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Nitric oxide-mediated inhibition of follicular apoptosis is associated with heat shock protein 70 induction and bax suppression.
produced in media #1 and #2 was similar as judged by cell count of differentially stained blastocysts (78 ⫾ 12 and 66 ⫾ 10 of inner cell mass cells and 241 ⫾ 39 and 217 ⫾ 32 of total cells for media #1 and #2, respectively, P ⬎ 0.05). Conclusions: In vitro development of monkey embryos to the blastocyst stage cultured in CMRL/BRL or G1.2/G2.2/BSA was comparable suggesting the possibility of using sequential media for production of monkey blastocysts without the technical complications of co-culture. Supported by: NIH grant RR12804 and Ares Advanced Technology, Inc. G1.2/G2.2 were kindly supplied by Dr. D. Gardner.
P-464 Sperm-binding to the human zona pellucida (hZP) and calcium influx in response to gonadotropin-releasing hormone (GnRH) and progesterone (P). P. J. Morales, E. Pizarro, M. Kong. Faculty of Health Science, Univ of Antofagasta, Antofagasta, Chile.
of healthy antral follicles. Extensive levels of DNA strand breaks were detected in situ in granulosa cells of atretic follicles by TUNEL analysis. However, in situ apoptotic cell death was not detected in the healthy preovulatory follicles. Granulosa cells within follicles incubated in medium alone for 24 hr exhibited extensive apoptosis by DNA ladder analysis. Treatment of SNP in the culture medium blocked this onset of apoptosis. Both mRNA and protein levels of HSP70 were highly increased with SNP than those of control group. On the contrary, those of Bax were suppressed with SNP treatment. Conclusions: Results of the present study strongly suggest that NO prevents rat preovulatory follicular apoptosis in vitro by stimulating HSP70 and suppressing Bax expression. Supported by: This work was supported by a grant (HMP-98-M-5-0054) of the Good Health R & D Project, Ministry of Health & Welfare, Republic of Korea.
P-466 Objective: Recently we reported that GnRH increases sperm binding to the hZP. This effect was mediated by a transient increase in the intracellular calcium concentration (Biol Reprod 63:635, 2000), similar to that produced by P. In this work we have evaluated the effect of the sequential exposure of the sperm to P and GnRH upon sperm hZP binding and intracellular calcium concentration. Design: Sperm aliquots were treated as follows: a) 0.7 M P or DMSO (solvent for P) for 5 min followed by 50 nM GnRH for 5 min; b) 50 nM GnRH or deionized water (dH2O, solvent for GnRH) for 5 min followed by 0.7 M P for 5 min; c) 50 nM GnRH or 0.7 M P for 5 min. Materials/Methods: Motile sperm, obtained through a percoll gradient, were incubated in modified Tyrode’s medium (2.6% BSA), at 37°C, 5% CO2, 10 ⫻ 106 sperm/ml, for 4.5 hr. Then, the intracellular calcium concentration was evaluated in sperm previously loaded with fura-2AM using a spectrofluorometer and the sperm-zona binding was evaluated using the hemizona assay. Results: Both GnRH and P increased sperm-zona binding and caused a transient increase in the intracellular calcium concentration. Regarding sperm-zona binding, the effect of GnRH was significantly greater when the sperm were previously treated with P (GnRH after P ⫽ 185 ⫾ 33 versus GnRH after DMSO ⫽ 104 ⫾ 19, P ⬍ 0.0001) (X ⫾ EE, n ⫽ 8). On the other hand, the effect of P was not significantly affected by the previous treatment of the sperm with GnRH (P after GnRH ⫽ 117 ⫾ 27 versus P after dH2O ⫽ 122 ⫾ 26, NS). The results regarding intracellular calcium concentration showed a similar pattern. Conclusions: This results suggest a synergistic effect between P and GnRH upon sperm-zona binding and intracellular calcium concentration. Moreover, the sequence of presentation of the stimulus seems to be important in this response. Supported by: WHO A05139.
P-465 Nitric oxide-mediated inhibition of follicular apoptosis is associated with heat shock protein 70 induction and bax suppression. S. Yoon, J. Ko, Y. Cho, T. Yoon, K. Cha, K. Lee. Infertility Medical Ctr, CHA Gen Hosp, Seoul, Korea. Objective: Nitric oxide (NO) has recently emerged as a potential regulator of follicular development because of its involvement in the regulation of several physiological functions of the ovary. Nitric oxide acts as a follicle survival factor to inhibit apoptotic cell death of the follicular cells. The present study was conducted to investigate the mechanism involved in the protective effect of NO on spontaneously induced follicular apoptosis in serum-free condition. Design: Follicle isolation, In vitro follicle culture, RT-PCR, Western blotting. Materials/Methods: Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 hr in serum-free medium with or without sodium nitroprusside (SNP), a NO generator. After culture, ovarian follicular apoptosis was assessed by DNA ladder analysis and concurrent expression of heat shock protein 70 (HSP70) and Bax mRNA and protein was measured by RT-PCR and Western analysis, respectively. Results: In vivo treatment with PMSG promoted the growth of a cohort
FERTILITY & STERILITY威
The expression of prostaglandin H synthase-2 and the profile of arachidonic acid metabolism by human fallopian tubes. K. J. Tumbusch, J. Goldsby, F. Arbab. N. Matijevic-Aleksic, J. Huang, Univ of Texas Medical Sch at Houston, Houston, TX; Dept of Ob/Gyn Univ of Texas Medical Sch at Houston, Houston, TX; Dept of Pathology Baylor Coll of Medicine, Houston, TX; Dept of Internal Medicine Univ of Texas Medical Sch at Houston, Houston, TX. Objective: To determine the expression of prostaglandin H synthase-2 (PGHS-2) and the profile of arachidonic acid (AA) metabolism by human fallopian tubes Design: Prospective study. Materials/Methods: Segments of fallopian tubes were obtained from patients undergoing postpartum tubal ligation (within 18 hours of delivery) or hysterectomy for non-tubal diseases. Samples were placed on ice and brought to the laboratory immediately. Depending on the nature of the experiment, they were used immediately, or stored at ⫺70°C or in formalin. For whole tissue experiments, the samples were minced into 1 ⫻ 1 mm pieces. For other experiments, microsomes were prepared. Our institutional review board approved the use of human tissue in this project. The microsome protein thus prepared was used for Western blot analysis using a PGHS-2 monoclonal antibody that does not cross-react to prostaglandin H synthase-1 (PGHS-1). PGHS-2 was immunohistochemically localized in paraffin sections using a rabbit polyclonal antibody, also not cross-reactive to PGHS-1. The metabolism of [1-C14]AA by microsome protein, whole tubes, isolated tubal smooth muscle, and isolated tubal lumen was investigated. The metabolites were separated and identified using high-pressure liquid chromatography (HPLC) equipped with a radio-detector. To confirm that PGHS-2 was functionally active, we used NS-398 (5 M) to block its activity. To determine the rate of prostaglandin I2 (PGI2) synthesis, samples of microsome protein were incubated with AA (20 M) at 37°C for 4 minutes and the reaction stopped by adding methanol. The stable metabolite of PGI2, 6-keto prostaglandin F2␣, was determined using enzyme immunoassay. The rate of PGI2 synthesis was expressed as pg PGI2/g microsome protein/minute. Results: Western blot analysis confirmed the presence of a 72 kDa protein, reactive to PGHS-2 antibody, in microsome fractions prepared from fallopian tubes (n ⫽ 10). Immunoreactive PGHS-2 was localized in luminal epithelia, smooth muscle cells and vascular endothelial cells (postpartum, follicular and luteal phases, n ⫽ 6 each). HPLC analysis showed PGI2 and prostaglandin E2 (PGE2) were the major metabolites, accounting for 55.8 ⫾ 11.4% and 34.5 ⫾ 11.1% (mean ⫾ SD, n ⫽ 4), respectively, of total prostaglandins (PGs). On the other hand, prostaglandin D2, prostaglandin F2␣, and thromboxane A2 accounted for less than 5% of total PGs. Similar patterns of metabolites were observed in samples obtained from follicular and luteal phases (n ⫽ 2 and 3, respectively) of menstrual cycle or immediately postpartum (n ⫽ 4). Isolated smooth muscle (n ⫽ 3) and tubal lumen (n ⫽ 3) metabolized [1-C14] AA the same way as did the whole tube. PGHS-2 inhibitor, NS-398 (5 M), reduced the PGs converted from [1-C14] AA by 84% (n ⫽ 2). The rate of PGI2 synthesis was 77.6 ⫾ 12.3 pg/g microsome protein/minute (mean ⫾ SD, n ⫽ 10). Conclusions: Luminal epithelia and smooth muscle cells of human fallopian tube express functionally active PGHS-2, which is critical for the synthesis of PGs. The major PGs formed from AA are PGI2 and PGE2.
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P-464 Sperm-binding to the human zona pellucida (hZP) and calcium influx in response to gonadotropin-releasing hormone (GnRH) and progesterone (P). P. J. Morales, E. Pizarro, M. Kong. Faculty of Health Science, Univ of Antofagasta, Antofagasta, Chile.
of healthy antral follicles. Extensive levels of DNA strand breaks were detected in situ in granulosa cells of atretic follicles by TUNEL analysis. However, in situ apoptotic cell death was not detected in the healthy preovulatory follicles. Granulosa cells within follicles incubated in medium alone for 24 hr exhibited extensive apoptosis by DNA ladder analysis. Treatment of SNP in the culture medium blocked this onset of apoptosis. Both mRNA and protein levels of HSP70 were highly increased with SNP than those of control group. On the contrary, those of Bax were suppressed with SNP treatment. Conclusions: Results of the present study strongly suggest that NO prevents rat preovulatory follicular apoptosis in vitro by stimulating HSP70 and suppressing Bax expression. Supported by: This work was supported by a grant (HMP-98-M-5-0054) of the Good Health R & D Project, Ministry of Health & Welfare, Republic of Korea.
P-466 Objective: Recently we reported that GnRH increases sperm binding to the hZP. This effect was mediated by a transient increase in the intracellular calcium concentration (Biol Reprod 63:635, 2000), similar to that produced by P. In this work we have evaluated the effect of the sequential exposure of the sperm to P and GnRH upon sperm hZP binding and intracellular calcium concentration. Design: Sperm aliquots were treated as follows: a) 0.7 M P or DMSO (solvent for P) for 5 min followed by 50 nM GnRH for 5 min; b) 50 nM GnRH or deionized water (dH2O, solvent for GnRH) for 5 min followed by 0.7 M P for 5 min; c) 50 nM GnRH or 0.7 M P for 5 min. Materials/Methods: Motile sperm, obtained through a percoll gradient, were incubated in modified Tyrode’s medium (2.6% BSA), at 37°C, 5% CO2, 10 ⫻ 106 sperm/ml, for 4.5 hr. Then, the intracellular calcium concentration was evaluated in sperm previously loaded with fura-2AM using a spectrofluorometer and the sperm-zona binding was evaluated using the hemizona assay. Results: Both GnRH and P increased sperm-zona binding and caused a transient increase in the intracellular calcium concentration. Regarding sperm-zona binding, the effect of GnRH was significantly greater when the sperm were previously treated with P (GnRH after P ⫽ 185 ⫾ 33 versus GnRH after DMSO ⫽ 104 ⫾ 19, P ⬍ 0.0001) (X ⫾ EE, n ⫽ 8). On the other hand, the effect of P was not significantly affected by the previous treatment of the sperm with GnRH (P after GnRH ⫽ 117 ⫾ 27 versus P after dH2O ⫽ 122 ⫾ 26, NS). The results regarding intracellular calcium concentration showed a similar pattern. Conclusions: This results suggest a synergistic effect between P and GnRH upon sperm-zona binding and intracellular calcium concentration. Moreover, the sequence of presentation of the stimulus seems to be important in this response. Supported by: WHO A05139.
P-465 Nitric oxide-mediated inhibition of follicular apoptosis is associated with heat shock protein 70 induction and bax suppression. S. Yoon, J. Ko, Y. Cho, T. Yoon, K. Cha, K. Lee. Infertility Medical Ctr, CHA Gen Hosp, Seoul, Korea. Objective: Nitric oxide (NO) has recently emerged as a potential regulator of follicular development because of its involvement in the regulation of several physiological functions of the ovary. Nitric oxide acts as a follicle survival factor to inhibit apoptotic cell death of the follicular cells. The present study was conducted to investigate the mechanism involved in the protective effect of NO on spontaneously induced follicular apoptosis in serum-free condition. Design: Follicle isolation, In vitro follicle culture, RT-PCR, Western blotting. Materials/Methods: Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 hr in serum-free medium with or without sodium nitroprusside (SNP), a NO generator. After culture, ovarian follicular apoptosis was assessed by DNA ladder analysis and concurrent expression of heat shock protein 70 (HSP70) and Bax mRNA and protein was measured by RT-PCR and Western analysis, respectively. Results: In vivo treatment with PMSG promoted the growth of a cohort
FERTILITY & STERILITY威
The expression of prostaglandin H synthase-2 and the profile of arachidonic acid metabolism by human fallopian tubes. K. J. Tumbusch, J. Goldsby, F. Arbab. N. Matijevic-Aleksic, J. Huang, Univ of Texas Medical Sch at Houston, Houston, TX; Dept of Ob/Gyn Univ of Texas Medical Sch at Houston, Houston, TX; Dept of Pathology Baylor Coll of Medicine, Houston, TX; Dept of Internal Medicine Univ of Texas Medical Sch at Houston, Houston, TX. Objective: To determine the expression of prostaglandin H synthase-2 (PGHS-2) and the profile of arachidonic acid (AA) metabolism by human fallopian tubes Design: Prospective study. Materials/Methods: Segments of fallopian tubes were obtained from patients undergoing postpartum tubal ligation (within 18 hours of delivery) or hysterectomy for non-tubal diseases. Samples were placed on ice and brought to the laboratory immediately. Depending on the nature of the experiment, they were used immediately, or stored at ⫺70°C or in formalin. For whole tissue experiments, the samples were minced into 1 ⫻ 1 mm pieces. For other experiments, microsomes were prepared. Our institutional review board approved the use of human tissue in this project. The microsome protein thus prepared was used for Western blot analysis using a PGHS-2 monoclonal antibody that does not cross-react to prostaglandin H synthase-1 (PGHS-1). PGHS-2 was immunohistochemically localized in paraffin sections using a rabbit polyclonal antibody, also not cross-reactive to PGHS-1. The metabolism of [1-C14]AA by microsome protein, whole tubes, isolated tubal smooth muscle, and isolated tubal lumen was investigated. The metabolites were separated and identified using high-pressure liquid chromatography (HPLC) equipped with a radio-detector. To confirm that PGHS-2 was functionally active, we used NS-398 (5 M) to block its activity. To determine the rate of prostaglandin I2 (PGI2) synthesis, samples of microsome protein were incubated with AA (20 M) at 37°C for 4 minutes and the reaction stopped by adding methanol. The stable metabolite of PGI2, 6-keto prostaglandin F2␣, was determined using enzyme immunoassay. The rate of PGI2 synthesis was expressed as pg PGI2/g microsome protein/minute. Results: Western blot analysis confirmed the presence of a 72 kDa protein, reactive to PGHS-2 antibody, in microsome fractions prepared from fallopian tubes (n ⫽ 10). Immunoreactive PGHS-2 was localized in luminal epithelia, smooth muscle cells and vascular endothelial cells (postpartum, follicular and luteal phases, n ⫽ 6 each). HPLC analysis showed PGI2 and prostaglandin E2 (PGE2) were the major metabolites, accounting for 55.8 ⫾ 11.4% and 34.5 ⫾ 11.1% (mean ⫾ SD, n ⫽ 4), respectively, of total prostaglandins (PGs). On the other hand, prostaglandin D2, prostaglandin F2␣, and thromboxane A2 accounted for less than 5% of total PGs. Similar patterns of metabolites were observed in samples obtained from follicular and luteal phases (n ⫽ 2 and 3, respectively) of menstrual cycle or immediately postpartum (n ⫽ 4). Isolated smooth muscle (n ⫽ 3) and tubal lumen (n ⫽ 3) metabolized [1-C14] AA the same way as did the whole tube. PGHS-2 inhibitor, NS-398 (5 M), reduced the PGs converted from [1-C14] AA by 84% (n ⫽ 2). The rate of PGI2 synthesis was 77.6 ⫾ 12.3 pg/g microsome protein/minute (mean ⫾ SD, n ⫽ 10). Conclusions: Luminal epithelia and smooth muscle cells of human fallopian tube express functionally active PGHS-2, which is critical for the synthesis of PGs. The major PGs formed from AA are PGI2 and PGE2.
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