• Development and Reproduction
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• v.14 no.2
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• pp.59-63
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• 2010

This study was performed to examine the influence of water temperature on egg developmental speed for determining the required time and optimum water temperature for hatching of olive flounder Paralichthys olivaceus eggs. The fertilized eggs were collected from the naturally spawned adults in November 2007. The eggs were randomly divided into 6 groups of temperature (5, 10, 15, 20, 25 and $30^{\circ}C$) and transferred in $1{\ell}$ beaker, respectively. The fertilized eggs of the olive flounder did not hatched at $5^{\circ}C$ and $30^{\circ}C$ and hatching rates at 10, 15, 20 and $25^{\circ}C$ were 3, 12, 25 and 50%, respectively. The relationships between the water temperature (T, $^{\circ}C$) and required time (1/t, hour) from egg to each developmental stage were given as follows ; Blastula: 1/t=0.0208T-0.0951 ($r^2$=0.8593) Kupffer's vesicle: 1/t=0.0052T-0.0176 ($r^2$=0.9819) Myotome: 1/t=0.0034T-0.0172 ($r^2$=0.8508) Hatching: 1/t=0.0016T-0.0068 ($r^2$=0.9915) Biological minimum temperature in egg development was calculated to be $4.3^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1271-1279
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• 2009

To develop low endotoxic and multi-immunogenic outer membrane vesicles (OMVs), a deletion mutant of the msbB gene in Salmonella enterica serovar Typhimurium (S. Typhimurium) was used as a source of low endotoxic OMV, and an expression vector of the canine parvovirus (CPV) VP2 epitope fused to the bacterial OmpA protein was constructed and transformed into the Salmonella ${\Delta}msbB$ mutant. In a lethality test, BALB/c mice injected intraperitoneally with the Salmonella ${\Delta}msbB$ mutant survived for 7 days, whereas mice injected intraperitoneally with the wild type survived for 3 days. Moreover, all mice inoculated orally with the ${\Delta}msbB$ mutant survived for 30 days, but 80% of mice inoculated orally with the wild type survived. The OmpA::CPV VP2 epitope fusion protein was expressed successfully and associated with the outer membrane and OMV fractions from the mutant S. Typhimurium transformed with the fusion protein-expressing vector. In immunogenicity tests, sera obtained from the mice immunized with either the Salmonella msbB mutant or its OMVs containing the OmpA::CPV VP2 epitope showed bactericidal activities against wild-type S. Typhimurium and contained specific antibodies to the CPV VP2 epitope. In the hemagglutination inhibition (HI) assay as a measurement of CPV-neutralizing activity in the immune sera, there was an 8-fold increase of HI titer in the OMV-immunized group compared with the control. These results suggested that the CPV-neutralizing antibody response was raised by immunization with OMV containing the OmpA::CPV VP2 epitope, as well as the protective immune response against S. Typhimurium in BALB/c mice.

• Korean Journal of Animal Reproduction
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• v.23 no.3
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• pp.263-270
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• 1999

This study was conducted to investigate effects of L-ascorbic acid and selenium on maturation, fertilization, and development ablity of porcine follicular oocytes in vitro. When the follicular oocytes were cultured in the media containing 0, 62.5, 100 and 300 $\mu$M of L-ascorbic acid for 40~44h, the percentages of germinal vesicle breakdown were 86.8, 92.9, 91.7 and 92.6% respectively, and the nuclear maturation rates (M II) were 44.7, 57.1, 52.8 and 53.7%. The nuclear maturation rates of treated groups were significantly higher than those of non-treated group (p＜0.05). When the follicular oocytes were cultured at 0, 0.4, 0.8, and $1.5\mu$M of selenium for 40~44h, the nuclear maturation rates of treated groups were significantly higher than those of non-treated group (p＜0.05). The addition of L-ascorbic acid or selenium to the maturation medium, the incidence of male pronuclear formation was significantly increased (p＜0.05) and polyspermy rate was significantly decreased (p＜0.05). The addition of L-ascorbic acid or selenium to the maturation medium increased the clevage rate, morula and blastocyst rate (p＜0.05). These results suggested that the addition of L-ascorbic acid and selenium to maturation medium increase the nuclear maturation rates, male pronuclear formation and normal embryonic development: in porcine oocytes matured and fertilized in vitro.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.221-225
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• 2011

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide ($H_2O_2$), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decrease with addition of $H_2O_2$, and were significantly (p<0.05) lower in medium with 0.1 mM $H_2O_2$ than control group. Also, the rate of degenerated oocytes was increased in as $H_2O_2$ concentration in eased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When $H_2O_2$ concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM $H_2O_2$ and (3) control medium with 1.0 mM $H_2O_2$ along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. $H_2O_2$ decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity, against oxidative stress caused by $H_2O_2$. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM $H_2O_2$ alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.

• The Korean Journal of Pharmacology
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• v.28 no.1
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• pp.103-114
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• 1992

Selective quenching of 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups was utilized to examine the transbilayer fluidity domains of the model membranes of total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from synaptosomal plasma membrane vesicles. At $37^{\circ}C$, all anisotropy (r), limiting anisotropy $(r_{\infty})$, and order parameter (S) values of DPH in the SPMVTL were larger than those in SPMVPL. The anisotropy, limiting anisotropy, and order parameter of DPH in the inner monolayer were 0.025, 0.033, and 0.070, respectively, greater than calculated for the outer monolayer of SPMVTL. In SPMVPL, the anisotropy, limiting anisotropy, and order parameter of DPH in the inner monolayer were 0.014, 0.018, and 0.047, respectively, greater than calculated for the outer monolayer. Selective quenching of DPH by trinitrophenyl groups was also utilized to examine the effects of barbiturates on the transbilayer fluidity domains of SPMVTL and SPMVPL. Barbiturates did not affect the anisotropy of DPH in the transbilayer domains of SPMVTL. In contrast, barbiturates increased the fluorescence anisotropy, limiting anisotropy, and order parameter of DPH in the SPMVPL in a dose-dependent manner. Barbiturates showed a greater ordering effect on the outer monolayer as compared to the inner monolayer of SPMVPL. Hence, it has been demonstrated for the first time that the Sheetz-Singer hypothesis (1974) may be valid for phospholipid model membranes.

• The Korean Journal of Pesticide Science
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• v.12 no.4
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• pp.342-350
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• 2008

This study was carried out to investigate the suitability of the pubertal assay and the enhanced TG 407 as methods for detection of endocrine-mediated effects. Thyroid function was also considered. Male and female Sprague-Dawley rats were gavaged daily with 0, 25, 50, 100 mg/kg metribuzin in corn oil during 30 days. The effects of metribuzin on thyroid gland, the genital organs and thyroid hormone were measured in male and female rats. Dose of metribuzin 50 mg/kg/day increased relative weight of testis, prostate, and seminal vesicle in male rats but relative weight of thyroid gland was not significantly different from control group. Dose of metribuzin 25 mg/kg/day decreased relative weight of thyroid gland in female rats. Another purpose of this study was to investigate the effects of endocrine-disruptors as like thyroid hormone in vitro. Luciferase activity was measured to detect reaction of test chemicals and thyroid hormone response elements in HeLaTRE cell. Dose of metribuzin from 1 to 1,000 nM increased to 106-122% of luciferase activity.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.231-236
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• 2000

This study was conducted to examine the effects of exogenous gonadotropins (PMSG+hCG) and an antioxidant (cysteine) on in vitro maturation of bovine follicular oocytes. Cumulus-oocyte complexes (COCs) aspirated from 2 to 5 mm ovarian follicles were cultured for 22 to 24 hours in a modified bovine embryo culture medium (mBECM) supplemented with 3 mg/mL bovine serum albumin, to which PMSG (10 IU/mL) + hCG (10 IU/mL) and/or cysteine (0.6 mM) were added. When examined the expansion of cumulus ce1ls at the end of maturation culture, greater (p<0.05) expansion was found after addition of PMSG+hCG (79 to 96%) to mBECM than after no addition (0%), regardless of the presence or absence of cysteine in the medium. The addition of cysteine did not stimulate cumulus expansion, but a high proportion (92%) of expansion was achieved when COCs were cultured after the addition of PMSG+hCG and cysteine to the medium. No difference in the proportion of oocytes underwent germinal vesicle breakdown (initiation of maturation) was found after the addition of PMSG+hCG and/or cysteine to mBECM. However, nuclear maturation (development to the metaphase-II stage) of oocytes was significantly stimulated by the combined addition of PMSG+hCG and cysteine, compared with no addition. In conclusion, both exogenous gonadotropins and an antioxidant are important for nuclear maturation of bovine immature oocytes and these factors have a cell-specific stimulatory action.

• The Korean Journal of Pharmacology
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• v.26 no.1
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• pp.35-49
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• 1990

A highly purified $(Na^+,\;K^+)-ATPase$ from the rectal gland of Squalus acanthias and from the electric organ of Electrophorus electricus has been used to raise antibodies in rabbits. The 97,000 dalton catalytic subunit and glycoprotein derived from the rectal gland of spiny shark were also used as antigens. The two $(Na^+,\;K^+)-ATPase$ holoenzymes and the two shark subunits were antigenic. In Ouchterlony double diffusion experiments, these antibodies formed precipitation bands with their antigens. Antibodies prepared against the two subunits of shark holoenzyme also formed precipitation bands with their antigens and shark holoenzyme, but not with eel holoenzyme. These observations are in good agreement with inhibitory effect of these antibodies on the catalytic activity of $(Na^+,\;K^+)-ATPase$ both from the shark and the eel, since there is very little cross-reaction between the shark anticatalytic subunit antibodies and the eel holoenzyme. The maximum antibodies titer of the anticatalytic subunit antibodies is found to be 6 weeks after the initial single exposure to this antigen. Multiple injections of the antigen increased the antibody titer. However, the time required to produce the maximum antibody titer was approximately the same. These antibodies also inhibit catalytic activity of $(Na^+,\;K^+)-ATPase$ vesicles reconstituted by a slow dialysis of cholate after solubilization of the enzyme in a presonicated mixture of cholate and phospholipid. In these reconstituted $(Na^+,\;K^+)-ATPase$ vesicles, effects of these antibodies on the fluxes of $Na^+$, $Rb^+$, and $K^+$ were investigated. Control or preimmune serum had no effect on the influx of $^{22}Na^+$ or the efflux of $^{86}Rb^+$. Immunized sera against the shark $(Na^+,\;K^+)-ATPase$ holoenzyme, its glycoprotein or catalytic subunit did inhibit the influx of $^{22}Na^+$ and the efflux of $^{86}Rb^+$. It was also demonstrated that these antibodies inhibit the coupled counter-transport of $Na^+$ and $K^+$ as studied by means of dual labeling experiments. However, this inhibitory effect of the antibodies on transport of ions in the $(Na^+,\;K^+)-ATPase$ vesicles is manifested only on the portion of energy and temperature dependent alkali metal fluxes, not on the portion of ATP and ouabain insensitive ion movement. Simultaneous determination of effects of the antibodies on ion fluxes and vesicular catalytic activity indicates that an inhibition of active ion transport in reconstituted $(Na^+,\;K^+)-ATPase$ vesicles appears to be due to the inhibitory action of the antibodies on the enzymatic activity of $(Na^+,\;K^+)-ATPase$ molecules incorporated in the vesicles. These findings that the inhibitory effects of the antibodies specific to $(Na^+,\;K^+)-ATPase$ or to its subunits on ATP and temperature sensitive monovalent cation transport in parallel with the inhibitory effect of vesicular catalytic activity by these antibodies provide direct evidence that $(Na^+,\;K^+)-ATPase$ is the molecular machinery of active cation transport in this reconstituted $(Na^+,\;K^+)-ATPase$ vesicular system.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.159-166
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• 2002

연구목적: 본 연구에서는 생쥐 Dazla 유전자의 난소내 발현 위치를 확인하고, 배아의 발달에 따른 Dazla 유전자의 발현 양상을 관찰하고자 하였다. 연구재료 및 방법: 임신 제 7일, 10일, 11일, 14일의 태자 (각각 n=9)와 생후 27일된 암컷 미성숙 생쥐 (n=32), 8주령의 암컷 성숙 생쥐 (n=9)로 부터 난자, 과립막세포, 난소 조직을 획득하였으며, 9주령의 수컷 성숙 생쥐 (n=3)로부터 고환 조직을 획득하였다. 각각의 획득된 조직과 세포에서 Dazla mRNA의 발현 여부를 RT-PCR, in situ hybridization (ISH) 방법 등으로 확인하였다. 성선자극호르몬을 투여하지 않은 미성숙 및 성숙 생쥐에서 획득한 미성숙 난자 (GV)와 PMSG와 hCG를 투여한 미성숙 및 성숙 생쥐에서 획득한 성숙 난자 (MII)에서 RT-PCR로 Dazla 유전자의 발현 여부를 확인하였다. 미성숙 및 성숙 생쥐의 난소 조직과 성숙 생쥐의 고환 조직에서 RT-PCR 및 ISH 방법으로 Dazla 유전자의 발현 여부를 확인하였다. 결 과: 난소의 과립막세포에서는 미성숙 및 성숙 생쥐, PMSG와 hCG 투여 여부 등과 상관없이 모두 Dazla 유전자의 발현은 음성으로 판정되었다. PMSG 및 hCG를 투여하지 않은 난소에서 획득한 미성숙난자 (germinal vesicle, GV) 또는 PMSG 및 hCG 투여 후 채취한 성숙 난자 (metaphase II, MII) 모두 Dazla 유전자가 발현되었다. Dazla 유전자의 발현은 수정 직후 (2PN) 음성으로 전환되었으며, 착상 직전의 배반포 시기까지 유전자 발현이 음성으로 지속되었다. Dazla 유전자는 임신 제 7일 (PCD 7), 10일 (PCD 10), 11일 (PCD 11)의 태자에서도 유전자 발현이 계속 음성으로 관찰되었으나, 성 분화가 일어나기 시작하는 임신 제 $12{\sim}14$일 (PCD $12{\sim}14$)의 태자에서 유전자 발현이 다시 관찰되었다. 결 론: Dazla 유전자는 난소 내 난자에서만 특이적으로 발현하는 난자 특이 유전자 (oocyte specific factor)로서 Dazla 유전자가 난소 내 난포 생성과 관련성이 있음을 제시하고 있다. 향후 조기 폐경 환자에서의 연관성 등을 확인한다면 임상적으로 유용한 지표가 될 수 있을 것으로 사료된다.

• Journal of Yeungnam Medical Science
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• v.3 no.1
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• pp.41-48
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• 1986

Band 3, the predominent 95,000 dalton anion transport protein, is the major intrinsic glycoprotein of the human erythrocyte membrane. This anion carrier exists as a dimer and binds the cytoskeletons such as spectrin, ankyrin and actin. And the liposomes are vesicular structures which form spontaneouly upon hydration of phospholipids. These artificial lipid vesicles have been investigated as model of the biological membranes and as a mean of improving the delivery of nucleic acids, drugs, proteins and biological substances to specific target tissues and cells. In this study, we were purified Band 3 from the human erythrocyte membrane(ghost) was prepared by hemolysis of intact human erythrocyte with weak alkali-hypotonic solution. Band 6 was removed from ghost by extracting with solution of an ionic strength of 0.15. Band 3 and Band 4 were solubilized selectively by extracting Band 6-depleted ghosts with Triton X-100 under nondenaturing conditions. Band 3 was then purified from Triton X-100 extract treated with p-chloromercuribenzoate by sucrose density gradient ultracentrifugation. This purified Band 3 was incorporated into liposomes prepared by reverse-phase evaporation. Phosphatidyl L-serine and cholesterol(1 : 1 molar ratio) were dissolved in chloroform and then chloroform was removed by rotatory evaporation under reduced pressure. Band 3 solution without Triton X-100 was introduced into a mixture of lipids and diethylether. Diethylether was subsequently removed by evaporation. This purified Band 3 and its incorporation into liposomes were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.