- Email: [email protected]
Developmental potential of ICSI-produced rhesus monkey embryos on co-culture or in sequential media.
P-461 Mitochondrial function and the effect of oocyte aging on parthenogenetic activation of mouse oocytes. K. T. McGinnis, M. P. Diamond, D. I. Dozortsev. Wayne State Univ, Detroit, MI. Objective: To examine the relationship of mitochondrial mass and function to parthenogenetic activation and subsequent development of mouse oocytes as a function of oocyte age. Design: In-vitro study. Materials/Methods: Two groups of B6D2 F1 mice were superovulated 1 day apart. After receiving human chorionic gonadotropin (hCG), half of the second group was allowed to mate. All mice were sacrificed on the same day resulting in normal zygotes, fresh eggs (15 hours post hCG), and aged eggs (34 hours post hCG). After removing the cumulus, zygotes were incubated overnight. Eggs were exposed to 7% ethanol and then incubated overnight. The next day, the following groups were identified: (A) normally fertilized 2-cell embryos; (B) activated 2-cell embryos from fresh eggs; (C) fresh eggs not activated; (D) aged eggs activated but not cleaved; (E) aged eggs activated and cleaved to the 2-3 cell stage; (F) aged oocytes not activated. All were stained with Mitotracker Red™ (mitochondrial function) and MitoFluor Green™ (mitochondrial mass) following a standard protocol. After staining, embryos were imaged using a computerized fluorescent microscope. Cell area and fluorescence representing mitochondrial mass and function were measured. Measurements were compared using Student’s t-test. Results: Comparing groups F and D demonstrates that aged eggs expand on activation (p ⬍ .00002). Total mitochondrial mass was higher in activated cells (p ⬍ .02) while total mitochondrial function (p ⬍ .03) and function/mass ratio (p ⬍ .001) was less. Comparing groups A and B shows that normally fertilized embryos and oocytes which activated and cleaved are similar in area (p ⫽ .33), mitochondrial mass (p ⫽ .15), and function/ mass ratio (p ⫽ .12), but differ in total mitochondrial function (p ⬍ .03). Comparing groups C and F shows that eggs that do not activate contract with age (p ⬍ .000003), have similar mitochondrial mass (p ⫽ .06) and function (p ⫽ .32), but a higher function/mass ratio (p ⬍ .004). When groups E and D are compared, aged oocytes which cleave have higher mitochondrial function (p ⬍ .004) and function/mass ratios (p ⬍ .03) in spite of similar areas (p ⫽ .17) and mitochondrial mass (p ⫽ .52). Conclusions: As oocytes age, their sensitivity to parthenogenetic activation increases. The underlying mechanisms for this have not been identified. Prior to activation, a large calcium gradient is maintained in the endoplasmic reticulum of the cell. This and other energy dependent activities are required to prevent spontaneous activation of the egg. Our data suggest that oocytes with decreased energy competence are more susceptible to activation, and variation in mitochondrial function may be responsible for a variation in response to treatment with ethanol. Our data also show that once activated, parthenogenetic embryos that cleave tend to demonstrate greater energy competence. Supported by: Dept of OB/GYN.
P-462 Stability of the meiotic spindle following rapid cryopreservation and thawing of Syrian hamster eggs. R. W. McGaughey, J. S. Nemiro, R. U. Trivedi. Arizona State Univ, Tempe, AZ; Arizona Ctr for Fertility Study, Scottsdale, AZ. Objective: Cryopreservation of unfertilized eggs can result in damage to the meiotic spindle. This study used a simple method of rapid freezing and analysis of meiotic spindles of thawed eggs to assess such damage. Design: Eggs were treated with cryoprotectants, frozen and thawed for analysis by laser confocal microscopy (LCM), to image meiotic spindles and chromosomes. Analyses were compared for untreated control eggs, eggs subjected to cryoprotectants (CPA) and eggs after cryopreservation. Materials/Methods: Eggs collected from 6 – 8 week old hamsters in human tubal fluid (HTF containing hepes, Irvine Scientific) with BSA (4.5 mg/ml) were denuded in hyaluronidase (300 IU/ml) and divided into three groups. Groups 1 and 2 were transferred from collection medium into pre-cooled medium and held in an ice-bath for 5 min. Cooled eggs were transferred and held for 10 min each in the second (6.0 M 1,2-propanediol) and final (6.0 M 1,2-propanediol/0.1 M sucrose) CPA. Group 1 eggs were sealed in cryovials with 1.0 ml of the final CPA at 4°C, held in liquid
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nitrogen (LN2) vapor for 15 sec and plunged. Groups 2 and 3 were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS). After holding in LN2 for 50 –100 min, group 1 eggs were placed in a 37°C water bath until thawed. Thawed eggs were transferred to thaw media and held at room temperature for 5 min each, consisting of 1.5 M, 1.0 M, 0.75 M, 0.5 M, and 0.25 M 1,2-propanediol/0.2 M sucrose. From the final thaw medium, 0.2 M sucrose in culture medium, eggs were fixed in 2% paraformaldehyde in PBS. Fixed eggs were detergent extracted, stained with monoclonal anti-␣tubulin in PBS/BSA, counterstained with anti-mouse IgG-TRITC conjugate and washed in PBS/BSA; then stained with Hoechst trihydrochloride in PBS/BSA and post-fixed in 2.5% gluteraldehyde in PBS. Eggs on waxspotted slides were analyzed using a Leica LCM, allowing simultaneous imaging of spindles and chromosomes. Results: A normal spindle, 18 m by 24 m, had two spindle poles joined by uniform microtubules in a barrel shaped configuration with a metaphase plate of low TRITC signal. Of 24 group 1 eggs, 8 (33.3%) exhibited subtle to dramatic structural spindle damage, while 5 (17.1%) of 35 group 2 eggs and 2 (5.8%) of 34 group 3 eggs exhibited such damage. While six (25.0%) group 1 eggs exhibited chromosomal dislocation, 9 (25.7%) group 2 eggs and 5 (14.7%) group 3 eggs showed this damage. For group 1, 5 (20.8%) eggs exhibited both structural spindle damage and chromosomal dislocation, while only 3 (8.6%) group 2 eggs and 1 (2.9%) group 3 egg exhibited both types of damage. Although 4 (16.7%) group 1 eggs exhibited both structural spindle damage and a small spindle, no (0%) group 2 eggs and only 1 (2.9%) group 3 egg fit these criteria. For group 1 eggs, 1 (4.2%) was parthenogenetically activated while 2 (5.7%) group 2 and 6 (17.6%) group 3 eggs were activated. Conclusions: Cryopreservation by rapid freezing, although damaging to the spindles of some hamster eggs, holds promise for improvement over currently employed methods and may eventually allow successful freezing of unfertilized human eggs. LCM proved highly effective in diagnosing subtle as well as marked spindle damage and chromsomal dislocation due to treatment with cryoprotectants and cryopreservation. Supported by: The Arizona State University Foundation.
P-463 Developmental potential of ICSI-produced rhesus monkey embryos on co-culture or in sequential media. S. M. Mitalipov, R. R. Yeoman, H. C. Kuo, D. P. Wolf. Oregon Regional Primate Research Ctr, Oregon Health Science Univ, Beaverton, OR. Objective: Compare the in vitro developmental potential of rhesus macaque embryos cultured in a complex medium on co-culture or in sequential media G1.2/G2.2. Design: MII oocytes were fertilized by ICSI and allocated to different culture conditions. Embryonic development was monitored daily over an 8 day period. Materials/Methods: ICSI was performed on Day 0 on mature oocytes retrieved by laparoscopic follicular aspiration from females stimulated with recombinant human hormones. Injected oocytes were randomly assigned to three culture systems: (1) CMRL (Connaught Medical Research Laboratories) medium supplemented with 10% fetal bovine serum (FBS), 10 mM L-glutamine, 5 mM sodium pyruvate, 1 mM sodium lactate, 100 units/ml of penicillin and 100 g/ml of streptomycin at 37°C in 5% CO2 and buffalo rat liver (BRL) cell co-culture; (2) G1.2 medium supplemented with 8 mg/ml bovine serum albumin (BSA) at 37°C in 6% CO2, 5% O2, 89% N2 and transferred on Day 3 into G2.2/BSA medium; (3) G1.2 medium supplemented with 10% FBS at 37°C in 6% CO2, 5% O2, 89% N2 and transferred on Day 3 into G2.2/FBS medium. Embryos were transferred into fresh media every other day. Results, expressed as means and standard error of the mean, were analyzed using one-way ANOVA and Fisher’s Protected Least Significant Difference test with Statview software. Results: Following ICSI, 90, 72 and 71 oocytes (10 replications) were placed into culture media #1, #2 and #3, respectively. Similar pronuclear formation rates were observed (69 ⫾ 6%, 80 ⫾ 8% and 81 ⫾ 6% in culture media #1, #2 and #3, respectively, P ⬎ 0.05). However, development of embryos to the morula stage was significantly impaired in medium #3 (76 ⫾ 4%, 67 ⫾ 12% and 31 ⫾ 8% in media #1, #2 and #3, respectively, P ⬍ 0.05). Overall development to compact morula and blastocyst stages did not differ significantly among treatments (51 ⫾ 11%, 54 ⫾ 16%, 22 ⫾ 6% of compact morulae and 41 ⫾ 7%, 29 ⫾ 16%, 11 ⫾ 7% of blastocysts in culture media #1, #2 and #3, respectively, P ⬎ 0.05). Quality of embyos
Vol. 76, No. 3, Suppl. 1, September 2001
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-462 Stability of the meiotic spindle following rapid cryopreservation and thawing of Syrian hamster eggs. R. W. McGaughey, J. S. Nemiro, R. U. Trivedi. Arizona State Univ, Tempe, AZ; Arizona Ctr for Fertility Study, Scottsdale, AZ. Objective: Cryopreservation of unfertilized eggs can result in damage to the meiotic spindle. This study used a simple method of rapid freezing and analysis of meiotic spindles of thawed eggs to assess such damage. Design: Eggs were treated with cryoprotectants, frozen and thawed for analysis by laser confocal microscopy (LCM), to image meiotic spindles and chromosomes. Analyses were compared for untreated control eggs, eggs subjected to cryoprotectants (CPA) and eggs after cryopreservation. Materials/Methods: Eggs collected from 6 – 8 week old hamsters in human tubal fluid (HTF containing hepes, Irvine Scientific) with BSA (4.5 mg/ml) were denuded in hyaluronidase (300 IU/ml) and divided into three groups. Groups 1 and 2 were transferred from collection medium into pre-cooled medium and held in an ice-bath for 5 min. Cooled eggs were transferred and held for 10 min each in the second (6.0 M 1,2-propanediol) and final (6.0 M 1,2-propanediol/0.1 M sucrose) CPA. Group 1 eggs were sealed in cryovials with 1.0 ml of the final CPA at 4°C, held in liquid
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nitrogen (LN2) vapor for 15 sec and plunged. Groups 2 and 3 were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS). After holding in LN2 for 50 –100 min, group 1 eggs were placed in a 37°C water bath until thawed. Thawed eggs were transferred to thaw media and held at room temperature for 5 min each, consisting of 1.5 M, 1.0 M, 0.75 M, 0.5 M, and 0.25 M 1,2-propanediol/0.2 M sucrose. From the final thaw medium, 0.2 M sucrose in culture medium, eggs were fixed in 2% paraformaldehyde in PBS. Fixed eggs were detergent extracted, stained with monoclonal anti-␣tubulin in PBS/BSA, counterstained with anti-mouse IgG-TRITC conjugate and washed in PBS/BSA; then stained with Hoechst trihydrochloride in PBS/BSA and post-fixed in 2.5% gluteraldehyde in PBS. Eggs on waxspotted slides were analyzed using a Leica LCM, allowing simultaneous imaging of spindles and chromosomes. Results: A normal spindle, 18 m by 24 m, had two spindle poles joined by uniform microtubules in a barrel shaped configuration with a metaphase plate of low TRITC signal. Of 24 group 1 eggs, 8 (33.3%) exhibited subtle to dramatic structural spindle damage, while 5 (17.1%) of 35 group 2 eggs and 2 (5.8%) of 34 group 3 eggs exhibited such damage. While six (25.0%) group 1 eggs exhibited chromosomal dislocation, 9 (25.7%) group 2 eggs and 5 (14.7%) group 3 eggs showed this damage. For group 1, 5 (20.8%) eggs exhibited both structural spindle damage and chromosomal dislocation, while only 3 (8.6%) group 2 eggs and 1 (2.9%) group 3 egg exhibited both types of damage. Although 4 (16.7%) group 1 eggs exhibited both structural spindle damage and a small spindle, no (0%) group 2 eggs and only 1 (2.9%) group 3 egg fit these criteria. For group 1 eggs, 1 (4.2%) was parthenogenetically activated while 2 (5.7%) group 2 and 6 (17.6%) group 3 eggs were activated. Conclusions: Cryopreservation by rapid freezing, although damaging to the spindles of some hamster eggs, holds promise for improvement over currently employed methods and may eventually allow successful freezing of unfertilized human eggs. LCM proved highly effective in diagnosing subtle as well as marked spindle damage and chromsomal dislocation due to treatment with cryoprotectants and cryopreservation. Supported by: The Arizona State University Foundation.
P-463 Developmental potential of ICSI-produced rhesus monkey embryos on co-culture or in sequential media. S. M. Mitalipov, R. R. Yeoman, H. C. Kuo, D. P. Wolf. Oregon Regional Primate Research Ctr, Oregon Health Science Univ, Beaverton, OR. Objective: Compare the in vitro developmental potential of rhesus macaque embryos cultured in a complex medium on co-culture or in sequential media G1.2/G2.2. Design: MII oocytes were fertilized by ICSI and allocated to different culture conditions. Embryonic development was monitored daily over an 8 day period. Materials/Methods: ICSI was performed on Day 0 on mature oocytes retrieved by laparoscopic follicular aspiration from females stimulated with recombinant human hormones. Injected oocytes were randomly assigned to three culture systems: (1) CMRL (Connaught Medical Research Laboratories) medium supplemented with 10% fetal bovine serum (FBS), 10 mM L-glutamine, 5 mM sodium pyruvate, 1 mM sodium lactate, 100 units/ml of penicillin and 100 g/ml of streptomycin at 37°C in 5% CO2 and buffalo rat liver (BRL) cell co-culture; (2) G1.2 medium supplemented with 8 mg/ml bovine serum albumin (BSA) at 37°C in 6% CO2, 5% O2, 89% N2 and transferred on Day 3 into G2.2/BSA medium; (3) G1.2 medium supplemented with 10% FBS at 37°C in 6% CO2, 5% O2, 89% N2 and transferred on Day 3 into G2.2/FBS medium. Embryos were transferred into fresh media every other day. Results, expressed as means and standard error of the mean, were analyzed using one-way ANOVA and Fisher’s Protected Least Significant Difference test with Statview software. Results: Following ICSI, 90, 72 and 71 oocytes (10 replications) were placed into culture media #1, #2 and #3, respectively. Similar pronuclear formation rates were observed (69 ⫾ 6%, 80 ⫾ 8% and 81 ⫾ 6% in culture media #1, #2 and #3, respectively, P ⬎ 0.05). However, development of embryos to the morula stage was significantly impaired in medium #3 (76 ⫾ 4%, 67 ⫾ 12% and 31 ⫾ 8% in media #1, #2 and #3, respectively, P ⬍ 0.05). Overall development to compact morula and blastocyst stages did not differ significantly among treatments (51 ⫾ 11%, 54 ⫾ 16%, 22 ⫾ 6% of compact morulae and 41 ⫾ 7%, 29 ⫾ 16%, 11 ⫾ 7% of blastocysts in culture media #1, #2 and #3, respectively, P ⬎ 0.05). Quality of embyos
Vol. 76, No. 3, Suppl. 1, September 2001
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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