This study was performed to investigate the stimulating effect on oocyte maturation and cumulus cell expansion in TC199 media by human cord serum (HCS) supplementation. Immature mouse oocyte cumulus complexes (OCCs) were cultured in TC199 media supplemented with bovine serum albumin (BSA), HCS and human chorionic gonadotropin (hCG) instead of luteinizing hormone (LH) respectively, and the expression of cumulus expansion and oocyte maturation were observed. After 4hr and 24hr culture with or without OCCs, media containing 0.4% BSA, 10% HCS and 10 lU hCG respectively were collected and analyzed for changing concentrations of estradiol (E2), progesterone(P4), testosterone(T), and PGF2. There were no elevation of E2, T, and PGF2 by OCCs culture, but minute elevation of P4 level by 24hr OCCs culture in hCG supplementation (p=0.048). The stimulating pattern of cumulus expansion of OCCs by HCS and hCG supplementation was similar to our previously report using Ham's F-10 media, however oocyte maturation rates after 24hr OCCs culture in all media were increased by 20~30% compared to Ham's F-10 media. These results suggest that LH in HCS induce cumulus expansion probably by P4 secretion of OCCs, and TC199 is efficient media for immature mouse oocyte maturation.
Animals ; Chorionic Gonadotropin ; Cumulus Cells* ; Dinoprost ; Estradiol ; Humans* ; Luteinizing Hormone ; Mice ; Oocytes* ; Serum Albumin, Bovine

No abstract available.
Insemination* ; Spermatozoa*

No abstract available.
Animals ; Embryonic Structures* ; Fertilization in Vitro* ; Humans* ; Mice* ; Quality Control*

OBJECTIVES: This study was to investigate the fertilization rate after intracytoplasmic sperm injection (ICSI) or partial zona dissection (PZD) of human and hamster sperm into hamster oocyte in in vitro fertilization (IVF). In addition, the possibility of clinical application was evaluated by the comparison of usefulness and difference of these method. MATERIALS AND METHODS: Hamster immature oocytes were obtained from oviducts superovulated by PMSG and hCG, and hamster sperms were obtained from epididymis. The freezed human sperms were thawed before use. Fertilization were confirmed by two pronuclei, one pronucleus, swollen sperm head or/and two polar bodies at 7~8 hour after ICSI or PZD. RESULTS: The fertilization rates after ICSI and PZD of human sperm to hamster oocyte were 3.6%, 64.2%, 73.6%, and 55.6% for negative control, positive control, ICSI, and PZD respectively, suggesting that ICSI only showed improved fertilization rate (p<0.01). The fertilization rates after ICSI and PZD of hamster sperm to hamster oocyte were 11.1%, 51.2%, 39.6%, and 72.7% for negative control, positive control, ICSI, and PZD respectively, suggesting that PZD only showed improved fertilization rate (p<0.01). PZD showed significantly higher fertilization rate than ICSI (p<0.05). CONCLUSIONS: As for the fertilization rate by ICSI and PZD using hamster oocyte in IVF, ICSI technique was considered to be more useful for human sperm and PZD technique for hamster sperm. Therefore, ICSI technique was considered more appropriate for experimental application using human sperm and hamster oocyte.
Humans

OBJECTIVES: This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). MATERIALS AND METHODS: Two to eight cell embryos were obtained from oviducts of mated F1 hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH, DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow- cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. RESULTS: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1%, 79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4-8 cell embryos in PROH were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The development rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. CONCLUSIONS: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH showed better development rates of embryos in 2 and 4-8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.
Pregnancy ; Female ; Humans ; Mice ; Animals

OBJECTIVE: Nitric oxide (NO) produced in ovary may contribute to follicle maturation, ovulation, oocyte maturation and luteinization. In this study, the effect of nitric oxide on the spontaneous maturation of mouse oocyte was observed. Method: The index of oocyte maturation was checked by the germinal vesicle breakdown (GVBD) and appearance of polar body (PB) under microscope in the denuded oocytes and oocyte-cumulus complexes (OCCs) from mouse ovarian follicles after 24 hours pregnant-mare serum gonadotropin treatment. RESULTS: The GVBD appeared 50 %, 1 hour and 80 %, 2 hrs after changes of oocytes from dibutyryl cAMP (dbcAMP, 0.5 mM) contained media into dbcAMP-free media. dbcAMP (0.5 mM) completely blocked the GVBD until 24 hrs but dbcGMP (5 mM) delayed the GVBD by 1 hr. Sodium nitroprusside, the NO generator, inhibited the GVBD dose-dependently at 2 hr incubation in denuded and OCCs. The appearance of GVBD was not different between control and dbcGMP or SNP in denuded oocytes and OCCs at 24 hrs incubation. The guanylate cyclase activity in denuded oocyte cytosol was not detected whereas the guanylate cyclase activity in OCCs cytosol was 1.3 nmole/min/mg protein which was increased about 3 times by SNP (100 micrometer). CONCLUSION: These results suggest that the NO in ovary may delay the spontaneous oocyte maturation in early stage by acting on the maturation signaling protein as well as guanylate cyclase.
Animals ; Bucladesine ; Cytosol ; Female ; Gonadotropins ; Guanylate Cyclase ; Lutein ; Luteinization ; Mice* ; Nitric Oxide* ; Nitroprusside ; Oocytes* ; Ovarian Follicle ; Ovary ; Ovulation ; Polar Bodies ; Staphylococcal Protein A

OBJECTIVE: Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel hypothalamic neuropeptide, has been suggested to play a role in ovarian folliculogenesis. The present study evaluated the effect of PACAP on the growth of preantral follicles. METHODS: Preantral follicles were mechanically isolated from ovaries of 21-day-old rats and cultured in groups for 3 days in serum-free medium in the absence or presence of PACAP-38 (10-6 M). RESULTS: Treatment with PACAP-38 resulted in an increase in follicle diameter by 75% whereas treatment with follicle stimulating hormone (FSH) increased follicle diameter by 65%. PACAP-38 treatment enhanced the granulosa cell proliferation as measured by thymidine incorporation analysis. Furthermore, the production of progesterone by cultured granulosa cells and GFSHR-17 cell line was stimulated by PACAP-38. Interestingly, PACAP enhanced FSH action on stimulation of SF-1 and aromatase gene expression. CONCLUSION: The present results demonstrate that PACAP stimulated preantral follicle growth by potentiating proliferation and by stimulating steroidogenesis.
Animals ; Aromatase ; Cell Line ; Female ; Follicle Stimulating Hormone* ; Gene Expression ; Granulosa Cells ; Neuropeptides ; Ovarian Follicle* ; Ovary ; Pituitary Adenylate Cyclase-Activating Polypeptide* ; Progesterone ; Rats ; Thymidine

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient. The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of 55 degrees C. Phenylmethylsulfonylfluoride and 4- (2- Aminoethyl) -benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.
Acanthamoeba/*enzymology/isolation & purification/pathogenicity ; Acanthamoeba Keratitis/*parasitology ; Animals ; Cornea/parasitology ; Humans ; Hydrogen-Ion Concentration ; Korea ; Serine Endopeptidases/chemistry/*isolation & purification/*metabolism ; Substrate Specificity ; Temperature ; Virulence Factors

Objectives: This study was to evaluate whether Ham's F-10 used in assisted reproductive technology (ART) could be replaced with newly-introduced Medicult or Human Tubal Fluid (HTF) media, and the rate of embryo development could be enhanced by cumulus cell coculture. METHODS: Ham's F-10, Medicult, and HTF media supplemented with 0.4% bovine serum albumin (BSA) were used. Two-cell embryos were obtained from oviducts of mated F1 hybrid female mice superovulated by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Cumulus cells for coculture were obtained from oviducts of ICR female mice superovulated by PMSG and hCG. Two-cell embryos were cultured in Ham's F-10, Medicult, and HTF media respectively to observe and compare the rate of embryo development. In addition, two-cell embryos were cultured in these three media for 24, 48, 72, 96 hrs with or without cumulus cell, and rates of embryo development were investigated and compared. RESULTS: As for the rate of embryo development to hatched blastocyst after 96 hrs culture, HTF (87.5%) and Ham's F-10 (85%) were significantly higher than Medicult (70.5%). The beneficial effect of embryo development by cumulus cell coculture on two-cell mouse embryo among these three media was enhanced significantly in Medicult (control 88.5% versus coculture 98.5%) by 24 hrs, and was not enhanced statistical significantly but slightly elevated in Ham's F-10 (86.5% versus 95.5%) and HTF (91.3% versus 96.9%) by 48 hrs, but rates of embryo development were similar between control and coculture group in all three media by 96 hrs. Significant differences were not shown in three media, but HTF showed generally high tendency of the enhancing effect of embryo development and the beneficial effect of embryo development by coculture. CONCLUSIONS: As a result of culturing two-cell embryos in three media for 96 hrs, generally HTF and Ham's F-10 showed higher rate of embryo development than Medicult. As for the beneficial effect of coculture, Medicult only showed early transient significant improvement of embryo development. Considering that coculture effect of good quality media may be not so great, Ham's F-10 and HTF are more stable media than Medicult. Accordingly, HTF may be considered to be a medium to replace with Ham's F-10, however, the present study suggest that Medicult or HTF is not able to replace with Ham's F-10 in ART.
Animals ; Blastocyst ; Chorionic Gonadotropin ; Coculture Techniques* ; Culture Media* ; Cumulus Cells* ; Embryonic Development* ; Embryonic Structures* ; Female ; Gonadotropins ; Humans* ; Mice* ; Oviducts ; Pregnancy ; Reproductive Techniques, Assisted ; Serum Albumin, Bovine

Abrupt increase in the size of cervico-mediastinal tumor due to infection or spontaneous hemorrhage into cyst can induce severe tracheal compression and therefore sudden death. A 5 year old boy, who had a history of URI, had an enlarging cystic hygroma on the right side of the neck and anterior mediastinum. Under diagnosis of the cervico-mediastinal cystic hygroma, surgical removal was scheduled. After induction of anesthesia, intubation was done without any difficulty. A few minutes later, signs of partial airway obstruction were appeared. And within a very short period, total airway occlusion occurred. The tracheal tube was removed and manual ventilation was performed with positive airway pressure, but ineffective. We attempted to puncture cricothyroid membrane with 14 Gauge needle in order to ventilate manually. As soon as we puncture cricothyroid membrane, straw-colored fluid, not air, gushed out through a needle. After aspiration of about 200ml of cystic fluid, the obstructive signs disappeared and the patency of the airway was maintained. Intraoperatively, no more airway problems occured and vital signs were stable. And postoperatively, patient had no specific complications and discharged on the 7th day after operation.
Airway Obstruction* ; Anesthesia ; Child, Preschool ; Death, Sudden ; Diagnosis ; Hemorrhage ; Humans ; Intubation ; Intubation, Intratracheal* ; Lymphangioma, Cystic* ; Male ; Mediastinum ; Membranes ; Neck ; Needles ; Punctures ; Ventilation ; Vital Signs