• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.319-327
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• 2011

This study investigated whether the addition of porcine sperm cytosolic factor (SCF) at fusion/activation affects in vitro development of porcine parthenogenetic(PA) and nuclear transfer (NT) embryos. To determine the optimum concentration of SCF, control group of oocytes was activated with 0.3M mannitol (1.0 mM $CaCl_2{\cdot}2H_2O$), other three groups of oocytes were parthenogentically activated with the fusion medium (0.1mM $CaCl_2{\cdot}2H_2O$) supplemented with 100, 200 or 300 ${\mu}$g/ml SCF, respectively. Matured oocytes were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}$sec. The activated embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Oocytes activated in the presence of SCF showed a significantly higher blastocyst rate than control (p<0.05). Apoptosis rate was significantly lower in 100 ${\mu}$g/ml SCF group than other groups (p<0.05). Cdc2 kinase activity in control and SCF treatment group of oocytes was determined using MESACUP cdc2 kinase assay kit at 1, 5, 10, 15, 30, 45 and 60 min after activation. Cdc2 kinase activity was significantly decreased (p<0.05) in SCF group than MII oocytes or control within 5 min. For NT embryo production, reconstructed oocytes were fused in the fusion medium supplemented with 0.1 mM $CaCl_2{\cdot}2H_2O$ (T1), 1.0 mM $CaCl_2{\cdot}2H_2O$ (T2) and 0.1 mM $CaCl_2{\cdot}2H_2O$ with 100 ${\mu}$g/ml SCF (T3). Fused embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Developmental rate to blastocyst stage was significantly higher in T3 than other groups (23.0% vs. 13.5 to 15.2%) (p<0.05). Apoptosis rate was significantly lower in T3 than T1 or T2 (p<0.05). The relative abundance of Bax-${\alpha}$/Bcl-xl was significantly lower in in vivo or SCF group than that of control (p<0.05). Moreover, the expression of p53 and caspase3 mRNA was significantly lower in in vivo or SCF group than that of control (p<0.05). These results indicate that the addition of SCF at fusion/activation might improve in vitro development of porcine NT embryos through regulating cdc2 kinase level and expression of apoptosis related genes.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.118-118
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• 2002

Programmed cell death (PCD) is thought as a well-controlled process by which unwanted cells are selectively eliminated. During the last decade many researches have elucidated molecules and their interactions involved in cell death by using largely in vitro induction of cell death or survival signals in a more defined manner, While these critical information and novel findings provide us with clearer understanding of mechanisms underlying cell death, it does by no means explain how PCD occurs and which cells or tissues are affected during normal embryonic development in vivo. In this study, we used zebrafish to examine whether the PCD is occurring selectively or randomly in developing embryos by whole mount in situ TUNEL analysis with specific markers for neural cells. The result revealed that the degree and distribution of TUNEL staining varied considerably throughout gastrulation stage, and there was also a number of TUNEL-negative embryos. Most of TUNEL-positive cells were scattered randomly throughout the blastoderm. During the gastrulation stage about 75 % of the embryos analyzed exhibited more than 5 TUNEL-positive cells. As the dorsal epiblast begins to thicken rather abruptly near the end of gastrulation, TUNEL-positive cells were mainly located along the dorsal side. Although there were some variations in TUNEL staining during segmentation and pharyngeal stages, TUNEL staining continued to be localized to the central nervous system, and was also detected in the sensory organs, trigeminal ganglions, and the primary sensory neurons. High levels of the cell death in developing brain between 20-somite and prim-6 stages are thought to play a role in the morphogenesis and organization of the brain. At prim-16 stage, cell death is considerably reduced in the brain region. Dying cells are mainly localized to the prospective brain region where ectodermal cells are about to initiate neurogenesis. As development progressed, high levels and more reproducible patterns of cell death were observed in the developing nervous system. Intensive TUNEL staining was restricted to the trigeminal ganglions, the primary sensory neurons, and sensory organs, such as olfactory pits and otic vesicles. Thus, PCD patterning in zebrafish embryos occurs randomly at early stages and becomes restricted to certain region of the embryos. The spatio-temporal pattern of PCD during the early embryonic development in zebrafish will provide basic information for further studies to elucidate genes involved in. regulation of PCD largely unknown in vivo during vertebrate embryogenesis.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.293-301
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• 1999

This study was to examine whether Hanwoo IVM/IVF/IVC blastocyst can be successfully survived in vitro/in vivo after vitrification and one-step dilution. For vitrification, blastocysts were serially exposed in glycerol (G) and ethylene glycol(EG) mixtures［10% (v Iv) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.］ which is diluted in 10% FBS added D-PBS. And then they were loaded in the straw, placed in cold nitrogen vapor for 3 min. and plunged into L$N_2$(－196$^{\circ}C$). One-step dilution within the straw was done in $25^{\circ}C$ and 36$^{\circ}C$ water for about 5 min. and 3 min., respectively. Recovered embryos after one-step dilution were cultured in cumulus cell mono-layered drop for 48 h or were transferred into recipient cows. When the embryo survival in vitro was assessed to re-expanded and hatched rates at 24 hand 48 h after one-step dilution, the results of vitrified group (85.4, 43.8%) was high, although these results were significantly lower than normal development (100.0, 63.3%) of control group, respectively (P＜0.001, P＜0.05). When in vitro survival of vitrified groups according to developmental stage was compared, the results of fast developed embryos (expanded blastocyst and early hatching blastocyst stage) were significantly higher than those of delayed developed one (early blastocyst stage) after one-step dilution (early hatching: 88.0, 48.0%: expanded: 81.1, 45.3%; early: 66.7, 14.3%) (P＜0.05). Also, in case of in vitro survival of vitrified groups according to embryo age (day 7, 8 and 9), when embryo age was younger, in vitro survival was significantly higher (day 7: 67.3, 34.5%; day 8: 76.9, 40.7%; day 9: 60.9, 23.9%)(P＜0.05). Finally, when in vivo development potential of vitrified and one-step diluted Hanwoo blastocysts was examined, 4 of 8 recipient (50%) cows became confirmed pregnant. These results demonstrated that our vitrification and one-step dilution technique can be applied easily and effectively on field trial without the equipment and embryological skills required for conservative dilution and transfer.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.117-123
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• 1992

Development in vitro of 2-cell mouse embryos was examined after appropriate exposure to oviductal milieu to demonstrate biological activity present in the oviducts. ICR and ($C57Bl/6{\times}Balb/c$) $F_1$ hybrid mice were superovulated and mated for the recovery of early embryos. Embryos were recoverd at every 2h intervals from 32h post-hCG(hph) to 56 hph. The proportions of developmental stages were determined in the recovered embryos. Development in vitro of 2-cell embryos was more rapid in $F_1$ hybrid than in ICR, showing high proportions of 4-cell embryo and blastocyst at 120 hph. 100% of blastocyst development was obtained at 38hph in $F_1$ hybrid and at 50 hph in ICR when 2-cell embryos were cultured upto 120hph in vitro. Moreover, in vitro culture of oviducts containing 2-cell embryos in ICR mice for 12h from 34hph to 46hph increased developmental capacity of ICR mouse embryo in vitro. The results indicate that oviductal environment contains substances having mitogenic activity and overcoming early cell block in vitro. The mitogenic activity is effective in vitro as well as in vivo.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.229-235
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• 2011

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. Ski protein is implicated in proliferation/differentiation in a variety of cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of this study was, by means of immunohistochemical methods, to locate Ski protein in the rat ovaries during ovulation and corpora lutea (CL) formation to predict the possible involvement of Ski in luteinization. In addition, we performed to examine whether the initiation of luteinization with luteinizing hormone (LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vivo models. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rat, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum (CL). These results indicate that Ski is profoundly expressed in the luteinized granulosa cells and luteal cells of CL during luteinization, and suggest that Ski may play a role in luteinization of granulosa cells.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.237-244
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• 2011

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to examine whether the initiation of luteinization with luteinizing hormone (LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vitro models. RT-PCR and real time PCR analysis respectively revealed that LH had no effect on c-Ski mRNA expression in the cultured granulosa cells regardless of LH treatment. Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally. Moreover, expression of mRNA of Arkadia, an E3 ubiquitin ligases, in luteinizing granulosa cells in vivo was assessed by realtime-PCR. The levels of Arkadia mRNA expression were unchanged during follicular growth and postovulatory luteinization. These findings suggest that Ski protein level may be regulated during luteinization at translational and/or post-translational level but not by Arkadia.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.229-237
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• 2008

Recently, parabens have been believed to act as xenoestrogens, an identified class of endocrine disruptors (EDs). These environmental compounds are the most well-known as preservatives in many commercial products, including food, cosmetics and pharmaceutical industries. It has been demonstrated that the human health risks of parabens result from a long-term exposure to skin in which this chemical group is rapidly absorbed through the skin. On the other hand, parabens are also completely absorbed from gastrointestinal tract. It has reported that these substances possess several biological effects in which inhibitory property involved in membrane transports and mitochondrial functions is considered to be important for their action. Testing of parabens has revealed that estrogen-like activities of these chemicals are much less potent than natural estrogen, $17{\beta}$ estradiol (E2). Additionally, the estrogenicity of individual paraben- compounds is distinct depending upon their biochemical structure. Recent findings of paraben-estrogenic activities have shown that these compounds may affect breast cancer incidence in women, suggesting adverse ecological outcomes of this environmental group on human and animal health. Although the biological and toxicological effects of parabens have been demonstrated in many previous studies, possible mechanism(s) of their action are required to be explored in order to bring the better understanding in the detrimental impacts of parabens in human and wildlife. There have several different types of parabens which are the most widely used as preservatives. These include methyl-paraben, ethylparaben, propylparaben, butylparaben and p-hydroxybenzoic acid, a major metabolite of parabens. In this review, we summarize current database based on in vitro and in vivo assays for estrogenic activities and health risk assessment of paraben- EDs which have been published previously.

• Reproductive and Developmental Biology
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• v.41 no.1
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• pp.17-23
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• 2017

The oocyte undergoes various events during in vitro maturation (IVM) and subsequence development. One of the events is production of reactive oxygen species (ROS) that is a normal process of cell metabolism. But imbalances between ROS production and antioxidant systems induce oxidative stress that negatively affect to mammalian reproductive process. In vitro environments, in vitro matured oocytes have many problems, such as excessive production of ROS and imperfect cytoplasmic maturation. Therefore, in vitro matured oocytes still have lower maturation rates and developmental competence than in vivo matured oocytes. In order to improve the IVM and in vitro culture (IVC) system, antioxidants, vitamins were added to the IVM, IVC medium. Antioxidant supplementation was effective in controlling the production of ROS and it continues to be explored as a potential strategy to overcome mammalian reproductive disorders. Based on these studies, we expect that the use of antioxidants in porcine oocytes could improved maturation and development rates.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.347-353
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• 1997

This study was to test whether in vitro/in vivo survival of vitrified mouse blastocysts was influenced by culture conditions and ET method. Mouse blastocysts were obtained from in vitro fertilization and cultured for 4 days in M16 medium, and they were vitrified in EFS40 which contained 40% ethlyene glycol, 18% Ficoll and 0.5 mol sucrose in PBS. In experiment I, in vitro and in vivo survival rate of these embryos were evaluated in different culture condition after thawing. When thawed embryos were cultured in M16 medium as a control, m-CR1 medium contained 20 amino acids (2% BME amino acis and 1% MEM non-essential amino acids solution) and 4 mg/ml BSA and cumulus monolayer cell co-cultured condition in mCR1 medium (10% FBS), their in vitro survival at 24 hr after thawing was not affected by culture condition (75.6, 83.1, 82.4%). However, in vivo survival rates of implantation in m-CR1 medium (80.4%) were significantly higher than those of M16 medium (51.2%), co-culture (57.1%) condition, although there was no difference in live fetuses rates on day 15 gestation (39.0, 49.0, 38.1%). In experiment II, the in vivo development potential of embryos by ET methods was examined. When blastocysts were transferred to the day 2, 3 pseudopregnant recipient without culture soon after thawing, no pregnant recipient was obtained on the day 2 pseudopregnancy, and 50% of pregnancy rates and 15.4% of live fetus rates were obtained on the day 3 pseudopregnant recipients. These results were significantly lower than those of transferred group (day 3 pseudopregnant recipients) after culture for 16 hr post thawing (73.5, 57.1%) (p<0.05). In experiment III, to elevate usability of delayed embryos in vitro/in vivo survival of vitrified embryos (day 4 early, day 5 early and expanding blastocyst) were examined. in vivo survival rates (live fetus, total implantation) were higher in day 4 early blastocysts (33.3, 66.7%) than in day 5 expanding blastocysts (29.0, 38.7%), although the highest in vitro survival rates were obtained in the day 5 expanding brastocysts (78.3%). Therefore, these results suggest that the in vitro/in vivo survival rates of vitrified embryos could be improve by the culture condition and ET method and that the in vivo development rates of delayed embryos were decreased with longer culture duration in vitro. It means that more effective cryopreservation was obtained in day 4 early blastocysts than in day 5 expanding blastocysts.

• Korean Journal of Medicinal Crop Science
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• v.14 no.5
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• pp.303-306
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• 2006

Seed of Codonopsis lanceolata exhibits low germination due to impermeable seed coat. Prolonged seed dormancy can be overcome by the application of gibberellins, as it promotes growth of the embryo and weakens the structures surrounding of embryo. The effects of photoperiod, sugar and gibberellin concentration were investigated at constant temperature for 12 days and 22 days in vitro and invivo conditions respectively. The rate of germination of seeds of Codonopsis lanceolata in wet filter paper in both complete dark and light treatments was significantly lower than that of seed treated with $GA_3$. It clearly indicates that there is significant coat imposed dormancy in the seed of Codonopsis lanceolata. The rate of germination in vivo condition was lower than that of the in vitro condition supplemented with $GA_3$ Thus, the physical dormancy due to impermeable seed coat and low level of endogenous gibberellins in the seed was the cause of poor germination rate in Codonopsis lanceolata.