• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.77-82
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• 2004

The purpose of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of antioxidants(aesculetin, taurine and melatonin) in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation in NCSU 23 mediumand matured oocytes were inseminated with frozen semen. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of antioxidants in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. Aesculetin were added to NCSU 23 medium at concentration of 1 ug, 5 ug, and 10 ug, when treated with 10 ug(35.7%) of aesucletin at the rate of embryos of the morula plus blatocsyts were higher than those of any other groups (30.2%, 29.5% and 29.2%)(P<0.05). The developmental rates beyond morula stage of porcine embryos in NCSU 23 medium supplemented with taurine 0, 2.5 and 5.0 mM were 26.1%, 26.9% and 31.7%, respectively. The addition of 5.0 mM taurine was higher the developmental rate beyond morula stage than in any other groups. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectively. The developmental rate of morula and blascytocys treated with 1nM melatonin was higher than in any other groups(P<0.05). Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10nM were 41.0, 42.6, 39.6 and 33.0, respectively. These results indicate that aesculetin, taurine and melation can increase the developmental rate beyond the morulae and blastocysts in porcine embryos.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.215-221
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• 2002

Antioxidants(N-acetyl-1-cysteine, ebselen and glutathione) and growth factors(EGF, PDGF) were studied as a mean of increasing the development of porcine embryos produced by in vitro maturation (IVM) and in vitro fertilization(IVF). Porcine embryos developed to the 2~8 cell stage after IVF were cultured fer 6 to 7 days at 38.5$^{\circ}C$ in NCSU 23 medium containing antioxidants plus growth factors. Cell numbers of blastocysts were counted by fluorescence staining method. The developmental rate beyond morula stages in NCSU 23 containing NAC(1nm) or NAC(1nm) plus EGF(100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 28.1, 32.3 and 35.3%, respectively. NAC plus PDGF group was slightly higher than control group(P>0.05). The developmental capacity in NCSU 23 containing ebselen(10 $\mu$M) or ebselen(10 $\mu$M) plus EGF(100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 17.8, 36.9 and 40.3%, respectively Ebselen plus growth factor groups were significantly higher than control group(P<0.05). The developmental capacity in NCSU 23 containing glutathione(100 $\mu$M) or glutathione(100 $\mu$M) plus EGF (100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 24.1, 30.5 and 27.7%, respectively. There were not difference in all experimental groups(P>0.05). In all experimental groups, there was no significantly differences on the cell number of blastocysts, but ebselen plus growth factor groups were significantly higher than control group. These studies indicate that antioxidants plus growth factors can increase the proportion of embryos that developed beyond morulae stage.

• IMMUNE NETWORK
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• v.3 no.1
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• pp.47-52
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• 2003

Background: The megakaryopoiesis and platelet production is regulated by several hematopoietc factors such as thrombopoietin (TPO), interleukin-11 (IL-11) and interleukin- 3 (IL-3). IL-11 is a potent stimulator of megakaryopoiesis in vivo, and acts primarily as a megakaryocyte maturation factor in vitro and it can act synergistically with IL-3 and TPO. We performed this study to investigate the effects of recombinant human IL-11 (rhIL-11) with other hematopoietic factors on megakaryocyte colony formation in vitro. Methods: CD34+ cells were separated from umbilical cord blood and megakaryocyte colonies using MegaCult Assay Kit were cultured with rhIL-11, recombinant human IL-3 (rhIL-3), and recombinant human TPO (rhTPO) for 7 and 14 days. The number and percentage of CD34+ and CD41a+ cells were determined by flowcytometry. Results: The number of CD41a+ cells were $0.54{\pm}0.05{\times}10^4$ (rhIL-11 100 ng/ml), $5.32{\pm}0.23{\times}10^4$ (rhIL-3 100 ng/ml), and $8.76{\pm}0.15{\times}10^4$ (rhTPO 50 ng/ml) of total expanded cells during the culture of the purified CD34+ cells in liquid phase for 7 days. The number of CD41a+ cells were increased to $7.47{\pm}0.69{\times}10^4$ (rhIL-3+ rhIL-11), $11.92{\pm}0.19{\times}10^4$ (rhTPO+rhIL-11) of total expanded cells, respectively, during the culture of the purified CD34+ cells in liquid phase for 7 days in the presence of rhIL-11 (100 ng/ml). When the purified CD34+ cells were cultured in semisolid mediaincluding various concentration of rhIL-11, the megakaryocyte colonies were not formed. When the purified CD34+ cells were cultured with rhIL-11 and rhTPO or with rhIL-11 and rhIL-3, the number of megakaryocyte colonies were increased compared with rhTPO or rhIL-3 alone. Conclusion: These results indicate that IL-11 exerts a potent proliferative activity to colony forming unit-megakaryocyte from human umbilical cord blood, and it acts with other hematopoietic factors synergistically.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.155-163
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• 2004

The aims of this study are 1) to test oocytes and embryos collected from in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to Funahashi et al (1994). Glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$, and 10% fetal bovine serum albumin was added to the culture medium thereafter. Embryos were treated with 7.5 ${\mu}g/ml$ cytochalasin-B for 30 min, centrifuged at 13,000 rpm for 13 min and then exposed sequentially to an ethylene glycol(EG) vitrification solution, aspirated into OPS, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three dornors after AI for control group. Forty-nine embryos were washed 3 times in mPBS + 10% FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients were transferred individually with 100, 100 frozen embryos derived from abattoir and 34 fresh embryos by surgically, and another three recipients were transferred individually with 150, 150 frozen embryos and 100 fresh embryos by nonsurgically, respectively. all recipient sows exhibited delayed returns to estrus. To our knowledge, theses results suggest that required an improved techniques, more vigorous embryos preparation and substitute to gilt with cleaner uterous condition.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.407-417
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• 1999

Objective: Mammalian follicle cells are the most important somatic cells which help oocytes grow, mature and ovulate and thus are believed to provide oocytes with various functional and structural components. In the present study we have examined whether cumulus or granulosa cells might playa role in establishing the plasma membrane structure of mouse oocytes during meiotic maturation. Design: In particular the differential resistances of mouse oocytes against chymotrypsin treatment were examined following culture with or without cumulus or granulosa cells, or in these cell-conditioned media. Results: When mouse denuded oocytes, freed from their surrounding cumulus cells, were cultured in vitro for $17{\sim}18hr$ and then treated with 1% chymotrypsin, half of the oocytes underwent degeneration within 37.5 min ($t_{50}=37.5{\pm}7.5min$) after the treatment. In contrast cumulus-enclosed oocytes showed $t_{50}=207.0$. Similarly, when oocytes were co-cultured with cumulus cells which were not associated with the oocytes but present in the same medium, the $t_{50}$ of co-cultured oocytes was $177.5{\pm}13.1min$. Furthermore, when oocytes were cultured in the cumulus cell-conditioned medium, $t_{50}$ of these oocytes was $190.0{\pm}10.8min$ whereas $t_{50}$ of the oocytes cultured in M16 alone was $25.5{\pm}2.9min$. Granulosa cell-conditioned medium also increased the resistance of oocytes against chymotrypsin treatment such that $t_{50}$ of oocytes cultured in granulosa cell-conditioned medium was $152.5{\pm}19.0min$ while that of oocytes cultured in M16 alone was $70.0{\pm}8.2min$. To see what molecular components of follicle cell-conditioned medium are involved in the above effects, the granulosa cell-conditioned medium was separated into two fractions by using Microcon-10 membrane filter having a 10 kDa cut-off range. When denuded oocytes were cultured in medium containing the retentate, $t_{50}$ of the oocytes was $70.0{\pm}10.5min$. In contrast, $t_{50}$ of the denuded oocytes cultured in medium containing the filtrate was $142.0{\pm}26.5min$. $T_{50}$ of denuded oocytes cultured in medium containing both retentate and filtrate was $188.0{\pm}13.6min$. However, $t_{50}$ of denuded oocytes cultured in M16 alone was $70.0{\pm}11.0min$ and that of oocytes cultured in whole granulosa cell-conditioned medium was $156.0{\pm}27.9min$. When surface membrane proteins of oocytes were electrophoretically analyzed, no difference was found between the protein profiles of oocytes cultured in M16 alone and of those cultured in the filtrate. Conclusions: Based upon these results, it is concluded that mouse follicle cells secrete a factor(s) which enhance the resistance of mouse oocytes against a proteolytic enzyme treatment. The factor appears to be a small molecules having a molecular weight less than 10 kDa.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.287-294
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• 2011

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs, we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at $39^{\circ}C$ 5% $CO_2$ in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control ($6.0{\pm}1.0$ vs $3.3{\pm}0.6$, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ER-stress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.223-231
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• 2001

The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 $\mu$M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.228-233
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• 2011

Objective: To investigate the effectiveness of GnRH antagonist multiple-dose protocol (MDP) with oral contraceptive pill (OCP) pretreatment in poor responders undergoing IVF/ICSI, compared with GnRH antagonist MDP without OCP pretreatment and GnRH agonist low-dose long protocol (LP). Methods: A total of 120 poor responders were randomized into three groups according to controlled ovarian stimulation (COS) options; GnRH antagonist MDP after OCP pretreatment (group 1), GnRH antagonist MDP without OCP pretreatment (group 2) or GnRH agonist luteal low-dose LP without OCP pretreatment (group 3). Patients allocated in group 1 were pretreated with OCP for 21days in the cycle preceding COS, and ovarian stimulation using recombinant human FSH (rhFSH) was started 5 days after discontinuation of OCP. Results: There were no differences in patients' characteristics among three groups. Total dose and days of rhFSH used for COS were significantly higher in group 3 than in group 1 or 2. The numbers of mature oocytes, fertilized oocytes and grade I, II embryos were significantly lower in group 2 than in group 1 or 3. There were no significant differences in the clinical pregnancy rate and implantation rate among three groups. Conclusion: GnRH antagonist MDP with OCP pretreatment is at least as effective as GnRH agonist low-dose LP in poor responders and can benefit the poor responders by reducing the amount and duration of FSH required for follicular maturation.

• Journal of Periodontal and Implant Science
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• v.28 no.4
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• pp.769-786
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• 1998

The purpose of the present study is to examine the effect of cell proliferation and alkaline phosphatase activity in osteoblastic cells and to compare the bone healing ability of rat calvarial defects between the control group and the safflower seed extract treated group. Osteoblastic cells were obtained from calvariae of a fetal rat. Cells were cultured containing DMEM and safflower seed extract ($10^{-6}g/ml$, $10^{-3}g/ml$) at $37^{\circ}$ with 5% $CO_2$ in 100% humidity for 3 days. MTT was performed to examine the viability of the cells, and alkaline phosphatase activity was analyzed to examine the mineralization in vitro. Rat calvarial defects($5{\times}5mm$) in 250g Sprague-Dawly were made using round bur. Rats were administrated with safflower seed extract(0.35g/kg/day) for experimental periods. Calvarial defects were studied histopathologically and immunohistochemically at 1,4, and 8 weeks. High concentration group($10^{-3}g/ml$) of safflower seed extract significantly increased in the cell proliferation and alkaline phos phatase synthesis in osteoblastic cells. The infiltration of inflammatory cells and osteoclastic activities were decreased in the safflower seed extract treated group as compared with control group. Bone maturation was accelerated in the safflower seed extract treated group as compared to control group. No difference in osteoinductive process was observed between the control and the safflower seed extract treated group. Immunohistochemical observation revealed that protein expression of TGF-$\beta$and osteonectin during early healing phase in the safflower seed extract treated group was slightly increased as compared to control group. These results indicate that safflower seed extract promotes the healing process in bony defect of rat calvariae, and retains a potential applicability as an adjuvant therapeutic modality for regeneration of periodontal bony defect.

• Journal of Animal Science and Technology
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• v.51 no.5
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• pp.413-420
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• 2009

The research has been aimed to investigate the effect of different sera including fetal bovine serum (FBS), male bovine serum (MS), female bovine serum (FS), and castrated-male bovine serum (C-MS) on cell proliferation, follicular maturation and ovulation in vitro. Established cell lines and primary cells were cultured in the culture media supplemented with different sera and cells proliferation was observed by cell counting and MTT assay. The results indicated that cell proliferation was significantly different for different serum source. Proliferation of bovine and human myogenic satellite cells was highest in MS. In contrast, proliferation of breast cancer cells and immune cells were the highest in FS and FBS, respectively. There was no difference in the rate of follicular growth, whereas the rate of ovulation was higher in FBS and C-MS. Finally, the wound healing effect and cell proliferation of 3T3-L1 cells showed that wound healing was fastest in FS and cell proliferation was higher in MS. These results suggest the importance of an optimal serum selection in the experiments involving cell culture system, and gender-specific Hanwoo sera may be used as a substitute to FBS.