This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.

The studies were carried out to investigate the effects of co-culture with cumulus cells and oviduct epithelial cells on the in vitro fertilization and cleavage rate of bovine follicular cocytes and to determine the optimum thawing temperature and equilibration time on in vitro developmental rate of frozen bovine embryos. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TGM-199 medium containing 10 IU /ml의 PM SG, 10 IU /ml의 hCG, ip g/ml의 -estradiol and 10% FCS for 24~48 hrs in incubator with 5% in air at 38.5. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquld nitrogen and thawed in 3 water. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are sunanarized as followes :1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM499 medium were 75.0~76.8% and 17.3~27.6%, respect-ively. And in-vitro fertilization rates of cumulus-enclosed oocytes(55.4%)were significantly(p<0.05) higher than cumulus-denuded oocytes (23.1%). 2. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with l l04cells /ml, 1 x l06cells /ml, lx l08cells /ml and 1 x l015cells /ml oviduct epithelial cells in TCM-199 medium were 74.5~77.8% and 15.7~21.20 respectively.3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes cocultured in '1CM-199 media containing PMSG, hCG, PMSG+hCG. PMSG+-estradiol, hCG+-estradiol 0 to 40 hrs after insemination were 74.0~77.4% and l8.9~23.l%, re-spectiv ely.4.The survival rates of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol,DMSO and propanediol were 23.5~31.4% and 20.6~34.l%, respectively. 5. The temperature thawed at 3 after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 2 and 35.6. The equilibration time on the survival rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20min.). (Key words : bovine embryos, co-culture, freezing, in vitro development)

The study was conducted to determine the optimal hormone and glucose levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for blastocyst development. Oocytes matured in TCM 199 + 10% FCS + hormones and glucose were fertilized in vitro in a TALP medium with swim up separated and heparin-treated epididymal cauda spermatozoa. Oocytes were cultured for 2~5 days in synthetic oviduct fluid medium (SOFM) supplemented with 10% FGS and with different hormone and glucose levels, and further cultured 5 days same medium in SOFM. The results are summarized as follows : The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + estradiol, PMSG + estradiol 0 to20 hours after insemination were 88.0% and 81.8%, 82.6% and 68.4%, 80.0% and 75.0%, 80.0% and 65.0%, 77.3% and 64.7%, respectively. The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + estradiol, PMSG + estradiol 20 to 40 hours after insemination were 92.0% and 87.0%, 92.0% and 82.6%, 91.3% and 81.0%, 85.2% and 73.9%, 87.5% and 81.0%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose lelvels 0~3 days after insemination were 31.5~48.1% and 10.0~16.7%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose levels 4~8 days after insemination were 30.0~53.8% and 8.7~19.2%, respectively. The cleavage and in vitro developmental rates to blastocyst were higher in TCM 199 media containing various glucose levels 0~3 days after insemination than 4~8 days.

재래산양(在來山羊)에 있어서 prostaglandin 의 투여(投與)가 황체퇴행(黃體退行)시에 야기되는 호르몬 함량의 변화(變化)를 구명(究明)하기 위하여 발정주기의 10일에 (Lutalyse) 5mg을 3시간 간격으로 2회 주사(注射)한 다음 경시적(經時的)으로 혈중(血中) 난소(卵巢) 및 뇌하수체(腦下垂體) hormone의 함량을 조사하였다. 본 연구에서 얻어진 결과(結果)를 요약(要約)하면 다음과 같다. 재래산양(在來山羊)에 있어서 발정주기(發情週期)의 10일에 를 주사한 결과 혈중(血中) progesterone의 평균농도는 주사직전에 였으며 주사후(注射後) 3시간에 로 약 60% 감소(減少)하였으며 12시간에는 로 감소(減少)하여 72시간까지 1.02ng/ml 이하로 감소(減少)하였다. 혈중(血中) estradiol- 함량은 투여후(投與後) 6시간부터 유의(有意)하게(p < 0.05) 증가(增加)하여 72시간까지 유지되었다. 혈중(血中) LH함량은 주사직전에 였으며 투여후 3~12시간에는 증가(增加)하였으나 그후 24~72시간 사이에는 에서 로 감소(減少)하는 경향(傾向)을 보였다. 혈중(血中) FSH 함량은 투여전 였으며 투여후(投與後) 3시간부터 감소하기 시작하여 72시간까지 유의차없이 약간 감소(減少)를 나타내었다. 혈중(血中) prolactin의 함량은 유의성(有意性)은 없었으나 투여후 약간 증가(增加)하는 경향(傾向)을 보였다. 이상의 결과(結果)에서 LH함량의 최초 증가는 혈장중(血漿中) progesterone의 감소로 기인된 것이며 성선척극(性腺刺戟) hormone의 분비형태(分泌形態)는 황체(黃體)의 퇴행시에 progesterone 또는 progesterone과 estradiol-의 함량비의 차이로 인하여 상위되었다고 본다.

These studies were undertaken to examine the interaction of tamoxifen with sex steroid hormones in rat uterine activity. The uterine wet weights of the immature Tat uterus were examined after the administration of estradiol-l7(1g), tamoxifen(50g), progesterone(lmg). The uterotropic activity in immature ovariectomized rats was observed under various treatment conditions following pretreatment with above drugs. The results obtained were as follows:1) Tamoxifen produced significant increase (p <0.01) in uterine wet weight compared with control group, although the increase was not as great as that seen with estradiol-17. Administration of estradiol-17 together with tamoxifen inhibited significantly the increase of uterine wet weight by estradiol-17 (p < 0.01). Coadministration of progresterone with tamoxifen partly blocked the increase of tamoxifen-induced uterine wet weights by progesterone. 2) Estradiol-17after the estradiol-17 pretreatment discontinued the declining uterine wet weights due to the absence of estrogen support, but uteri continued to increase in weight if daily estradiol-17 was maintained. Administration of tamoxifen on the fourth day of estradiol-17 treatment reduced uterine wet weights within 24 hours, and the weights continued to decline with additional tamoxifen. 3) The modest growth of the uterus induced by three daily injections of 5Opg tamoxifen remained stable for five days, with or without additional tamoxifen treatment. Coadministration of tamoxifen with estradiol17 increased slightly the increase of uterine wet weight by tamoxifen. Coadministration of tamoxifen with progesterone inhibited the increase of uterine wet weight by tamoxifen. 4) The modest growth of the uterus induced by three daily injections of lmg progesterone reduced uterine wet weight to the control level for five days. Commencement of tamoxifen or estadiol-17 injections on the fourth day of progesterone treatment rapidly elevated uterine wet weight.

The greater horseshoe bat (Rhinolophus ferrumequinum) is distributed throughout Europe, Africa, Australia, and South Asia. It habits mainly in the cave in small groups and forming communities in late spring. It has interesting reproductive behavior because it keeps sperm for a few months in female reproductive tracts and then those sperms attend in fertilization. This breeding pattern is a sperm storage type and belongs to Rhinolophidae or Hipposideridae. The greater horseshoe also habits in Korea. However, the reasons of reproductive behaviors has not much uncovered. In this study the characters of ovary and the levels of steroid hormones were investigated from September to November. The histological, ELISA, and immunohistochemical methods were employed. The pre-ovulatory follicle was detected only at October sample. On the other hand, the blood level of testosterone was not detectable but the levels of 17β-estradiol and progesterone were exist within the detectable range. E2 and P4 levels were peak in October. Besides, the key enzymes for estradiol synthesis, CYP17 and CYP19 were localized in the theca layer and granulosa cells, respectively. October is known as mating time in this species. However, progesterone receptors could not detect at this period. Put together, it is suggested that, the increase of estrogen and the absence of progesterone receptors on preovulatory follicle is the cause of the mating without ovulation. The understanding of the expression regulation in this system will be base of the understanding the anovulation in mammals.

The roots of Platycodon grandiflorum are known as traditional medicine, has been extensively used since ancient times as a therapeutic to treat cold, cough and asthma in Korean traditional medications. This study was conducted in order to profile proteins from the hormone induced diploid and tetraploid roots using high throughput proteome approach. Two dimensional gels stained with CBB, a total of 64 differential expressed proteins were identified from the diploid root using image analysis by Progenesis SameSpot software. Out of total differential expressed spots, 20 differential expressed protein spots (≥ 2-fold) were analyzed using MALDI-TOF-TOF mass spectrometry whereas a total of 13 protein spots were up regulated and 7 protein spots were down-regulated. However, in the case of tetraploid root, a total of 78 differential expressed proteins were identified from tetraploid root of which a total of 28 differential expressed protein spots (≥ 2-fold) were analyzed by mass spectrometry whereas a total of 16 protein spots were up regulated and a total of 12 protein spots were down-regulated. However, proteins identified using iProClass databases revealed that the identified proteins from the explants were mainly associated with the nucleic acid binding, oxidoreductase activity, transporter activity and isomers activity. The exclusive protein profile may provide insight clues for better understanding the characteristics of proteins and metabolic activity in various explants of the economically important medicinal plant Platycodon grandiflorum.

Platycodon grandiflorum, known as Doraji in Korea, is used in various medications and traditional cuisine in Korea. This study was conducted to characterize the hormonal effects of diploid and tetraploid roots of P. grandiflorum using proteomics technique. Prior to proteome analysis, different kinds of growth hormones; IBA (1mg/L), NAA (1mg/L) and IAA (1mg/L) were applied in the adventitious (Diploid and tetraploid) roots for investigation. Solid (1/4MS) and liquid (1/2MS) medium were performed in the present study to investigate the hormonal effects. In diploid roots, two dimensional gels stained with CBB, a total of 1154 protein spots were identified using image analysis by Ludesi REDFIN 3 programme (Ludesi AB, Lund, Sweden: www.ludesi.com). Out of 1154 differential expressed protein spots, a total of 33 protein spots (≥ 2-fold) were selected for mass spectrometry. Among the 33 protein spots, 7 protein spots were up-regulated in IBA, 12 proteins in NAA and 14 proteins in IAA. In the case of tetraploid roots that performed under solid medium, a total of 842 differentially expressed protein spots were identified of which 34 proteins spots (≥ 1.5-fold) were selected for mass spectrometry. Out of 34 protein spots, 11 proteins were up-regulated in IBA, 10 proteins in NAA and 13proteins in IAA. However, a total of 659 differentially expressed proteins were confirmed from the liquid medium of tetraploid roots from which 32 proteins spots (≥ 1.5-fold) were sorted for MS analysis. Out of these 32 proteins, a total of 3 proteins were up-regulated in IBA, 7 proteins in NAA and 22 proteins in IAA. The identified proteins may provide insight clues for better understanding of the characteristics of proteins and biological activity from adventitious roots of Platycodon grandiflorum.