• Journal of Embryo Transfer
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• v.19 no.3
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• pp.245-255
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• 2004

In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2％ versus 15.4％, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0％ versus 53.9％, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.2
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• pp.129-138
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• 2002

Objective: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Materials and Methods: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and $5{\mu}g$/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal sperm of hybrid F1 male mice ($1{times}10^6/ml$). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, b1astocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identify ES cells, the surface markers alkaline phosphatase, SSEA-1, 3,4 and Oct4 staining were examined in rep1ated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. Results: Although the cleavage rate (${\geq}$2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic b1astocysts ($9.6{\pm}3.1,\;35.1{\pm}5.2$) were signficantly lower than those of IVF blastocysts ($19.5{\pm}4.7,\;63.2{\pm}13.0$) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-l and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac cell differentiation derived from mES or P-mES cells was confirmed. Conclusion: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.133-142
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• 2002

The objective of this study was to examine maturation, fertilization and developmental rate of the in vitro-grown mouse oocytes, and to compare these results with those of oocytes grown and matured in vivo. The preantral follicles isolated from 12-day-old mice were cultured on Transwell-COL membrane inserts. After in vitro growth and maturation, 72.5 % of oocytes grown in vitro produced polar body which can be comparable to in vivo growth (70.5 %). However, the mean oocyte diameter of the in vitro group (69.6$\pm$2.1$\mu$m) was smaller than that of the in vivo group (73.3$\pm$3.0$\mu$m). The fertilization rate was significantly lower (p<0.05) in the in vitro group (76.5%) than in the in vivo group (90.2%), however, there was no difference in the percentage of monospermic and polyspermic oocytes between two groups. The capacities of in vitro grown ova to cleave and develop to blastocyst were (57.8 and 14.4%, respectively) significantly lower (p<0.001) than those of the in vivo counterpart (84.4 and 56.6%, respectively). Moreover, the mean number of cells per blastocyst was significantly lower (p<0.05) in the in vitro group (39.0$\pm$10.8) than in the in vivo group (60.5$\pm$12.5). Live young were produced from transferred 2-cell embryos derived from in vitro-grown and matured oocytes. In conclusion, the results show that in vitro-grown oocytes did not achieve the developmental capacity of in vitro-grown oocytes.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.51-51
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• 2001

Cocaine and amphetamine-regulated transcript (CART) is a satiety factor that is regulated by leptin. It was reported that the mice intracerebroventricularly injected with CART showed behavioral changes resembled with the typical behavioral alterations found in the mice carrying disorders in the brain serotonergic (5-HT) system. Hence, this study was conducted to find out the relationships between CART and 5-HT. We first examined the mRNA levels of CART after the injections of para-chlorophenylalanine (pCPA, 300 mg/kg i.p., single injection or daily for three consecutive days) in the rat brains by in situ hybridization using the mouse CART cDNA probe cloned in our laboratory. Systemic administrations of pCPA, a potent inhibitor of tryptophan hydroxylase, the rate limiting enzyme of 5-HT biosynthesis, acutely depletes the brain 5-HT transporter (5-HTT) in the dorsal raphe nucleus (DRN), which reuptakes terminal 5-HT. Results indicated that the mRNA level of CART significantly decreased in the arcuate nucleus, paraventricular nucleus, and lateral hypothalamic nucleus by three days of daily injection with pCPA with no noticeable change detected 24 hrs after the single injection. The message levels of 5-HTT in DRN decreased in both single and three days of injections. Secondly, to investigate whether CART affect to 5-HT, mouse genomic CART gene, which is consist of 3 exons and 2 introns and mouse neurofilament light (NF-L) chain promoter were cloned. Then, we constructed neuron specific expression vector, which was transfected into HeLa cell using lipid-mediated transfection system. Expression of GFP and CART linked to NF-L-chain promoter in the transfected HeLa cell were detected by using fluorescent microscope and RT-PCR. These results confirmed normal expression of DNA constructs in vitro. Then, to increase brain specific expression of CART in vivo transgenic mice carrying CART gene controlled the deleted NF-L-chain promoter were generated by the DNA microinjection into pronuclei of fertilized embryos. Transgenic mice were detected by Southern blot. Further study is necessary to examine CART expression and 5-HTT in these transgenic mice. Therefore, these results suggest that there maybe a positive molecular correlation between CART and 5-HT in responding to the stimuli.

• Development and Reproduction
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• v.4 no.2
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• pp.161-173
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• 2000

The aim of this study was to assess the effect of the frequency of the L$N_2$ infusion on the ultrastructure, metabolism and development of the embryo after freezing and thawing by computerized cell freezer. Two-cell embryos of ICR mouse were randomly allocated into fresh (control), high-frequency freezing (group 1) and low-frequency freezing (group 2). For fresh and frozen-thawed intact 2-cell embryos, total ceil number in the blastocyst was counted by fluorescent microscope after Hoechst 33258 staining. Relative amount of $H_2O$$_2$ was measured by DCHFDA. Intracellular location and membrane potential of the mitochondria were evaluated by staining with rhodamine 123 and JC-1. The structure of actin filament was also evaluated by confocal microscope. DNA fragmentation was assessed by TUNEL method after development into blastocyst. The survival rate of intact embryo was higher in group 1 than group 2 (50.7％ vs. 34.6％ respectively, p<0.05). The blastocyst developmental rate was significantly low in group 2 (86.7％, 76.7％ vs. 44.0％ for control, group 1 and group 2 respectively, p<0.05). Total cell number in the blastocyst was also significantly lower in group 2 than control (79.5$\pm$12.9, 71.6$\pm$8.0, and 62.5$\pm$4.7 for control, group 1 and group 2 respectively, p<0.05). The relative amount of $H_2O$$_2$ was higher in group 2 than other groups (15.3$\pm$3.0, 16.6$\pm$1.6 vs. 23.4$\pm$1.8, p<0.05). After JC-1 staining, relative intensity of mitochondria with high membrane potential was significantly lower in group 2 than control and group 1 (17.2$\pm$3.8, 17.4$\pm$1.3 vs. 13.2$\pm$2.0, p<0.05). In group 2, partial deletion and aggregation of the actin filament was found. DNA fragmentation rate was also hieher for group 2 versus other groups (30.8％, 36.0％ vs. 65.6％, p<0.05). The frequency of the L$N_2$ infusion is an important factor for the development of frozen-thawed mouse embryo. High-frequency infusion may prevent damages of cytoskeleton and mitochondria in the embryo probably by preventing the temperature fluctuation during dehydration phase. We speculate that the application of high-frequency infusion method in human embryo may be promising.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.275-283
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• 2008

Objective: This study was conducted to investigate the effects of the artificial shrinkage and assisted hatching (PZD; patial zona dissetion) before vitrification on the development of vitrified mouse expanding blastocyst. Methods: Mouse 2-cell embryos were collected and cultured in G1.1 and G2.2 to expanding blastocyst. For artificial shrinkage (AS) the micro injection pipette was inserted into blastocoele cavity and blastocoele fluid was aspirated. For assisted hatching (AH) PZD method was used. Control group was -AS/-AH and treatment groups were -AS/+AH, +AS/-AH and +AS/+AH. After AS and AH mouse blastocysts were equillibrated in G10 and G10E20 for 3 mins, respectively, and vitrified in G25E25 after loading on capped pulled-straw. Vitrified mouse blastocysts were thawed and cultured for 24 hrs. The survival and hatching rate was compared among one control and three treatment groups. Results: The survival rates were 99%, 92% in +AS/+AH and +AS/-AH groups and 54%, 58% in -AS/-AH and -AS/+AH group, respectively. The survival rate was significantly higher in +AS group than in -AS group (p<0.01). Hatching rates were 34%, 96% in -AS/-AH and -AS/+AH groups and 41%, 100% in +AS/-AH and +AS/+AH, respectively. The hatching rates was higher in +AH group than in -AH group (p<0.01). After thawing recovery rates were 100%. Loading on capped pulled-straw, that is effective and useful method on vitrification. Conclusion: This study showed that artificial shrinkage of blastocoele cavity and assisted hatching (PZD) significantly improved the development of the vitrified mouse expanding blastocysts.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.10
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• pp.1412-1421
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• 2002

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. In situ hybridization demonstrated that the transfection rate of sperm cells with and without BSA was 41 and 68% respectively, when the cells were treated with liposome/DNA complexes and 13% for DNA alone. LSC analysis showed that the binding of exogenous DNA was greatly reduced by DNase I treatment which digests DNA bound onto spermatozoa, suggesting that some of the DNA was internalized into the sperm membrane. To find out whether transfected DNA was internalized into sperm intracytomembrane, sperm DNA was amplified by inverse PCR. No PCR products were detected from sperm cells, indicating that the foreign DNA was simply bound onto the sperm membrane. To investigate transfer rates of exogenous DNA into oocytes via sperm cells, we used immunofluorescene method to follow the distribution of foreign DNA via spermatozoa: a few exogenous DNA was located in the cytoplasm of early embryos (13/60, 21.7% for DNA+/liposome+/BSA) and was not located in the pronucleus and/or nucleus. These results suggest that most of the transfected sperm cells could carry the foreign DNA into the egg by in vitro fertilization, but that the transferred DNA is degraded in the developing embryos without stable integration into the zygote genome. Therefore, we have directly injected with transfected sperm cell into oocyte cytoplasm and observed that some of the exogenous DNA was detected in preimplantation embryonic cytoplasm and expressed at preimplantation stages, suggesting that exogenous DNA in early zygote has their integrity. In this study, we have not identified a noble mechanism that interfering transportation of foreign DNA into zygote genome via spermatozoa. Our data, however, demonstrated that inverse PCR and immunofluorescene methods would be used as a new tool for follow-up of gene distribution in oocyte via sperm cells.

• Development and Reproduction
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• v.13 no.4
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• pp.305-312
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• 2009

Multipotent spermatogonial stem cells (mSSCs), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESCs). The aim of this study was to evaluate the potential for transgenesis of mSSC derived from outbred mice and the production of transgenic animal by the mSSC-insertion into embryo. mSSCs, established from outbred mice (ICR strain) in the previous study, were maintained and then transfected with a lenti-viral vector expressing green fluorescent protein (GFP), CS-CDF-CG-PRE. Embryonic stem cells (ESCs) were derived from inbred transgenic mice (C57BL/6-Tg (CAG-EGFP)) and were used as an experimental control. Transfected mSSCs were well proliferated in vitro and maintained their characteristics and normal karyotype. Ten to twelve mSSCs and ESCs were collected and inserted into perivitelline space of 8-cell mouse embryos, and then transferred them into uteri of poster mothers after an additional 2-days of culture. Percentage of mSSC-derived offsprings was 4.8% (47/980) and which was lower than those (11.7% (67/572)) of ESC-derived ones (P<0.05). However, even though different genetic background of mSSC and ESC origin, the production efficiency of coat-colored chimeric offspring in mSSC group was not different when compared it with ESC (6.4% (3/47) vs. 7.5% (5/67)). From these results, we confirmed that mSSC derived from outbred mice has a pluripotency and a potential to produce chimeric embryos or mice when reaggregatation with mSSC is performed.

• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.237-242
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• 2001

We recently demonstrated that both linear- and supercoil-form B1/B2 SINE (short interspersed elements) sequences could increase an integration frequency of a reporter gene in preimplatation mouse embryos. In those reports, when either a control or SINE-flanked DNA was separately applied to microinjection, the proportions of $\beta$-gal positives were 16% and 63%, respectively, in linear-form DNA, and 6% and 25%, respectively, in circular-form DNA. Here, we examined the contribution of a circular-form DNA moiety to integration frequency by using a mixed-farm (linear and circular-form) DNA in microinjection. When examined in the blastocyst stage, the proportion of $\beta$-gal-positive embryos was 17.3% and 46.6% in control and SINE-flanked DNA, respectively. These results suggest that there is little contribution of circular-form DNA moiety to the resultant integration frequency, and that the majority of the integration events are mediated through a linear conformation of vector DNA. In addition, some clues on integration process could be obtained from the analysis of microinjection results.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.189-197
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• 2009

Two-pore domain $K^+(K_{2P})$ channels contribute to setting the resting membrane potential in excitable and nonexcitable cells. However, the cellular or tissue distribution and function of $K_{2P}$ channels expressed in mammalian germ cells and reproductive organs have not yet been reviewed by researchers. In this review, we focus on expression, localization and expected properties of $K_{2P}$ channels in germ cells and reproductive organs. The $K_{2P}$ channels are expressed in human cytotrophoblast cells, myometrium, placental vascular system, uterine smooth muscle, and pregnant term tissue, suggesting that $K_{2P}$ channels might be involved in the processes of pregnance. The $K_{2P}$ channels are also expressed in mouse zygotes, monkey sperm, ovary, testis, germ cells, and embryos of Korean cattle. Interestingly, $K_{2P}$ channels are modulated by changes in temperature and oxygen concentration which play an important role in embryonic development. Also, $K_{2P}$ channels are responsible for $K^+$ efflux during apoptotic volume decreases in mouse zygotes. These expression patterns and properties of the $K_{2P}$ channels in reproductive organs and germ cells are likely to help the understanding of ion channel-related function in reproductive physiology.