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Human Amniotic Fluid Induces Spontaneous Hardening of the Zona Pellucida of Mouse Immature Oocytes During Maturation In Vitro (인간양수에 의한 생쥐 난자 투명대의 정자수용능력 억제의 관찰)

  • Park, Kee-Sang;Lee, Taek-Hoo;Song, Hai-Bum;Chun, Sang-Sik
    • Clinical and Experimental Reproductive Medicine
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    • v.27 no.1
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    • pp.23-29
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    • 2000
  • Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

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Treatment of Extremely High Risk and Resistant Gestational Trophoblastic Neoplasia Patients in King Chulalongkorn Memorial Hospital

  • Oranratanaphan, Shina;Lertkhachonsuk, Ruangsak
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.2
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    • pp.925-928
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    • 2014
  • Background: Gestational trophoblastic neoplasia (GTN) is a spectrum of disease with abnormal trophoblastic proliferation. Treatment is based on FIGO stage and WHO risk factor scores. Patients whose score is 12 or more are considered as at extremely high risk with a high likelihood of resistance to first line treatment. Optimal therapy is therefore controversial. Objective: This study was conducted in order to summarize the regimen used for extremely high risk or resistant GTN patients in our institution the in past 10 years. Materials and Methods: All the charts of GTN patients classified as extremely high risk, recurrent or resistant during 1 January 2002 to 31 December 2011 were reviewed. Criteria for diagnosis of GTN were also assessed to confirm the diagnosis. FIGO stage and WHO risk prognostic score were also re-calculated to ensure the accuracy of the information. Patient characteristics were reviewed in the aspects of age, weight, height, BMI, presenting symptoms, metastatic area, lesions, FIGO stage, WHO risk factor score, serum hCG level, treatment regimen, adjuvant treatments, side effects and response to treatment, including disease free survival. Results: Eight patients meeting the criteria of extremely high risk or resistant GTN were included in this review. Mean age was 33.6 years (SD=13.5, range 17-53). Of the total, 3 were stage III (37.5%) and 5 were stage IV (62.5%). Mean duration from previous pregnancies to GTN was 17.6 months (SD 9.9). Mean serum hCG level was 864,589 mIU/ml (SD 98,151). Presenting symptoms of the patients were various such as hemoptysis, abdominal pain, headache, heavy vaginal bleeding and stroke. The most commonly used first line chemotherapeutic regimen in our institution was the VAC regimen which was given to 4 of 8 patients in this study. The most common second line chemotherapy was EMACO. Adjuvant radiation was given to most of the patients who had brain metastasis. Most of the patients have to delay chemotherapy for 1-2 weeks due to grade 2-3 leukopenia and require G-CSF to rescue from neutropenia. Five form 8 patients were still survived. Mean of disease free survival was 20.4 months. Two patients died of the disease, while another one patient died from sepsis of pressure sore wound. None of surviving patients developed recurrence of disease after complete treatment. Conclusions: In extremely high risk GTN patients, main treatment is multi-agent chemotherapy. In our institution, we usually use VAC as a first line treatment of high risk GTN, but since resistance is quite common, this may not suitable for extremely high risk GTN patients. The most commonly used second line multi-agent chemotherapy in our institution is EMA-CO. Adjuvant brain radiation was administered to most of the patients with brain metastasis in our institution. The survival rate is comparable to previous reviews. Our treatment demonstrated differences from other institutions but the survival is comparable. The limitation of this review is the number of cases is small due to rarity of the disease. Further trials or multicenter analyses may be considered.

Effects of PMSG Treatment on Reproductive Organs and Serum Steroid Hormone Level in Adrenalectomized Immature rats (부신척출 미성숙 흰쥐에 PMSG투여가 생식기관 및 혈청중 Steroid Hormone 농도에 미치는 영향)

  • 한찬규;정영채;김창근
    • Korean Journal of Animal Reproduction
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    • v.7 no.1
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    • pp.41-51
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    • 1983
  • The purpose of this experiment was to investigate the effects of adrenalectomy and PMSG treatment on reproductive organs and serum steroid hormone level in immature female rats. The animals used in this experiment were 25 days old female rats weighing a, pp.oximately 70g. They were randomly divided into two groups of intact rat group (Int-) and adrenalectomized rat group (Adx-) and each group were subdivided into two groups of Non-PMSG (-Cont) and PMSG treated (-PMSG) group. The rat of PMSG-treated group (-PMSG) was administered subcutaneously with 25 IU PMSG on first day (9 a.m.) after adrenalectomy. The adrenalectomized rat groups were su, pp.ied with saline solution through the experiment period. The rate of ovulation and vaginal opening and reproductive organ weights were observed at 8, 32, 56, 80 and 104 hours after PMSG treatment. At the same time, the serum level of estradiol-17${\beta}$ and progesterone were measured by the radioimmunoassay. The results obtained were as follows: 1. Ovulation was shown at 56 hours after treatment in Int-PMSG group and Adx-PMSG group and Adx-PMSG group. The rate of ovulation was very low in PMSG-treated groups, but it was increased in 80 to 90% at 104 hours after treatment. However, there was no ovulation in Int-Cont group and Adx-Cont group. 2. Vaginal opening was shown at 56 hours after treatment in Int-PMSG group and Adx-PMSG group and a, pp.ared in 80% at 104 hours after treatment. The rate of vaginal opening in PMSG-treated groups was very low, but Int-Cont group and Adx-Cont group had no vaginal opening. 3. The weight of ovary and uterus in two PMSG-treated groups were increased with the elapse of time after treatment and were significantly heavy in all observation time, but changes in Int-Cont group and Adx-Cont group were not recognized. The weights of ovaries and utera in Adx-Cont group were increased with the elapse of time. 4. The level of serum estradiol-17${\beta}$ was remarkably increased in PMSG-treated groups (Int-PMSG and Adx-PMSG groups) compared with Int-Cont and Adx-Cont group, and significant difference was recognized between Non-PMSG group and PMSG-treated group in the experimental period. Especially, the highest levels of Int-PMSG groups and Adx-PMSG groups were shown at 80 and 56 hours after treatment and after ward estradiol-17${\beta}$ levels of PMSG-treated groups were decreased. However, changes of the levels did not a, pp.ared in Non-PMSG groups at 104 hours after treatment. 5. The level of serum progesterone in PMSG-treated groups was significantly increased between 80 and 104 hours after treatment. With the elapse of time, the level was increased in all observed groups except for Int-Cont and Adx-Conx group. And the order from the highest level at 104 hours after treatment was Int-PMSG, Adx-PMSG, Int-Cont and Adx-Cont group.

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Effect of Garlic Oil (diallyl disulfide)/ Vitamin A( retinol acetate on Heat Shock Protein Induction in Cadmium Treated Rats. (마늘유(diallyl disulfide)와 비타민 A(retinol acetate)가 카드뮴 투여 랫드에 미치는영향)

  • 김판기
    • Journal of Food Hygiene and Safety
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    • v.13 no.2
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    • pp.171-187
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    • 1998
  • Garlic occupies a special position among the many foods of vegetable origin because it is the sole food for Koreans during the their lives. And vitamin A has been ingested by forms of food or additives. Cadmium has been described as one of the most dangerous trace elements in the food and environment of man and livestocks. Since the de novo synthesis of stress proteins can be detected early after exposure to some agents, analysis of cadmium-induced changes in gene expression , ie. alterations in patterns of protein synthesis, may be useful to develop as biomarkers of exposure and damage for food hygiene. He acute and chronic combine effects of cadmium (Cd, CdCl2 20mg/kg), garlic oil(Dds: diallyl disulfide 50mg/kg, 3 times a week) and vitamin A(Ra: retinol acetate 50,000 IU/kg, 3 times a week) on Wistar male rats were evaluated concerning cadmium contents, tissues enzyme activity, HSP expression histopathological and electron microscopical examinations. The results of the study are as follows ; 1. Less cadmium was absorbed through the digestive tracts, but the ratio of contents in tissue were not changed by the simultaneous adminstration of diallyl disufide or retinol acetate. 2. ALT(alanine aminotransferase) , AST(aspartate aminotransferase), glucose, BUN (blood urea nitrogen), creatinine, the key indices of the clinical changes in hepatic and renal function were significantly hanged by the cadmium treatment after 1 week in liver, after 4 weeks in kidney. 3. Histopathological changes in cadmium treated rats were appeared at 8 weeks age treatment in kidneys. Homogenous eosinophilic material was accumulated in cortical and collecting tubular lumens at 16 weeks. Degenerated or necrotized tubular cells were observed in cortex and medulla. Degenerated seminiferous tubules and homogeneous eosinophilic material was seen in interstitial tissue of rat treated with cadmium for 16 weeks. Calcium deposits were seen in degenerated seminiferous tubules and the tubules showed severe calcification of rat treated with cadmium for 16 weeks. Electron microscope changes in kidney were observed in rats treated with CdCl2 20 mg/kg. Proximal convoluted tubule cells showed selling of cytoplasm and narrow lumen. Capillary endothelial cells showed cytoplasmic vacuoles and swelling. Degenerated epithelial cells were accumulated in tubular lumen of kidney. 4. Enhanced synthesis of 70 KDa relateve molecular mass proteins were detected in 2 hours after cadmium, exposure, with maximum activity occurring at 8~48 hours. Induction of HSP 70 was evident at proximal tubules and glomeruli in kidney. Testicular cells produced enough HSP to be detected normally. From the above results, it could be concluded that HSP70 induction by the cadmium treatment was a rapid reaction to indicated the exposure of xenobiotics, and retinol acetate reduced the cadmium induced nephrotoxicity.

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The Enhancing Effect of Atopic Dermatitis by cosmetics containing estern medical herbs (한방소재 화장품의 아토피 피부염 개선 효과)

  • Kang, Shin-Jyung;Kim, Ae-Jung;Lee, Yeon-Hee;Lee, Myoung-Sook;Joung, Kyung-Hee;Cho, Nam-Ji
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.10 no.11
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    • pp.3500-3505
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    • 2009
  • Sixteen moderate atopic dermatitis patients were treated with applying toner and cream using estern medical herbs[Estern medical complex(Radix Glycyrrhizae, Radix Angelicae Dahuricae, Rhizoma Ligustici Chuanxiong, Fructus ponciri Seu Aurantii Immaturus, Radix Rehmanniae Praeparata, Radix Puerariae, Rhizoma Corydalis), Cypress, Green tea, Radix Astragali, Red ginseng, Syrup ferment, Apple ferment filtrate] on the atopic area 2-3 times per day for 6 weeks. After 6 weeks of study, the result was a decrease in SCORAD index($26.9{\pm}11.8$ to $14.9{\pm}9.7$(p<0.05)), IgE($641.9{\pm}1294.6$ to $565.8{\pm}1076.8$), Eosinophil count($246.7{\pm}203.5$ to $203.3{\pm}130.7$(p<0.05)) and TEWL($16.7{\pm}5.1$ to $15.4{\pm}8.7$(p<0.05)). And skin hydration was increased($36.6{\pm}10.0$ to $44.0{\pm}10.3$(p<0.01)) maintaining skin pH level. The patient;s and physician's global assessment also improved. Therefore, estern medical herbs may play a role in treatment of atopic dermatitis.

Influence of Interferon-${\tau}$ on the Production of Prostaglandins, Cyclooxygenase-2 Expression In Vitro and Release of Progesterone in Bovine Endometrial Cells

  • Lee, Ji-Eun;Lee, Yong-Seung;Yoo, Han-Jun;Lee, Kyoung-Jin;Park, Joung-Jun;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Choon-Keun
    • Journal of Embryo Transfer
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    • v.27 no.4
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    • pp.245-252
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    • 2012
  • The purpose of the present study was to investigate the effect of IFN-${\tau}$ on prostaglandin synthesis, cyclooxygenase-2 (COX-2) gene expression in vitro and concentration of progesterone (P4) in endometrial cells. Epithelial and stromal cells cultured in vitro were isolated from bovine endometrium and stimulated with increasing doses of IFN-${\tau}$ (0, 0.02, 0.2 and 2 ug/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin $E_2$ and $F_{2{\alpha}}$ levels in the culture media were analyzed by enzyme immunoassays and total RNA was extracted from the cells for RT-PCR. P4 concentrations of blood samples were assayed by chemiluminescent immuno assays system. In epithelial cells, COX-2 gene expression was increased in the presence of IFN-${\tau}$ (p<0.05), but it was not significantly different in all groups of stromal cells except for 2 ug/ml IFN-${\tau}$ group (p<0.05). Although IFN-${\tau}$ did not affect $PGE_2$ and $PGF_{2{\alpha}}$ production in epithelial cells, it decreased $PGE_2$ and $PGF_{2{\alpha}}$ production significantly in stromal cells (p<0.05). In vivo experiment, blood concentration of P4 was significantly increased after addition of IFN-${\tau}$ (1 ug/ml). The results indicate that PG production was mediated by COX-2 expression in stromal cells but it was not affected in epithelial cells and this suggest that treatment of IFN-${\tau}$ could improve the implantation environment of uterine by maintenance of high P4 concentration.

Isotopic Differences among Zooplankton Taxa and Seasonal Variation of Zooplankton Community Coexisting with Microcystis (Microcystis와 공존하는 동물플랑크톤 군집의 계절변화 및 안정동위원소비의 차이)

  • Lee, Jae-Yong;Kim, Jai-Iu;Jung, Yu-Kyong;Kim, Bom-Chul
    • Korean Journal of Ecology and Environment
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    • v.43 no.1
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    • pp.1-10
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    • 2010
  • We used stable isotope analysis to investigate the hypothesis that zooplankton were not able to utilize Microcystis as a food source. We also studied seasonal variation of the zooplankton community in a eutrophic Wangsong reservoir. The dominant copepod species changed from Cyclops vicinus to Thermocyclops taihokuensis. Density of zooplankton was suppressed by high density of Microcystis in June and the density of phytoplankton and chlorophyll a concentration was lowest in July. The difference in $\delta^{13}C$ and $\delta^{15}N$ between cladocerans (Daphnia galeata and Bosmina longirostris) and copepods showed the maximum difference of $12^{\circ}/_{\circ\circ}$ and $4^{\circ}/_{\circ\circ}$, respectively, indicating different energy sources and trophic positions between two taxonomic groups. The difference in $\delta^{13}C$ between Microcystis and zooplankton ranged from $3{\sim}7^{\circ}/_{\circ\circ}$, supporting the hypothesis that Microcystis could not be used as food source of zooplankton.

The effect of adding ethanolic saffron petal extract and vitamin E on growth performance, blood metabolites and antioxidant status in Baluchi male lambs

  • Alipour, Fereshteh;Vakili, Alireza;Mesgaran, Mohsen Danesh;Ebrahimi, Hadi
    • Asian-Australasian Journal of Animal Sciences
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    • v.32 no.11
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    • pp.1695-1704
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    • 2019
  • Objective: This study investigated the effects of the administration of ethanolic saffron petal extract (SPE) and vitamin E (Vit E) on growth performance, blood metabolites and anti-oxidant status in Baluchi lambs. Methods: Thirty-two Baluchi male lambs ($35.22{\pm}5.75kg$) were randomly divided into 4 groups. The 1st (control), 2nd (injectable saffron petal extract [ISPE]), and 3rd (Vit E) groups were respectively injected subcutaneously with either physiological saline (5 mL), SPE (25 mg/kg body weight [BW]) or DL-${\alpha}$-tocopheryl acetate (225 IU) once a week. An oral dose of SPE (500 mg/kg BW) was also administered to the 4th group (oral saffron petal extract [OSPE]). Feed intake and BW were measured for 42 days and blood samples were taken on days 1, 14, 28, and 42. The lambs were slaughtered, and tissue samples were taken. Results: Growth performance and many blood metabolites were not affected (p>0.05) by the treatments. Cholesterol of plasma in the ISPE and Vit E groups was similar and less (p<0.01) than both the OSPE and control groups. Although there was no significant difference between the control and other groups for plasma triglyceride, the ISPE group showed lower (p<0.05) triglyceride than the OSPE and Vit E groups. The highest (p<0.01) plasma glutathione peroxidase (GPx) was detected in the OSPE group, while the ISPE and Vit E groups showed higher (p<0.01) superoxide dismutase (SOD) of plasma than the control. Malondialdehyde of plasma in the ISPE group was lower (p<0.05) than the OSPE. No differences (p>0.05) were observed among the groups for antioxidant status of both longissimus dorsi muscle and liver. However, the activity of GPx in the kidney and heart, as well as SOD activity in the kidney, were influenced ($p{\leq}0.01$) by the treatments. Conclusion: Adding ethanolic SPE improved antioxidant status and lowered lipids oxidation in lambs. The SPE and Vit E demonstrated similar effects on antioxidant status in lambs.

Development and characterization of hyaluronic acid-based orally disintegrating film containing vitamin D (히알루론산 기반 비타민 D 함유 구강용해필름의 제조 및 특성평가)

  • Kang, Seo-Yeon;An, Da-Yeon;Han, Jung-Ah
    • Korean Journal of Food Science and Technology
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    • v.54 no.3
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    • pp.327-333
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    • 2022
  • An orally disintegrating film (ODF) based on hyaluronic acid (HA) containing vitamin D was developed. The vitamin D content in the ODF was set based on the adequate intake (AI) of vitamin D from 0 to 10 AI (0, 1, 4, 7, and 10AI). The control (0AI) had the highest thickness and showed the longest disintegration time among the samples. The moisture content of the ODFs was significantly lower in those with vitamin D compared to the control. As the amount of vitamin D increased, the water vapor permeability (WVP) of the ODFs decreased, and the opacity significantly increased. The tensile strength was higher in the films containing vitamin D compared to the control films. However, the elongation at the break showed no significant difference among the films. The vitamin D content in the film was reduced by 25.7-44.2% during processing compared to the amount that was originally added. Based on the above results, a new and convenient vitamin D delivery system, an ODF, could be successfully produced.

Improvement of Reproductive Efficiency of Artificial Insemination Following Estrus Induction in Dog II. Semen Freezing and Artificial Insemination in Dog

  • Lee, Y. R.;S. Y. Choe
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2002.11a
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    • pp.92-92
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    • 2002
  • Considerable attention has been focused on the cryopreservation of semen and estrus induction in dog, as consequence of poor productivity caused by long anestrus period, in order to enhance the productivity of youngs and to preserve the breeds. The objectives of this study were to evaluate semen quality after cryopreservation and to evaluate the Pregnancy rate after insemination (AI). Fifty infertilie dogs (age 2∼3 years) were selected for the study and divided into three different estrus induction treatment groups. Group 1: dogs (n=15) were given clomifene (0.1 mg/kg) orally for five days at 12 hr intervals. Group 2: dogs (n=15) were given bromocriptine (50 $\mu\textrm{g}$/kg) orally for five days at 12 hr intervals, followed by single injection intravenously of 500 IU GnRH (Group 3, n=20) when pro-estrus occurred. The rates of pregnancy in estrus inducted dogs mated naturally compared to those inseminated artificially with ejaculated fresh semen and frozen-thawed semen. Estrus detection was performed using the method of vaginal smear and confirmed by the plasma progesterone assay. The ejaculated semen to freeze was exposed to a mixture of Tris extender with cryoprotectant (Trisma, 81 mM: TES, 209 mM: citric acid, 6 mM; glucose, 5 mM; glycerol, 8%) and cryopreserved gradually by slow-cooling at 17 cm above the surface of liquid nitrogen (LN$_2$) for 23 min. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Pregnancy was confirmed by ultrasonograpy on day 25, 35 and 55 post insemination. The use of fresh semen, the pregnancy rates were observed 66.6, 66.6, 75.0 and 83.3% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. The use of frozen-thawed semen, the pregnancy rates were observed 66.6, 33.3, 50.0 and 60.0% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. No difference was observed in the number of offspring produced among natural estrus and treated groups inseminated with fresh or frozen-thawed semen. In conclusion, the pregnancy rate of dogs treated with a combination of GnRH and bromocriptine was more effective than use of clomifene or bromocriptine only. In addition, frozen-thawed semen can be used successfully far artificial insemination in dog.

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