hFSH is a glycoprotein secreted from anterior pituitary and consists of α and β subunits. Because of its major biological functions including sperm formation in the male and for follicular growth, FSH is used to cure woman's sterility. In this study we tried to produce recombinant hFSH in vitro using a retrovirus expression vector. Two major components of the vector we constructed are: (ⅰ) a DNA fragment containing α and β genes fused by a DNA sequence coding carboxyl terminal peptide (CTP) of human chorionic gonadotropin, (ⅱ) a DNA fragment corresponding woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Evaluation of expression profile of the recombinant FSH using reverse transcription PCR and enzyme-linked immunosorbent assay (ELISA). Among three cell lines tested, HeLa cells were the best for hFSH expression (5,395 mIU/ml), then followed by chicken embryonic fibroblast (CEF) cells and Chinese hamster ovary (CHO) cells in the order of hFSH production. In addition to the amount, the FSH produced from HeLa cells was highest in terms of biological activity which was determined by measuring cAMP.

Transducin-like enhancer protein 1(TLE-1) is protein associated with cell proliferation. This study analyzed change of TLE-1 mRNA expression during in vivo and in vitro maturation in porcine oocytes. Oocytes and granulose cells were collected from follicles of <2 mm, 2～6 mm and >6 mm in diameter in slaughtered pig’s ovaries. Oocytes collected from follicles of 2～6 mm in diameter were used after in vitro maturation for 0, 10, 20 and 44 h. Cumulus cells and granulose cells were collected after treatment with hyaluronidase. In results, TLE-1 mRNA expression in oocytes collected from follicle >6 mm in diameter is increased, TLE-1 RNA expression in cumulus cells and granulosa cells from follicles <2 mm, 2 mm 6 mm and >6 mm in diameter. However, there is no significant difference. On the other hand, TLE-1 mRNA expression from oocytes cultured for 10 h and 44 h is increased, TLE-1 mRNA in cumulus cells cultured for 10 h is significant increased(p<0.05) than other culture periods. In conclusion, these results show that TLE-1 is expressed in all cell types of oocytes, cumulus cells and granulose cells, and associated with oocyte maturation.

The objective of this study was to evaluated the efficiency on sperm cryosurvival and ability of in vitro fertilization using Triladyl and Lactose Egg-Yolk(LEY) as extenders for cryopreservation of separated sperm by 65% percoll in miniature pig. Sperm viability was measured with SYBR-14/PI double stained sperm by flow cytometry. Ability on embryo cleavage rate and blastocyst development were observed by in vitro fertilization after frozen-thawing of sperm separated by 65% percoll. The experimental groups were designed that separated sperm by 65% percoll with Triladyl (ST) or LEY(SL) and unseparated sperm with Triladyl(UT) or LEY(UL) for cryopreservation. As a results, the viability was significantly(p<0.05) higher in ST(55.1%), SL(63.1%), UL(58.8%) than UT(38.2%) group. Sperm viability in SL(63.1%) group was significantly(p<0.05) higher than other experimental groups. On the other hand, embryo cleavage rate was significantly(p<0.05) higher in ST(79.1%), SL(83.2) than UT(74.1%) and UL(75.7%) groups at 96h after in vitro fertilization. Blastocyst development was also significantly(p<0.05) higher in ST(21.5%), SL(20.9%) than UT(17.0%) and UL (18.8%) groups. In conclusion, cryopreservation of miniature boar sperm separated by 65% percoll were beneficial to viability and capacity on in vitro fertilization.

본 연구는 우리나라 무스카리 우점품종인 ‘Early Giant’ 의 대량증식 체계를 구축하기 위하여 잎 절편으로부터 소인경 형성과 비대에 미치는 MS 무기염 농도, 당 농도 및 광조건의 영향을 조사하였다. 무스카리의 잎 절편을 2주간 암 배양 후 MS 기본배지에 NAA 0.01mg·L−1, kinetin 0.2 m·L−1, 자당 30 g·L−1와 gelrite 3 g·L−1가 첨가된 배지에 16시간 일장으로 명 배양하였을 때 자구형 성 및 생육이 가장 양호하였다. 다만, 자구 비대는 MS 배지 내 60 g·L−1 당 농도에서 촉진되었다. 온실에서 순 화된 식물체는 정상적으로 개화하였으며 정식 2년차에 1.5개 내외의 자구를 가진 구를 생산할 수 있었다.

남해 약쑥을 새로운 기능성 식품의 재료로 활용하기 위하여 각 부위별 60% 에탄올 추출물에 대 한 항산화 활성을 In vitro 시스템에서 조사하였다. 남해약쑥의 부위별 총 페놀성 화합물 함량은 잎 60% 에탄올 추출물에서 23.08 mg/g으로 가장 높은 함량을 보였다. 남해 약쑥의 부위별 60% 에탄올 추출물로 DPPH 및 ABTS 라디칼 소거 활성을 측정한 결과, 추출물의 농도가 증가함에 따 른 DPPH 및 ABTS 라디칼 소거 활성이 증가하는 경향을 나타내었고, 잎에서 가장 높은 DPPH 및 ABTS 라디칼 소거 활성을 나타내었다. 환원력도 추출물의 농도가 증가함에 따라 환원력이 증가하 는 경향을 나타내었으며, 환원력도 잎에서 가장 높게 나타났다. Linoleic acid를 이용한 자동산화 억제활성을 실험한 결과, 다른 부위보다 잎에서 가장 높은 자동 산화 억제활성을 나타내었고, MDA assay를 이용하여 항산화 활성을 측정한 결과, 일부 농도 의존적인 경향을 보였으며, 잎 200 μ g/mL 농도에서 71.38%의 항산화 활성을 보였다. 따라서 남해 약쑥 잎은 높은 항산화 활성을 바탕 으로 천연 항산화제로서의 활용가능성이 매우 높을 것으로 판단된다.

An efficient method for the rapid micropropagation of Camptotheca acuminata from axillary buds was established by application of various plant growth regulators. Among various cytokinins, 0.5 mg L-1 BA showed the best performance on shoot multiplication, number average multiple shoots up to 10.8. The propagated shoot cuttings in vitro were elongated on NN basal medium without plant growth regulators. The secondary multiple shoots were induced at the site of initially induced buds. Rooting was induced directly near the base of the shoot on half-strength NN medium containing 0.5 mg L-1 of IBA, whereas high concentration of 1.0 mg L-1 IBA could induce callus at the base of the shoot. The camptothecin content, anticancer compound of the micropropagated plants was contained in various tissues. Camptothecin contents were 1.8 and 2.5 mg g-1 dry weight in stems from propagated in vitro and mother plant, respectively. This result may be used to develop strategies for large-scale propagation of elite C. acuminata trees.

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of MⅡ stage.

The present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen in our laboratory for in vitro fertilization (IVF) experiments due to lack of fresh semen. Antibiotics were added in the frozen semen extender (kanamycin and gentamicin) and in vitro culture (IVC) medium (gentamicin) for further inhibiting growth of microorganisms. Nevertheless, proliferations of microorganisms were observed in IVC culture drop during culturing of IVF embryos using frozen semen. Randomly 3 samples were taken from the liquid semen, frozen semen and egg yolk. Contaminated IVC medium, frozen-thawed semen, liquid semen and egg yolk were cultured in de Man, Rogosa and Sharpe (MRS) agar medium. Whitish colonies were detected in contaminated IVC drop, frozen-thawed semen samples and egg yolk but no colonies were formed in liquid semen samples. Gram-negative and rod-shaped identical bacteria were found in both frozen-thawed semen sample and contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to manufacturer's instruction with identification value (% ID) 94.3% and T index 0.88. Antibiotic susceptibility tests were done according to Clinical and Laboratory Standards Institute (CLSI) by using ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin test. Among them Enterobacter cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, kanamycin but susceptible to tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin. From these findings it could be suggested that this contamination sources might be from egg yolk.

Prediction of semen's fertilizing ability used in artificial insemination (AI) is one of very important factors on pig reproductive performance. In vitro fertilization (IVF) has been used for indirect evaluation of sperm's fertilizing ability and it has been showed as highly correlated index. In swine industry, increasing interest in preservation of boar semen raises questions on the sperm motility from semen used in commercial AI centers. Mitochondria in sperm mid-piece generate the energy to support motility and could be an explanation of impaired fertility. Objective of this study was to suggest usable sperm motility to farms in measuring the effect of sperm motility and sperm abnormality on in vitro production of embryo in which sperm's fertilizing ability can be determined indirectly. Semen samples were provided from local AI center and used within 3 days after collection. Semen samples were divided by 4 different motile groups (>70%; 61~70%; 51~60%; <50%) using CASA (computer-assisted sperm analysis) on the days of IVF. Developmental rate to the blastocyst stage from over 61% motile sperm group showed significantly higher rate than below 60% motile sperm group ( vs , p<0.05). In experiment to determine the relationship between sperm motility and viability and abnormality, over 61% motile sperm groups showed significantly higher viability rate compared to below 60% motile sperm groups ( vs , p<0.05). On the other hand, morphological sperm abnormality showed significantly higher in over 70% motile sperm group ( vs , p<0.05). In experiment to find the correlation between sperm motility of 4 different motile groups and amount of mitochondria, lower motility group also showed lower level of mitochondria (p<0.05). The mitochondria parameter used in this study showed another possibility to differentiate the sperm motility. Taken together, because below 60% motile semen used in AI reduce the fertility, AI centers should provide the over 60% motile sperm to the farms at the time of AI.

Optimization of the preimplantation mammalian embryo culture condition was widely focused on refining medium composition under the name of chemically defined media. However, recent research revealed that the alteration of physical environment can be a crucial factor to a successful embryo development. In this study, under the same embryo density, a novel culture device named oil-free micro tube culture (MTC) system was evaluated using porcine parthenogenetic embryos. The activated oocytes were placed into the 0.2 ml thin-wall flat cap PCR tube and cultured to the blastocyst stage. As a preliminary step, embryo density and culture medium volume were optimized under a standard drop culture system. The optimal embryo density range for in vitro culture was 0.5 embryos per in drop (20.5%) and 1.0 embryos per in drop (20.6%). Based on these results, we compared drop culture system and 'MTC' system in terms of the developmental rate to the blastocyst stage. In medium volume, the 'MTC' system showed similar blastocyst formation rate when compared with drop culture system (20.2% versus 20.5%, respectively) while the 'MTC' system showed lower blastocyst formation rate than drop culture system in one (12.7% versus 20.0%, respectively). Therefore the MTC system may be an alternative incubation system for short-distance embryo transport without carrying the incubator and this provides novel embryo culture device to clinical veterinary embryologists.

Thousands of new chemicals have been introduced to environment during last decades. Many of them and common consumer products have been shown to be the endocrine disrupting chemicals. One such chemical group is the phthalates, used in soft poly vinyl chloride (PVC) material and in a huge number of consumer products. The prevalence of these modem chemicals have a remarkable increase. Approximately 3.5 million tons of the main phthalate, di-(2-ethylhexyl) phthalate (DEHP), are produced annually worldwide and indeed, DEHP is considered a ubiquitous environmental contaminant. It has been demonstrated that high doses of phthalate can adversely affect adult and developing animals. In this review, we critically discuss the conclusions of recently original research papers and provide an overview of studies on reproductive disrupting effects of phthalates. In addition, we review the reproductive toxicity data of phthalates in some in vitro research and in both male and female reproductive systems in experimental and domestic animals. Finally, we point out some critical issues that should be addressed in order to clarify the implication of phthalates for human reproduction.

Auricularia auricula-judae has long been used as food and traditional remedies in Asian countries such as Korea and China. In this study, we evaluated the in vitro anti-tumor activity of various fractions from the ethanol extracts of Auricularia auricula-judae using various tumor cell lines. To do this, the mesh of Auricularia auricula-judae was mixed with 70% ethanol and heated at 1000C for 6 hrs and ethanol extract (ETOH) was collected. Ethanol extract was fractionated with dichloromethane (DCM), ethyl acetate, n-butanol and a water extract at room temperature as well as concentrated in a vacuum concentrator at a controlled temperature(<500C). The P388D1 macrophage and Sarcoma 180, human NSCLC NCI H358 (bronchioalveolar) and SNU1 cells (Gastric carcinoma) were cultured in RPMI. As the results, the cytotoxicity of the fractional extracts decreased significantly (P<0.05) in a dose-dependent manner. Dichloromethane extract (1 mg/ml) was the highest (P<0.05) in all experimental cell lines. There was also a significantly different sensitivity (P<0.05) among the P388D1, Sarcoma 180, NCI H358 and SNU1 cells for the fractional extracts. According to IC50 values, the most potent cytotoxic activity of dichloromethane fraction was found in Sarcoma 180 and NCI H358 cell lines. Butanol fraction appeared more cytotoxic to SNU1 cell line and water fraction had the highest cytotoxicity in P388D1 cell line. We did not find any significant difference between MTT and SRB assays in their ability to estimate cytotoxicity in all cell lines. Our findings suggest a potent antitumor activity of various fractions from the ethanol extracts of Auricularia auricula-judae depending on the solvent fractions and tumor cell lines. Further in vitro and in vivo studies will provide more information on the active compounds responsible for these activities and their potential as an anti-cancer remedy.

Phellinus gilvus(PG) is a medicinal mushroom belonging to the Hymenochaetaceae basidiomycetes, and has advantages over many Phellinus species due to its short growth period (3 mo), making it cheaper to produce. In the current investigation, we determined the major components of the ethyl acetate extract of PG responsible for its biological activities and further compared the magnitude of the antioxidant/anti-inflammatory activities of components with the various fractional extracts of PG. As the results, the average total DPPH radical scavenging activities of both Fd and Fc of PG was 10 mg/mL, > 95%. Among the fractional extracts of PG, Fd had the greatest inhibitory activity with an IC50 value of 36.70㎍/mL, whereas Fb showed the lowest activity. PCA had even greater activity of NO inhibition than Fd with an IC50 value of 19.46㎍/mL. The mRNA expression of iNOS or COX-2 was nearly undetectable in the absence of LPS. However, LPS- stimulation markedly increased the expression of both iNOS and COX-2 genes. Fd inhibited the effect of LPS in a concentration-dependent manner. Six major compounds were identified from the ethyl acetate extract of PG, and protocatechualdehyde (PCA) was supposed to be the major phenolic compound of PG responsible for its DPPH free radical scavenging activity and its inhibitory effects on LPS-induced NO production in RAW264.7 cells. Further in vitro and in vivo experiments are currently underway to confirm this observation and to investigate the detailed molecular mechanisms involved in the process as well as the biological activities of other fractions of Fd.

The objective of this study was to evaluate the effects of co-culture of bovine oocytes with cumulus cells on in vitro maturation and development following in vitro fertilization in bovine oocytes. Bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DO) were co-cultured with the cumulus cells in TCM199 for 20~22 hr, and evaluated the nuclear type of oocyte. After in vitro maturation, oocytes were coincubated for in vitro fertilization with frozen-thawed spermatozoa selected by 65% percoll in DM-Heparin and DM-Caffeine for 15~18 hr. Presumptive zygotes were cultured for 48 hr in CR1aa in vitro culture medium with 10% FBS, and evaluated the cleavage rates. The results confirmed that the highest percentage of metaphase II (M-II) stage was observed in COCs (30.1±3.5%, 24.2±1.8%) as compared to DO (7.1±1.3%, 17.4±13.9%) (p<0.05). In addition, the increased cleavage rates were obtained from COCs (69.6±2.1%, 75.6±2.9%) when compared to DO (21.6±7.5%, 29.5±12.6%) (p<0.05). In conclusion, this study suggested that cumulus cells secreted positive factors during in vitro maturation of oocytes and early embryonic development after in vitro fertilization of bovine oocytes.

The purpose of current study was to examine bioaccessibility of antioxidant activity and total phenolic content in each part of water spinach (Ipomoea aquatic Forsk.). In vitro biomimicking system simulated human digestive fluid was employed in order to measure bioavailable anti-oxidative effect and phenolic content. Antioxidant activity and total phenolic content was measured by using the DPPH method and the Folin-Ciocalteu assay, respectively. Stem of water spinach had a higher DPPH free radical scavenging effect (5.43 mg/mL for IC50) than leaf (5.95 mg/mL for IC50), while leaf had a greater level of total phenolic content (287.45 μg GAE/mL) than stem (216.45 μg GAE/mL). Bioaccessible antioxidant capacity and digestive stability of total phenolic content showed a similar pattern to what found in raw materials. Our result also indicated that total phenolic content was not found to be a major marker for prediction of antioxidant activity. It is plausible that other constituents such as vitamin E and C in water spinach could be contributors for antioxidant activities.