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: 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

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

Eight cases of combine hepatocellular-cholangiocarcinoma(HCC-CC) of the liver were analysed along with their growth patterns and histologic subtypes to draw a possible implication in understanding of their histogenesis. The relative incidence of combined HCC-CC among the surgically resected primary carcinomas of the liver(485 cases) was 1.6%. The combination patterns varied and were classified as follows; the multinodular tumor, each consisting of HCC or CC element(type 1) was found in 1 case, the single tumor mass with two distinct compartments of HCC and CC(type 2) in 3, and the single tumor with random mixture of two elements(type 3) comprised the remaining 4 cases. Of the 7 cases of single tumor mass(type 2 and 3) HCC and CC comprised the major component in 5 and 2 cases, respectively. The histologic features of both HCC and CC elements were not different from those of isolated HCC and CC, except for two of CC element in type 3 which disclosed the intermediated differentiation or socalled cholangiolocellular carcinoma in part. We conclude that there is no significant difference in the relative incidence of combined HCC-CC among primary carcinomas of the liver and their subtypes compared to that in other countries, regardless of high incidence of both HCC and CC in Korea. Also, we discussed a possible histogenisis along a hypothesis that some of the combined HCC-CC be the consequence of interposition of different cell type from a new subclone into the growth of an initial single cell type of primary carcinoma of the liver.
Incidence ; Carcinoma, Hepatocellular

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

OBJECTIVE: Immunofluorescence microscopy including confocal laser scanning microscopy and electron microscopy were used to study the production of fibronectin, tenascin, and laminin in the cumulus-corona (CC) cells surrounding mature, unfertilized oocytes after ovulation in view of their presumptive importance in the coordination of the processes leading to fertilization and early embryo cleavage, including the final maturation of the ovum, the sperm-egg interaction, and the complex biochemical mechanism between the ovum and the oviduct. METHODS: Mature oocyte-cumulus complex (OCC) was cultured for 24 and 48 hour and fixed in 3.7% formaldehyde. Specimens were incubated with a mixture of primary monoclonal antibodies recognizing different epitopes of fibronectin, tenascin, and laminin, and then with a mixture of secondary antibodies containing FITC, TRITC, and Cy-5 conjugated antibodies. Observation was made by confocal laser scanning microscope equipped with epifluorescece optics. Transmission electron microscopy were used to observe the OCC at 24 and 48 hours after cultrue. RESULTS: The immunocytochemical date demonstrated that CC masses are capable of producing fibronectin and tenascin but their production is heterogeneous in the CC population. Immunoreactivity to fibronectin and tenascin was shown mostly by inner corona cells, and the intensity of immunofluorescence decreased from the central corona cells to the peripheral cumulus cells. Colocalization of fibronectin and tenascin was evident in most CC cells. Moreover, fibronectin and tenascin immunoreactive material was observed in the intracytoplasmic areas, at the plasma membrane level as well as in the extracellular matrix. Whereas, laminin immunofluorescence was found around plasma membrane and extracellular area, but a intracytoplasmic reaction was rarely observed. The distribution of laminin immunofluorescence was similar to that of fibronectin and tenascin, but in some cumulus cells, colocalization between them was not found. Ultrastructurally, cumulus cells projected numerous long, thin microvilli into the intercellular area and some micovilli penetrated into zona pellucida. The inner layer of the cumulus mass was loose arrangement of relatively uniform, small cells with widened intercellular spaces, whereas in the outer layer, cumulus cells are rather larger in size and compact arrangement by narrow, irregular spaces. A small and large linear gap junctions were easily found at cell contacts. The cytoplasm of most cells had abundant organelles typical of steroidogenesis: numerous mitochondrias, a well-developed smooth endoplasmic reticulum, electron dense lipid droplets, and bundles of microtubules and microfilaments. Rudimentary disrupted basal lamina along the cytoplasmic border was rarely seen in a few inner conora cells. CONCLUSION: Even though the functional role of these extracellular matrix proteins remains still unclear, it is reasonable to suggest that they are necessary in various steps of the reproductive process. Cumulus cells appears to be a heterogeneous and dynamic system for suitable microenviroment of fertilization. And functional differences between corona and cumulus cells during the oocyte denudation may be accounted for particular distribution of these adhesive proteins and steroidogenesis-related organelles.
Actin Cytoskeleton ; Adhesives ; Animals ; Antibodies ; Antibodies, Monoclonal ; Basement Membrane ; Cell Membrane ; Cumulus Cells ; Cytoplasm ; Embryonic Structures ; Endoplasmic Reticulum, Smooth ; Epitopes ; Extracellular Matrix ; Extracellular Matrix Proteins ; Extracellular Space ; Female ; Fertilization ; Fibronectins* ; Fluorescein-5-isothiocyanate ; Fluorescent Antibody Technique ; Formaldehyde ; Gap Junctions ; Immunohistochemistry ; Laminin* ; Microscopy, Confocal ; Microscopy, Electron* ; Microscopy, Electron, Transmission ; Microscopy, Fluorescence ; Microtubules ; Microvilli ; Mitochondria ; Oocytes ; Organelles ; Oviducts ; Ovulation ; Ovum ; Sperm-Ovum Interactions ; Tenascin* ; Zona Pellucida