineal Gland ; Polar Bodies ; Seasons

No abstract available.
Animals ; Mice* ; Microinjections* ; Zygote*

OBJECTIVE: In muscle and neuronal cells, calcium channels have been classified by electrophysiological and pharmacological properties into (1) voltage-dependent Ca2+-channel(1) P/Q-type Ca2+-channel (2) N-type Ca2+-channel R(3) L-type Ca2+-channel (4) T-type Ca2+-channel (5)R-type Ca2+-channel. The present study was done in order to investigate whether there is any difference in Ca2+-channel distribution between activated and normally fertilized embryos. METHODS: The immunocytochemical method was used to identify the existence of voltage-dependent Ca2+-channels in parthenogenetically activated 2-cell embryos by ethanol and SrCl2 treatment. These 2-cell embryos were obtained by exposure to 6% ethanol for 6 min and to 10 mM SrCl2 for 2h. RESULTS: P/Q-type Ca2+ channels and L-type Ca2+-channels have been identified. Whereas, three type of Ca2+-channel P/Q-type, N-type, L-type have been identified in 2-cell embryos fertilized in vivo. CONCLUSION: Activation by ethanol was faster than those by SrCl2. However, there was difference in DAB staining of the embryos between ethanol and SrCl2 treatment (87.7% and 54.1%). Intensity of staining was also different between ethanol- and SrCl2-treated group. However, it has not been known why there was some difference in DAB staining and staining intensity in the present study.
Mice ; Animals

Female ; Humans ; Mice ; Animals

No abstract available.
Animals ; Gonadotropins* ; Mice* ; Oocytes*

No abstract available.
Carcinoma, Papillary* ; Thyroid Gland* ; Tretinoin*

OBJECTIVE: The study was performed to compare the survival rate and the development of day 2 mouse embryos which had freezing procedures done. METHODS: We used three different vitrification solutions (EFS, VS14, DPS) and a ultrarapid freezing solution (UFS) for cryopreservation of day 2 mouse embryo. RESULTS: We tested toxicity by exposing embryos to vitrification solutions and a ultrarapid freezing solution. The survival rates are 100%, 97.8%, 95.6% and 100% (EFS, VS14, DPS and UFS). After cultured for 96 hours, hatching rates of each group are 93.5% (no freezing), 95.6% (EFS), 86.4% (VS14), 93.0% (DPS), and 93.0% (UFS). There is no significant differences among groups. The survival rates after thawing cryopreserved embryos are 80.2%, 91.7%, 69.5%, 0% and 91.8% (slow freezing, EFS, VS14, DPS and UFS). Also cultured for 96 hours, the hatching rates are 93.5% (no freezing), 84.1% (slow freezing), 93.9%) (EFS), 48.5% (VS14) and 70.1% (UFS). CONCLUSION:The survival rates of vitrification in EFS solution and ultrarapid freezing are higher than slow freezing (p<0.05). The hatching rate of vitrification in EFS solution cultured for 96 hours is highest, so vitrification of day 2 mouse embryos in EFS solution considered as more effective for cryopreservation.
Animals ; Cryopreservation ; Embryonic Structures* ; Freezing* ; Mice* ; Survival Rate ; Vitrification*

The use of hormonal stimulation in human in vitro fertilization and embryo transfer (IVF-ET) leads to increased production of embryos for ET. So to avoid high pregnancies and to allow conception in future, unstimulated cycles, cryopreservation of spare embryos is desirable. One of the improvement of cryopreservation methods is vitrification. We cryopreserved mouse day 3 embryos by vitrification using the three different vitrification solution (EFS40, VSll and VS3a). EFS40 solution is consisted of 40% (v/v) ethylene glycol, Ficol170 30% (w/v) and 0.5M sucrose and VSll is 6.0M ethylene glycol and 1.8M glycerol. And VS3a is 6.5M glycerol and 6% (w/v) BSA (bovine serum albumin). First we tested the toxicity of three vitrification solution by exposure to these solution during 3 min. After washing by thawing solution, the survival rates of each groups are 95.5%, 90.9% and 84.4% (EFS40, VS11 and VS3a). High percentages of them developed to expanded blastocyst and hatching embryos in culture 48hrs 94.2%, 97.7%, 100% and 97.4% (no treatment group, EFS40, VS11 and VS3a). So there is no significant differences among the each group. Second, after thawing of vitirfied embryos, the survival rates of each groups are 96.8% (slow freeze), 94.1% (EFS40), 85.5% (VS11) and 80.0% (VS3a, P vs. no freeze or EFS40 is 0.01). Vitrified embryos exhibited a high rate of development in vitro after 48hrs culture. The percentages of each group to blastocyst and hatching embryos are 88.7% (no freeze), 91.8% (slow freeze), 93.4% (EFS40), 87.7% (VS11) and 73.0% (VS3a, P vs. other group is 0.01). The results suggest that there is no significant differences in exposure of various vitrification solution and day 3 mouse embryos can be vitrified in solution EFS40 and VS11 by simple procedure.
Animals ; Blastocyst ; Cryopreservation* ; Embryo Transfer ; Embryonic Structures* ; Ethylene Glycol ; Fertilization ; Fertilization in Vitro ; Glycerol ; Humans ; Mice* ; Pregnancy ; Sucrose ; Survival Rate ; Vitrification*

No abstract available.
Animals ; Calcium* ; Mice* ; Oocytes*