• Journal of Embryo Transfer
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• v.22 no.2
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• pp.121-126
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• 2007

The objective of this study was carried out to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as non-invasive marker to know the developmental competence in advance. The porcine oocytes matured for 48 h were examined the polar body extrusion. The examined oocytes were matured for additional $16{\sim}18h$ and activated with 7% ethanol and cultured in $5{\mu}g/ml$ cytochalasin B for 5 h for diploid formation. The treated oocytes were cultured and examined the cleavage after 48 h and continued culturing for 5 days. The oocytes of 21.9% were discarded in morphological selection and 32.1% oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated and then after 48 h the cleavage rates were examined. In morphologically selected oocytes, 15.8% oocytes were not cleaved and 52.6% oocytes were normally cleaved and 31.6% oocytes were hyper-cleaved over 8-cell stage. However in the first polar body extruded oocytes, 7.1% oocytes were not cleaved and 73.1% oocytes were normally cleaved and 19.8% oocytes were hyper-cleaved. The morphologically selected embryos that not cleavage-selected were developed in 16.7% up to blastocyst and the morphologically selected and cleavage-selected embryos were developed in 31.7%. The polar body extruded oocytes that were not carried out cleavage selection were developed in 39.0% and the polar body extruded and cleavage-selected embryos were developed 49.0%. The first cleavage timing was examined with 12 h interval after activation. In $0{\sim}12,\;12{\sim}24,\;24{\sim}36,\;and\;36{\sim}48h$ intervals, 4.1%, 68.6%, 19.1%, and 2.3% oocytes were cleaved and 5.9% oocytes were not cleaved until 48 after activation. The cleaved oocytes in each interval were cultured and developed upto blastocyst with 0, 39.1, 9.5, and 0%, respectively. This results suggests that polar body extruded and cleaved at $12{\sim}36h$ embryo has higher developmental potential than the others.

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.279-287
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• 2005

This study was carried out to investigate resumption of ovarian cyclicity and effect of BCS on estrous response by treatment of $PGF_2a+PGF_2a+CIDR$ program on day 40 postpartum in lactating dairy cow. First $PGF_2a$ was given on day 40 postpartum, second $PGF_2a$ was given 14 days apart to cows not-responded to 1st $PGF_2a$ and then CIDR was inserted for 7 days after 5 days in cows not-responded to 2nd $PGF_2a$. The $42.9\%$ of the cows showed more than 1 ng/mL milk progesterone concentration within 10 to 30 days postpartum. About $19\%$ of the cows exhibited more than 1 ng/mL milk pro-gesterone concentration between 31 to 50 days postpartum. However $38.1\%$ of the cows have not shown more than 1 ng/mL milk progesterone up to 50 days postpartum. Estrous response to the treatment of 1 st $PGF_2a$ and 2nd $PGF_2a$ was $47.5\%$ and $52.4\%$, respectively. Combination of 1 st $PGF_2a$ and 2nd $PGF_2a$ was $75\%$ and combination of 1st $PGF_2a$+2nd $PGF_2a$+CIDR was $87.5\%$. Estrous response to the treatment of $PGF_2a+PGF_2a$ program was $61.5\%$ in cows with less than 2.50 BCS and $81.5\%$ in cows with 2.75${\~}$3.50 BCS. Estrous response to the treatment of CIDR was $40\%$ in cows with less than 2.50 BCS and $80\%$ in cows with 2.75${\~}$3.50 BCS. Estrous response to the treatment of PPC on day 40 postpartum was $76.9\%$ in cows with less than 2.50 BCS and $96.3\%$ in cows with 2.75${\~}$3.50 BCS.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.89-99
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• 2004

Stretch-activated channels (SACs) responds to membrane stress with changes in open probability (Po). They play essential roles in regulation of cell volume and differentiation, vascular tone, and in hormonal secretion. SACs highly present in Xenopus oocytes and Ascidian oocytes are suggested to be involved in the regulation of pH and fluid transport to balance the osmotic pressure, but remain unclear in mammanlian oocytes. This study was investigated to find the presence of SACs in hamster oocytes and to examine their electrophysiological properties. To infer a role of SAC in relation to the development of early stage, we followed up to the stage of two-cell zygote with patch clamp techniques. Single channels were elicited by negative pressure (lower than ­15 cm$H_2O$). Interestingly, SACs were dependent on permeable cations such as $Na^+$ or $K^+$. As permeable cation removed from both sides across the membrane, SAC activity completely disappeared. When permeable cations present only in intracellular compartment, outward currents appeared at positive potentials. In contrast to this, inward currents occurred only at the negative voltage when permeable cation absent in cell interior. These result suggests that SAC carry cations through the nonselective cation channel (NSC channel). Taken together, we found that stretch activated channels present in hamster oocyte and the channel may carry cations through NSC channels. This stretch activated-NSC channels may play physiological role(s) in oocyte growth, maturation, fertilization and embryogenesis in fertilized oocytes to two-cell zygotes of hamster.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.101-112
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• 2004

Chloride($Cl^-$) channels play critical roles in cell homeostasis and its specific functions such as volume regulation, differentiation, secretion, and membrane stabilization. The presence of these channels have been reported in all kinds of cells and even in frog oocytes. These essential role of $Cl^-$­ channels in cell homeostasis possibly play any role in egg homeostasis and in the early stage of development, however, there has been no report about the presence of $Cl^-$­ channel in the mammalian oocyte. This study was performed to elucidate the presence of $Cl^-$­ channels in hamster eggs. When allowing only $Cl^-$­ to pass through the channel of the egg membrane by using impermeant cation such as N-methyl-D-glucamine(NMDG), single channel currents were recorded. These channel currents showed typical long-lasted openings interrupted by rapid flickering. Mean open $time({\tau}o)$ was 43${\pm}$10.14 ms(n=9, at 50 mV). The open probability(Po) was decrease with depolarization. The current-voltage relation showed outward rectification. Outward slop conductance(32${\pm}$5.4 pS, n=22) was steeper than the inward slop conductance(10${\pm}$1.3 pS). Under the condition of symmetrical 140 mM NaCl, single channel currents were reversed at 0 mV(n=4). This reversal potential(Erev) was shifted from 0 mV at 140 mM concentration of internal NaCl(140 mM [Na+]i) to ­9.8${\pm}$0.5 mV(n=4) at 70 mM [Na+]i and 11.5${\pm}$1.9 mV at 280 mM [Na+]i(n=4) respectively, strongly suggesting that these are single $Cl^-$­ channel currents. To examine further whether this channel has pharmacological property of the $Cl^-$­ channel, specific Cl­ channel blockers, IAA-94(Indanyloxyacetic acid-94) and DIDS(4, 4'-diisothiocyan ostillben- 2-2'disulfonic acid) were applied. IAA-94 inhibited the channel current in a dose-dependent manner and revealed a rapid and flickering block. From these electrophysiological and pharmacological resluts, we found the novel $Cl^-$­ channel present in the hamster oocyte membrane. The first identification of $Cl^-$­ channel in the hamster oocyte may give a clue for the further study on the function of $Cl^-$­ channel in the fertilization and cell differentiation.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.307-317
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• 1998

This study was to investigate the vitrification method for cryopreservation technique of bovine in vitro fertilization(IVF) embryos. The morphological appearance and viability following vitrification of IVF bovine blastocysts and expanded blastocysts were examined. Embryos obtained 6, 7, 8 or 9 days after IVF were vitrified in both 40% ethylene glycoI(EG) plus 0.3M trehalose and 20% polyvinylpyrrolidone(PVP) in DPBS(ETP, Exp. 1) and ETP solution added 0.375M dextrose (ETPD, Exp. 2). The viability of Days 6, 7, 8 and 9 vitrified /thawed embryos at 24∼48 h culture after thawing was 11.9%, 19.8%, 23.4% and 15.3% in ETP(Exp. 1), and 34.6%, 54.5%, 37.9% and 13.0% in ETPD(Exp. 2), respectively. The viability of vitrified embryos produced from the culture days after IVF did not differ in Exp. 1, but significantly differ in Exp. 2(P＜0.05). The viability of blastocysts and expanded blastocysts significantly differed(P＜0.05) in 15.2% and 23.3%(Exp. 1), and 25.0% and 45.8%(Exp. 2). The result of Exp. 1 was similar to that of Exp. 2 on the viability of embryo according to developmental stages, but ETP solution plus sugar(dextrose) was increased the viability of vitrified embryos. In Experiment 3, The viability of vitrified embryos was not different between 12% and 20% PVP concentrations in ETP solution according to culture days or developmental stages. To investigate the effect of addition of sugar, two type of carbohydrates and a mixture of cryoprotectants for vitrification on the survival of bovine IVF embryos, bovine Days 7 to 9 blastocysts and expanded blastocysts were cryopreserved in either 20% glycerol plus 20% EG, 0.375M sucrose and 0.375M dextrose (GESD, Exp. 4) or 20% glycerol plus 20% EG, 0.3M trehalose and 20% PVP(GETP, Exp. 5) in DPBS. Survival rates of Day 7, 8 and 9 embryos at 24∼48h culture after thawing were 71.4%, 94.6% and 40.5% in GESD, and 59.5%, 81.5% and 62.5% in GETP, respectively. Hatching rates of Day 7, 8 and 9 embryos after thawing were 28.6%, 35.1% and 16.2% in GESD, and 27.0%, 33.3% and 18.8% in GETP, respectively. These results indicates that a mixture of cryoprotectants(glycerol and EG) and addition of sugar can improve the survival rates of the bovine IVF embryos(Day 7 or 8) vitrified, and the expanded blastocyst embryos are more suitable for vitrification than early blastocysts stage.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.26 no.3
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• pp.251-256
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• 1981

Silage yield and nutritional values of a locally collected Korean corn line with high tillering and earing characteristics were investigated at plant densities from 2778 to 8333 plants/ l0a. This line and a non-tillering and nonprolific corn as a check were grown at four plant densities and at three planting dates. The Korean local line abbreviated as MET had the highest dry matter per l0a. The highest dry matter of the MET line compared with the check hybrid was due to the highest dry leaf weight of the MET line. The highest dry leaf weight of the MET line was due to the increased number of tillers of the MET line. Other plant parts such as ear weight, kernel weight and cob weight of the MET line were lower than those of the check hybrid. The dry husk weight per l0a of the MET line was higher than that of the check hybrid, probably due to the increased number of ears in the MET line. The total embryo production per 10 a of the MET line was significantly higher than that of the check hybrid. The increased portion of embryo of the MET line is probably responsible for the higher TON values of the MET line. No interaction between variety x planting dates or planting density was found, indicating that the MET line and check hybrid were both the same in effects of planting dates and densities. Both line and hybrid showed the highest dry matter production when the planting density was high and planting dates was early. When silage was made from either MET line or check hybrid the nutritional values in terms of crude protein, crude fat, fiber, and ash contents of the MET line were similar to those of the check hybrid. But the TON of the MET line was higher than that of the check hybrid, while the OCP of the MET line lower than that of the check hybrid. Amino acid contents of the MET line were also comparable to those of the check hybrid, while lysine content of the MET line was 10% higher than that of the check hybrid.

• Development and Reproduction
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• v.10 no.2
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• pp.105-113
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• 2006

Reactive oxygen species(ROS) generated in cellular metabolism have an effect on cell maturation and development. In human reproductive tract, oxidative injury by ROS may induce female infertility. Also, oxidative injury may be responsible for developmental retardation and arrest of mammalian preimplantation embryos. Activating transcription factor 4(ATF4) is a member of the cyclic-AMP response element-binding(CREB) familiy of basic region- leucine zipper(bZip). ATF4 is known to regulate stress response to protect cell from various stress factors and inducer of apoptisis. The purpose of this study was to investigate whether ATF4 is involved in the defensive mechanism in oxidative stress condition during the development of mouse preimplantation embryos. To verify the expression of ATF4 in oxidative stress condition, 2-cell stage embryos were cultured in HTF media containing 0.1mM, 0.5mM or 1mM hydrogen peroxide($H_2O_2$) for 1hr(2-cell), 8hr(4-cell), 17hr(8-cell), 24hr(morula), 48hr(early blastocyst) or 64hr(late blastocyst). The developmental rate decreased in the 0.1mM $H_2O_2$ treated group compared with control group. In embryos treated with 0.5mM and 1mM $H_2O_2$ showed 2-cell block. As a results of the semi-quantitative RT-PCR analysis of SOD1, ATF4 and Bax gene expression, SOD1, ATF4 and Bax genes were increased in 0.1mM, 0.5mM, 1mM $H_2O_2$ treated groups compared with control group. In 2-cell embryos, expression of SOD1, ATF4 and Bax genes were notably increased in 0.1mM, 0.5mM, 1mM $H_2O_2$ treated groups compared with control group. Immunofluorescence analysis showed that ATF4 protein was localized at the cytoplasm of preimplantation embryos. The increase in ATF4 immunoreactivety was observed in the 0.1mM, 0.5mM, 1mM $H_2O_2$ treated groups compared with control group. It suggests that oxidative stress by $H_2O_2$ induces expression of ATF4 and may be involved in protection mechanism in preimplantation embryos from oxidative injury.

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.293-300
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• 1998

The objective of this study was to determine effects of $\beta$-mercaptoethanol ($\beta$-ME) and cyst-eine (CYS) on the development of bovine em-bryos obtained from in vitro matured and fertil-ized oocytes. Cumulus-oocyte-complexes (COC-s) were matured in micro-drop of TCM-199 medium containing 10% FBS, 17$\beta$-Estradiol and FSH-p under paraffin oil at 39$^{\circ}C$ for 24 hrs. The fertilization of COC were induced in Fert-TALP medium supplemented with PHE, heparin, BSA and then the fertilized oocytes were cultured in CR1aa medium for 24 hrs. To investigate the effects of the agents on the development of the embryos, the embryos developed to the late 2-cell stage were cultured in the media with and without $\beta$-ME, CYS for 9 days. In experiment 1, to select appropriate concentration of $\beta$-ME and CYS during whole culture period (9 days), various concentrations of $\beta$-ME and CYS were add ded to the CR1aa medium. Addition of 25TEX>$\mu$M of $\beta$-ME and O.1mM of CYS to the culture medium 1 increase the incidence of embryos developed to the blastocyst. In experiment 2, we evaluated the effects of 25$\mu$M of $\beta$-ME and O.1mM of CYS addition on the blastocyst formation when emb bryos at different stages were exposed to 25$\mu$M $\beta$-ME and O.1mM of CYS. $\beta$-ME and CYS enhanced in vitro development of embryos to the blastocyst stage. The effect was greater in 8-ceII to morula embryos than in embryos fewer than 2-cells at the initiation of treatment. These results suggested that the addition of 25$\mu$M B-ME and O.1mM cysteine enhanced development to the blastocyst and hatching stage of in vitro derived bovine embryos, also addition of $\beta$-ME and cysteine were effective later stage embryo than early embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.281-292
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• 1997

The objective of this study was to compare retrospectively the survival and pregnancy rates(PR) of cryopresered-thawed embryos obtained from intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). Ninety-six cycles of cryopresered-thawed embryo transfer (ET) were performed in 79 patients from June, 1996 to September, 1997 and grouped as followings: 20 cycles (16 patients) inseminated by ICSI (ICSI Group) and 76 cycles (63 patients) by conventional IVF (IVF Group). Slow-freezing and rapid-thawing protocol was used with 1.5M propanediol (PROH) and 0.1M sucrose as cryoprotectant. All embryos were frozen-thawed at the two pronuclear (2 PN) stage excluding four cycles in which the early cleavage stage embryos were frozen, and allowed to cleave in vitro for one day before ET. The duration from freezing to thawing was comparable in both groups ($mean{\pm}SD$, $112.1{\pm}80.0$ vs. $124.8{\pm}140.1$ days). The age of female ($31.2{\pm}3.4$ vs. $32.6{\pm}3.3$ years) and the endometrial thickness prior to progesterone injection ($9.4{\pm}2.0$ vs. $9.3{\pm}1.8$ mm) were also comparable in both groups. There was no significant difference in the outcomes of cryopreserved-thawed ET between two groups: survival rate ($85.2{\pm}16.1%$ vs. $82.2{\pm}19.7%$), cleavage rate ($96.9{\pm}6.7%$ vs. $94.7{\pm}13.0%$), cumulative embryo score (CES, $54.5{\pm}31.1$ vs. $49.0{\pm}20.0$), preclinical loss rate (5.0% vs. 5.3%), clinical miscarriage rate (0% vs 29.4%), clinical PR per transfer (35.0% vs. 22.4%), implantation rate (9.9% vs. 5.6%), and multifetal PR (42.9% vs. 17.6%). In conclusion, human embryos resulting from ICSI can be cryopreserved-thawed and transferred successfully, and the survival rate and PR are comparable to conventional IVF.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.31 no.2
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• pp.129-142
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• 1986

Fifty-five local collections of buck wheat, Fagopyrum esculentum, were investigated their ratios of long-styled (LS) and short-styled (SS) flowers, fertility, meiosis of megaspore and microspore mother cell, female and male gametogenesis, and egg apparatus in accordance with the sowing seasons (spring, summer), altitudes (20m, 50-100m, 300m), and parent style types (L, S). Also they were embryologically investigated the fertility, fertilizing phenomenon and proembryogenesis by the legitimate and illegitimate pollination. There were no differences in the ratios of long-styled and short-5tyled flowers along with altitudes, but more irregularness was observed in plain area than that in the mountaineous or coastal area. LS versus SS ratios by sowing seasons were significantly separated into 1 : 1 in the summer sowing (P 0.1), but they were irregularly separated in the spring sowing. The segregating ratios by parent style types showed more number of short-styled flower in the spring sowing, and were statistically seperated into 1 : 1 in the summer sowing (P 0.25), regardless to parent style types. In the artificial legitimate union, the seed setting rates of the summer sowing (59-61%) were much higher than those of the spring sowing (about 30%), but in the artificial illegitimate union the seed setting rates were only fructified about 0.8-1.8% in the spring sowing. The seed setting rates in accordance with flowering stages were larger in turn early, middle, late, in the summer sowing. The grain number and grain weight per plant of short-styled flower were more than those of long-styled one regardless to style types. The 1,000 grain weight of long-styled flower was heavier than that of short-styled one in large grain, but it was lighter than that of short-styled flower in small or medium grain. The percentage of normal female and male gametogenesis in the summer sowing were higher than those in the spring sowing. The ovule was atropous and two polar nuclei were a synkarion before flowering. The pollens germinated at 30 minuts after pollination and the pollen tube grew continually and penetrated into micropyle at 1.5-2 hours and the two male nuclei fertilized with egg nucleus at 3 -5 hours after pollination. Flertilizing times in summer were shorter than in autumn. The fertilized egg was divided in a small apical cell toward the interior of the embryo sac and a large basal cell toward the micropyle cell at 15-24 hours after pollination, and division times in summer were shorter than in autumn. The proembryo began the embryogenesis at 7-8 days and formed itself into the perfect embryo at 15 days after pollination.