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Endothelin-I interaction with the ET-A receptor in the regression of the rat corpus luteum
end of experiment, total blastocyst cell numbers and blastocyst cells viability were evaluated using the staining with 1g/ml of Hoechst 33342 and 10g/ml of propidium iodide under fluorescent microscope. Additionally TUNEL reaction was performed to detect apoptosis within the blastocyst cells. Statistics was based on ANOVA and log-linear analysis with significance accepted at p⬍0,05. Results: Standard IVF medium enriched with SCF did not improve embryo growth and blastocyst quality (A vs. B). Freeze-thaw procedure (A vs. C), oxidative stress (A vs. E) and exposure to Fas-L (A vs. G) deteriorated growth of embryos. SCF improved the dynamism of growth of embryos exposed to cryopreservation (C vs. D) or oxidative stress (E vs. F), while it had no impact on blastocyst quality. SCF did not affect the dynamism of growth of embryos exposed to Fas-L (G vs. H). However, blastocysts grown in Fas-L with SCF had more cells (G vs H). Conclusion: SCF seems to improve quality of blastocysts and dynamism of growth of embryos exposed to freeze-thaw procedure, oxidative stress or Fas-L. P-424 High incidence of tetraploid male bovine blastocyst development from oocytes injected with sexed freeze-dried spermatozoa. Levent Keskintepe, Anna Machnicka, Gabriella Pacholczyk, Karen Norris, Benjamin G. Brackett, Geoffrey Sher. Sher Institute for Reproductive Medicine, Las Vegas, NV; Sher Institute for Reproductive Medicine, Glendale, CA; Medical Coll of Georgia, Augusta, GA; The Univ of Georgia, Athens, GA. Objective: To assess pronuclear formation, the chromosomal constitution, and the developmental capacity of bovine blastocysts formed by intracytoplasmic sperm injection (ICSI) with sexed freeze-dried (lyophilized) spermatozoa. Design: Prospective randomized study. Setting: Academic setting. Interventions: Fresh sperm collected from a registered bull were sorted for X and Y bearing spermatozoa by flow cytometry, and frozen with a conventional freezing method. Frozen samples were thawed in a water bath at 37°C, then freeze-dried, and stored at 4°C until use. After 22-24 h of in-vitro maturation, the bovine oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 M ionomycin (5min) and with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. The embryos were sexed using the polymerase chain reaction (PCR) technique on day 6. Day 6 Blastocysts were fixed for cytogenetic examination, stained with Giemsa and examined for chromosomal complements. Primary measures of outcome: Pronuclear formation (PN), morula and blastocyst development and cytogenetic normality on day 6. Results: There were statistically significant differences in pronuclear formation and morula development between those oocytes injected with X, Y and XY sorted spermatozoon (p⬍0.05). The development rates for 2PN and morula for X, Y, XY injected oocytes were 70.2% (66/94) and 46.8 (44/94), 85.1% (80/94) and 55.3% (52/94), 64.8% (68/105) and 37.1% (39/105), respectively. However, there was no statistically significant difference in blastocyst development from oocytes injected with X, Y or XY sorted spermatozoon (p⫽0.03). Cytogenetic examination of blastocysts revealed a significantly raised incidence of tetraploidy for Y injected embryos 85% (17/20) as compared to embryos injected with X 15% (3/20) or unsorted XY 5% (1/20). Conclusions: The results of this study suggests that in vitro matured bovine oocytes can be fertilized withy freeze-dried sperm cells such that the resultant zygotes are capable of developing to the blastocysts stage, but sperm sorting in combination with lyophilization can result in abnormal embryo development for Y sorted sperm. P-425 Endothelin-I interaction with the ET-A receptor in the regression of the rat corpus luteum. Teresita E. Tognetti, A. Estevez, Alicia Motta, Ariel Bello, Eduardo Lombardi, Carlos Sueldo. IFER, Capital Federal, Argentina; Ctr de Estudios Farmacologicos y Botanicos, Capital Federal, Argentina. Objective: The process of luteolysis is rather complex and only partially understood. Endothelin I (ET-1), a peptide released by the vascular endo-
FERTILITY & STERILITY威
thelium and identified as a potent vasoconstrictor appears to play an important role. The purpose of our study is to evaluate the effects of ET-1 on progesterone (P) and prostaglandin F2 a (PGF2a) production by the ovary and the regulation by its principal receptor (ET-A) in rodents. Design: Experimental animal study performed at an Academic Research Laboratory. Materials and Methods: Wistar female rats (28-31 days old) were injected with PMSG (SIGMA USA), 7 days post injection the rats were sacrificed and the ovarian tissue removed for the study. The explants were cultured in M199 for 24 hs and the supernatants individually isolated for measurement of P and PGF2a (by RIA) and ET-1 with an EIA kit (Assay Designs, USA) expressing the results in pg/mg of tissue. To study the effect of ET-1 in P and PGF2a production, ET-1 at 10-7 M was added to the culture media. To asses the mechanism of action, a specific receptor antagonist to ET- A (BQ123) was preincubated for 30⬘prior to adding ET1 at 10-7 M. Finally, by western blot we evaluated the expression of ET-A receptor throughout the luteal phase and the impact of ET-1 in the midluteal phase on the receptor expression. Results: ET-1 (10-7 M) significantly inhibited P production by the luteal cells (from baseline 400 ⫾ 93 pg/mg tissue down to 185 ⫾ 27 pg/mg tissue, p⬍0.05). Conversely, ET-1 (10-7 M) significantly increased PGF2a production by the luteal cells (from baseline 50 ⫾ 2.5 pg/mg of tissue up to 71 ⫾ 2.5 pg/mg tissue, p⬍ 0.05). The preincubation of the ovarian tissue with an specific antagonist to ET-A receptor (BQ123, 10-6 M) reversed the effects of ET-1, since P production increased to 308 ⫾ 65 pg/mg of tissue (not different that under baseline conditions 400 ⫾ 93 pg/mg of tissue), while the production of PGF2a went down to 55 ⫾ 1.8 pg/mg of tissue (not different that under baseline conditions 50 ⫾ 2.5 pg/mg of tissue). Also, we evaluated the effect of ET-1 on its own receptor (ET-A), by immunoblotting, observing a significant downfall in the expression of the ET-A receptor. Conclusions: Our data shows a regulatory interaction between ET-1 and its specific receptor ET-A, modulating the corpus luteum production of P and PGF2a, and by this mechanism allowing the maintenance of cyclicity in the ovarian function.
P-426 Inhibition of chemokines prevents intraperitoneal adhesions in mice. Murat Berkkanoglu, Lufang Zhang, Umit A. Kayisli, Sinan Kursun, Sarper Taskiran, Aydin Arici. Yale Univ Sch of Medicine, New Haven, CT. Objective: Intraperitoneal adhesions are a significant cause of morbidity among women of reproductive age. It is widely accepted that infection, ischemia, and trauma to the peritoneum are initiators of the inflammatory reaction of adhesion formation. Chemokines regulate leukocyte recruitment and play a role in the adhesion process. NR58-3.14.3 is a novel broadspectrum inhibitor of chemokines including monocyte chemoattractant protein-1, macrophage inflammatory protein-1␣, and RANTES (regulated on activation, normal T-cell expressed and secreted), and interleukin-8. The aim of the present study was to evaluate the efficacy of this broadspectrum chemokine inhibitor (BSCI) in the prevention of adhesion formation after intraperitoneal surgery in mice. Design: Prospective study. Materials and Methods: A total of 40 eight-week-old Balb/c mice were used. They all underwent laparotomy, where the ventral and dorsal surfaces of the cecum were stroked with dry gauze pads. The parietal peritoneum 5 mm to the right of the midline incision was also traumatized by clamping with a hemostat. After the closure of the abdominal wall, animals were randomly assigned to receive daily intraperitoneal injections of either PBS (control) or the BSCI. A set of 12 animals was sacrificed every other day to assess time course of adhesion formation. On days 6 and 8 after the operation, 16 and 12 additional animals were sacrificed, respectively. The number, extent, location, and type (filmy vs fibrous) of adhesions were recorded. Subsequently a cumulative adhesion score was derived for each animal. In addition, immunohistochemistry was done on slides obtained from day eight adhesion sites with leukocyte common antigen, CD45. Mann-Whitney U test and Student’s t-test were used for statistical analysis. Results: No abnormalities of healing of the anterior abdominal wall incision were noted in animals. Adhesion scores reached a peak at 6-8 days after the operation. On day 6, the adhesion size was smaller in BSCI treated group compared to control group (P⫽0.01). Furthermore, there was a
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FERTILITY & STERILITY威
thelium and identified as a potent vasoconstrictor appears to play an important role. The purpose of our study is to evaluate the effects of ET-1 on progesterone (P) and prostaglandin F2 a (PGF2a) production by the ovary and the regulation by its principal receptor (ET-A) in rodents. Design: Experimental animal study performed at an Academic Research Laboratory. Materials and Methods: Wistar female rats (28-31 days old) were injected with PMSG (SIGMA USA), 7 days post injection the rats were sacrificed and the ovarian tissue removed for the study. The explants were cultured in M199 for 24 hs and the supernatants individually isolated for measurement of P and PGF2a (by RIA) and ET-1 with an EIA kit (Assay Designs, USA) expressing the results in pg/mg of tissue. To study the effect of ET-1 in P and PGF2a production, ET-1 at 10-7 M was added to the culture media. To asses the mechanism of action, a specific receptor antagonist to ET- A (BQ123) was preincubated for 30⬘prior to adding ET1 at 10-7 M. Finally, by western blot we evaluated the expression of ET-A receptor throughout the luteal phase and the impact of ET-1 in the midluteal phase on the receptor expression. Results: ET-1 (10-7 M) significantly inhibited P production by the luteal cells (from baseline 400 ⫾ 93 pg/mg tissue down to 185 ⫾ 27 pg/mg tissue, p⬍0.05). Conversely, ET-1 (10-7 M) significantly increased PGF2a production by the luteal cells (from baseline 50 ⫾ 2.5 pg/mg of tissue up to 71 ⫾ 2.5 pg/mg tissue, p⬍ 0.05). The preincubation of the ovarian tissue with an specific antagonist to ET-A receptor (BQ123, 10-6 M) reversed the effects of ET-1, since P production increased to 308 ⫾ 65 pg/mg of tissue (not different that under baseline conditions 400 ⫾ 93 pg/mg of tissue), while the production of PGF2a went down to 55 ⫾ 1.8 pg/mg of tissue (not different that under baseline conditions 50 ⫾ 2.5 pg/mg of tissue). Also, we evaluated the effect of ET-1 on its own receptor (ET-A), by immunoblotting, observing a significant downfall in the expression of the ET-A receptor. Conclusions: Our data shows a regulatory interaction between ET-1 and its specific receptor ET-A, modulating the corpus luteum production of P and PGF2a, and by this mechanism allowing the maintenance of cyclicity in the ovarian function.
P-426 Inhibition of chemokines prevents intraperitoneal adhesions in mice. Murat Berkkanoglu, Lufang Zhang, Umit A. Kayisli, Sinan Kursun, Sarper Taskiran, Aydin Arici. Yale Univ Sch of Medicine, New Haven, CT. Objective: Intraperitoneal adhesions are a significant cause of morbidity among women of reproductive age. It is widely accepted that infection, ischemia, and trauma to the peritoneum are initiators of the inflammatory reaction of adhesion formation. Chemokines regulate leukocyte recruitment and play a role in the adhesion process. NR58-3.14.3 is a novel broadspectrum inhibitor of chemokines including monocyte chemoattractant protein-1, macrophage inflammatory protein-1␣, and RANTES (regulated on activation, normal T-cell expressed and secreted), and interleukin-8. The aim of the present study was to evaluate the efficacy of this broadspectrum chemokine inhibitor (BSCI) in the prevention of adhesion formation after intraperitoneal surgery in mice. Design: Prospective study. Materials and Methods: A total of 40 eight-week-old Balb/c mice were used. They all underwent laparotomy, where the ventral and dorsal surfaces of the cecum were stroked with dry gauze pads. The parietal peritoneum 5 mm to the right of the midline incision was also traumatized by clamping with a hemostat. After the closure of the abdominal wall, animals were randomly assigned to receive daily intraperitoneal injections of either PBS (control) or the BSCI. A set of 12 animals was sacrificed every other day to assess time course of adhesion formation. On days 6 and 8 after the operation, 16 and 12 additional animals were sacrificed, respectively. The number, extent, location, and type (filmy vs fibrous) of adhesions were recorded. Subsequently a cumulative adhesion score was derived for each animal. In addition, immunohistochemistry was done on slides obtained from day eight adhesion sites with leukocyte common antigen, CD45. Mann-Whitney U test and Student’s t-test were used for statistical analysis. Results: No abnormalities of healing of the anterior abdominal wall incision were noted in animals. Adhesion scores reached a peak at 6-8 days after the operation. On day 6, the adhesion size was smaller in BSCI treated group compared to control group (P⫽0.01). Furthermore, there was a
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