Apoptosis Induction and Clusterin Expression of NRP-152 Cells by Tamsulosin

PURPOSE: The aim of this study was to know whether and how tamsulosin induces apoptosis of normal rat prostate cells, and the relationship between apoptosis and clusterin expression. MATERIALS AND METHODS: We used a prostate cell line, NRP-152 cells which are the basal epithelium cell originated from rat prostate. The NRP-152 cells were treated with various concentrations(50, 100, 200, 400 uM) of tamsulosin for 24 h. To evaluate apoptosis, the cultured NRP-152 cells were stained with Heochst 33258 and Propidium Iodide (PI) without fixation. We also examined DNA fragmentation analysis to confirm apoptosis. In addition, to elucidate the signal transduction pathway by which apoptosis is induced, we examined Bcl-2 family proteins such as Bcl-2, Bax, Bad, Bcl-xL, and Bim by real-time RT-PCR. RESULTS: After tamsulosin treatment, the rate of apoptosis was 25% at 100 micrometer, 50% at 200 micrometer, and 63% at 400 micrometer, whereas the rate of necrosis was 10% at 100 micrometer, 38% at 200 micrometer, and 56% at 400 micrometer. DNA fragmentation was also gradually increased and the highest at 400 micrometer, similar to apoptotic cell rates. As a result of real-time RT-PCR, there was significant difference of Bcl-2 and Bim mRNA expression among the groups. Expression of clusterin protein was significantly increased after treatment of tamsulosin, even as low as 50 micrometer concentration. CONCLUSION: These results demonstrate that tamsulosin causes the cell death of NRP-152 cells, displaying low concentration of tamsulosin induces apoptosis, but high concentration occurs necrosis. Bim, a proapoptotic factor of the Bcl-2 family, expression was increased in the cells treated with tamsulosin, whereas Bcl-2 expression was decreased. The present study suggests that clusterin may play a role in the process of apoptosis induced by tamsulosin and Bim could be involved in the apoptosis.

Expression of Inhibin in the Whole-body gamma-irradiated Mouse Ovary / 대한불임학회지

OBJECTIVE: The purposes of the present study were to investigate the effect of gamma-radiation on the expression of inhibin-alpha proteins and genes for inhibin alpha, betaA, and betain the ovary. METHODS: Immature mice were whole-body gamma-irradiated with 25% of a lethal dose. At time 0, 3, 6, 12, and 24 hours after the irradiation,the ovaries were collected and used for immunohistochemistry for inhibin-alpha, and RT_PCR for inhibin-alpha, betaA, and betaB. RESULTS: The expression of the immunoreactive inhibins-alpha was maintained at 12 hours post-irradiation and reduced thereafter. The expression of inhibin-alpha mRNA was significantly increased with the time after the irradiation. However there were no significant changes in the expression of betaA and betaB mRNAs. CONCLUSION: It might be thought that inhibin acts as one of the regulatory factors in the gamma-radiation-induced follicular atresia in mice

Follicular Lactate Dehydrogenase Activity and Steroid Concentrations in the Immature Gilt Ovary / 대한불임학회지

No abstract available.

Effects of Tributyltin Acetate on the Testicular Expression of Steroidogenic Enzyme Genes in Immature Mouse Testes / 대한남성과학회지

PURPOSE: The present study was performed to evaluate the effects of tributyltin acetate(TBTA) on mouse testes. The effects of TBTA on mammalian reproduction are not well known. MATERIALS AND METHODS: Three-week-old male mice(ICR strain) were orally administered TBTA at doses of 0 (control vehicle, CV), 25(T25), 50(T50), and 100 mg/kg(T100). Serum and intratesticular concentrations of testosterone and estradiol were determined by conventional radioimmunoassays. RT-PCR analysis was also performed. RESULTS: Transcriptional activity of 3-hydroxysteroid dehydrogenase (3beta-HSD), 17-hydroxysteroid dehydrogenase(17 beta-HSD) and cytochrome P450 17alpha-hydroxylase/C17,20 lyase(P450 (17 alpha)) were decreased by treatment. whereas mRNA levels of P450 aromatase were unaffected. In addition, TBTA significantly decreased serum testosterone levels in T100, while estradiol levels were not affected significantly. CONCLUSIONS: Administration of TBTA decreases testosterone level in testes, and this effect might be due to the alteration of mRNA levels of steroidogenic enzymes. Taken together, these findings suggest that TBTA, impairs testicular functions in a dose-dependent manner. The present results can be used as basic data in the study of TBTA action on gonads.

Differential Expressions of Apoptosis Regulators and Protein Profiling by SELDI-TOF Mass Spectrometry in Human Testis with Obstructive and Non-obstructive Azoospermia / 대한불임학회지

No abstract available.

Apoptosis and Peripheral Benzodiazepin Receptor (PBR) Expression in Human Granulosa-Luteal Cells by GnRH-agonist / 대한불임학회지

OBJECTIVE: To investigate whether GnRH-agonist (GnRH-Ag) using in IVF-ET affects apoptosis of human granulosa-luteal cells and expression of peripheral benzodiazepine receptor (PBR) protein involved in the apoptosis of the cells. METHODS: Granulosa-luteal cells obtained during oocyte retrieval were cultured and treated with 10(-5) M GnRH-Ag. Apoptosis of the cells by the treatment was confirmed using DNA fragmentation analysis 24 h after culture. The presence of PBR protein within the cells was examined by immunofluorescence staining and the expression of the protein was analyzed by Western blotting. In addition, it was measured for progesterone and nitric oxide (NO) produced by granulosa-luteal cells after GnRH-Ag treatment. To evaluate the relationship between NO production and PBR expression, sodium nitroprusside (SNP) as a NO donor was added in media and investigated the expression of PBR protein by Western blotting. RESULTS: Apoptosis increased in the granulosa-luteal cells 24 h after GnRH-Ag treatment, whereas the expression of PBR protein significantly decreased. Furthermore, the production of progesterone and nitric oxide (NO) by the cells significantly fell from 12 h after the treatment. In the results of Western blotting after SNP treatment, the expression of PBR protein increased in the treatment with SNP alone to the granulosa-luteal cells, but was suppressed in the treatment with GnRH-Ag and SNP. Additionally, the staining result of PBR protein in the cells showed the even distribution of it through the cell. CONCLUSION: These results demonstrate that GnRH-Ag treatment induces apoptosis, decreasing expression of PBR protein and NO production in human granulosa-luteal cells. The present study suggests that one of the apoptosis mechanism of human granulosa-luteal cells by GnRH-Ag might be a signal transduction pathway via NO and PBR.

Research of New Biomarker for Breast Cancer Using Proteomic Patterns / 한국유방암학회지

PURPOSE: Early detection and treatment of cancer is a primary focus of health care. Many serum markers are available for breast cancer, but are not good enough for screening. Cancer antigen CA 15-3 is the most widely used biomarker for breast cancer. However, CA 15-3 has low sensitivity and specificity. This study was performed to analyze the serum proteomic pattern in breast cancer patients by surface-enhanced laser desoption/ionization time- of-flight (SELDI-TOF). METHODS: We screened for potential tumor biomarkers in 42 serum samples, including samples from a group of 23 breast cancer patients at different clinical stages [stage I (n=3), stage II (n=11), stage III (n=6), and stage IV (n=1)], and a control group of 19 healthy women. Diluted serum samples were applied to a C16 hydrophobic interaction chip (H4). Complex protein profiles of different groups were compared and analyzed using the Protein Chip software 2.1 (Ciphergen Biosystems). RESULTS: There were 7 significant protein peaks in the breast cancer group and 5 in the control group. Scoring the expression of each peak, the mean score was 8.5 in the cancer group and 3.5 in the control. The results of the combination of each peak were highly sensitive (91.2%) and specific (94.7%). These proteomic patterns did not correlate with tumor stage and hormonal receptor, c-erb B2. CONCLUSION: In this preliminary report, we identified protein profiles that were differentiated in breast cancer patients. After proper validation, serum proteomic pattern analysis may ultimately be applied in screening breast cancer as a stand-alone or combined with current options.

Expression of Steroidogenic Acute regulatory (StAR) Protein during Oocyte Apoptosis in the Gonadotropin-Stimulated Human Ovary / 대한산부인과학회잡지

OBJECTIVE: To determine the distribution and expression of steroid acute regulatory (StAR) protein in human oocyte and embryo in relation to apoptosis. METHODS: Immuno-labelling and confocal microscopy were applied to examine the localization of StAR protein in human oocytes and embryos. Western blot analysis was also used for qualitative and quantitative assessment of StAR protein expression. RESULTS: There were lipid droplet accumulation in fragmented human oocytes and embryos. StAR protein (30 kDa) expression was detected in human oocytes and embryos. The level of StAR protein expression was lower in the fragmented group than the normal group. CONCLUSION: The present study provides evidence for involvement of StAR protein in the apoptosis of fragmented oocytes and embryos during in vitro fertilization (IVF) program as well as in the normal development of human oocytes and embryos.

The Study on Apoptosis and Expression of Fas, Fas-ligand, Bax, and Bcl-2 in Human Fragmented Embryos / 대한불임학회지

OBJECTIVE: The present study was performed to investigate whether apoptosis occur in human embryos by annexin staining and detect the expression of Fas, Fas-ligand (FasL), Bax, and Bcl-2 in human fragmented embryos derived from IVF-ET by immunofluorescence and Western blot analysis. MATERIALS AND METHODS: Using annexin staining, immunofluorescence and Western blot analysis on normal and fragmented embryos, we were able to detect apoptotsis and apoptotic gene products in fragmented embryos. RESULTS: Phosphatidylserine (PS) translocation, the marker for apoptosis, were detected frequently in fragmented embryos. Bcl-2 and Bax protein were detected in both fragmented and non-fragmented embryos. When fragmented embryos compared to normal embryos, immunofluorescent intensity of Bcl-2 tended to be lower in fragmented embryos. Bax gene expression increased in the fragmented embryos compared to the normal embryos. This result supports a model in which the molar ratio of Bcl-2 to Bax determines whether apoptosis induced or inhibited in human embryo. Fas was highly expressed in human preimplantation embryos but not FasL. It suggests that embryo may undergo apoptosis by binding with FasL produced by follicular or immune cells. CONCLUSION: The over expression of Bax and Fas will trigger apoptosis to lead embryo fragmentation and change embryo to be nonviable.

Endocrine Disrupters and Reproduction / 대한내분비학회지

No abstract available.

Effects of GnRH Agonist Used for Ovarian Hyperstimulation in Human IVF-ET on the Apoptosis of Preovulatory Follicular Cells / 대한불임학회지

There have been many reports to date regarding the role of GnRH as a local regulatory factor of ovarian function as studies of human and rat ovaries revealed GnRH and its receptor. In recent studies it has been shown that GnRH directly causes apoptosis in the granulosa cells of the rat ovary, and such results leads to the suggestion that the use of GnRH agonist for more stable long term ovarian hyperstimulation in human IVF-ET programs causes granulosa cell apoptosis which may lead to follicular atresia. Therefore this study attempts to determine if granulosa-luteal cell apoptosis occurs in patients during IVF-ET programs in which GnRH agonist is employed for ovarian hyperstimulation. The quality of oocyte-cumulus complexes obtained during ovum pickup procedures were assessed morphologically and then the fertilization rate and developmental rate was determined. Apoptotic cells among the granulosa-luteal cells obtained during the same procedure were observed after staining with Hematoxylin-rosin. The fragmentation degree of DNA extracted from granulosa-luteal cells was determined and comparatively analyzed. There was no difference in the average age of the patients, the number of oocytes retrieved, and fertilization and developmental rates between the FSH/hMG group and GnRH-long group. There was also no difference in the apoptosis rate and pyknosis rate in the granulosa-luteal cells between the two groups. However, when the oocyte-cumulus complexes were morphoogically divided into the healthy group and atretic group without regard for the method of hyperstimulation, the results showed that the number of oocytes obtained averaged 11.09+/-8.75 and 10.33+/-4.53 per cycle, respectively, showing no significant difference, but the fertilization rate (77.05%, 56.99%, respectively, p<0.01) and developmental ,ate (65.96%, 41.51%, respectively, p<0.01) was significantly increased in the healthy group when compared to the atretic group. The degree of apoptosis in the granulosa-luteal cells showed that in the healthy group it was 2.25% which was not significantly different from the atretic group (2.77%), but the pyknosis rate in the atretic group (27.81%) was significantly higher compared to the healthy group (11.35%, p<0.01). The quantity of DNA fragmentation in the FSH/hMG group was 32.22%, while in the GnRH-long group it was 34.27%, showing no significant difference. On the other hand the degree of DNA fragmentation was 39.05% and 11.83% in the healthy group and atretic group, respectively, showing significantly higher increase in the atretic group (p<0.01). The above results suggest that death of granulosa-luteal cells according to the state of the oocyte-cumulus complex is more related to pyknosis rather than apoptosis. Also, the GnRH agonist used in ovarian hyperstimulation does not seem to directly affect the apoptosis of retrieved oocytes and granulosa-luteal cells, and which is thought to be due to the suppression of the apoptogenic effect of GnRH agonist as a result of the high doses of FSH administered.

Effects of Ketamine and Pentobarbitone on Degeneration of Oocyte and of Granulosa Cells in Mouse Ovary / 대한불임학회지

In mammal, lots of follicles start simultaneously their growth but only a few oocytes are ovulated in every sexual cycles. Most of matured and grown oocytes are destined to degenerate by atresia. However, the molecular and physiological mechanisms are not elucidated yet. The present study was designed to establish an induction method of follicular atresia with ketamine or pentobarbitone and evaluate the effect of these anesthetics on oocyte maturation and granulosa cell apoptosis of the mouse ovarian follicle. The percentages of degenerated oocyte and apoptotic granulosa cell in ketamine treated groups were significantly higher than that in controls (58.9% vs 33.5%, p<0.01, degeneration; 44.9% vs 26.6%, p<0.01, apotosis). Futhermore, it was revealed that the concentrations of progesterone in both groups were markedly higher than that in control. In conclusion, it is considered that ketamine induce an atresia as pentobarbitone, and may be useful for inducing follicular atresia.