P-690 Ammonium Alters the Proteome of Cultured Mouse Blastocysts. D. K. Gardner, D. W. Linck, W. B. Schoolcraft, M. G. Katz-Jaffe.

Colorado Center for Reproductive Medicine, Englewood, CO. OBJECTIVE: Ammonium has been shown to induce several pathologies in the preimplantation mouse embryo including; reduced blastocyst cell number and ICM development, altered gene expression and impaired metabolism. It was therefore the aim of this study to investigate the effects of exposing preimplantation mouse embryos to ammonium on the protein content (proteome) of resultant blastocysts. DESIGN: Experimental study MATERIALS AND METHODS: Zygotes were collected from superovulated CF1 (outbred) mice at 21 h post hCG. Embryos were cultured at 37oC, 5% O2 & 6% CO2, in groups of 10 in 20 l drops of sequential media G1/G2. Embryos were cultured in one of three groups: G1/G2 with no added ammonium, G1/G2 with 150 M ammonium and G1/G2 with 300 M ammonium. After 96h of culture the resultant blastocysts were extracted in groups of 5, processed and analyzed by time-of-ight mass spectrometry. RESULTS: The presence of ammonium at either concentration (150 or 300 M) had a signicant (P 0.05) negative effect on the levels of ve different proteins/biomarkers in the mouse blastocyst. These proteins had molecular weights between 1,200 to 6,650 Da. CONCLUSION: Consistent with the adverse effects of ammonium on embryo physiology, this study has shown that ammonium also alters the proteome of the mouse blastocyst in culture. Ongoing studies are therefore aimed at identifying the proteins/biomarkers within the proteome which are altered by the presence of ammonium during the preimplantation period. A promising candidate for one of the ve proteins/biomarkers signicantly affected by ammonium is cytochrome c oxidase polypeptide (with a molecular weight of 6,310 Da). Previous studies have revealed that ammonium impairs the oxidative capacity of the preimplantation mouse embryo. Supported by: None

CONCLUSION: Hormonal regulation of the studied genes expression was observed. TIMP-1 mRNA was highly expressed in all cells, consistent with the strong signal detected in cell media in our protein chip analysis (submitted ASRM 2005). Hormonal inuence on the expression of IGFBP-3 and -6 mRNA paralleled their protein secretion, as both were signicantly upregulated by P treatment (mRNA, p .001). We found signicantly increased TIMP-1 mRNA expression in P treated cells (p .001), contrasting with the nding of relatively decreased secretion of TIMP-1 by P cells compared to control. We also found signicantly increased expression of VCAM-1 mRNA in E2 treated cells (p .001), whereas secretion of VCAM-1 by all cell groups with hormonal treatment was relatively lower than that of control cells. These discrepancies between mRNA expression and hormonal secretion of TIMP-1 and VCAM-1 suggest that they may have post-transcription control mechanisms. The utilization of quantitative PCR to study hormonal inuence on gene expression in cultured endometrial cells shows promise as a method to better understand the regulation of genes that prepare the endometrium for implantation. Supported by: None P-692 Increase Abortion in Mice Exposed to Environmental Air Pollution in Sao Paulo. I. R. Silva Sr., A. Lichtenfels Sr., P. N. Saldiva Sr. FMUSP, Sao Paulo, Brazil. OBJECTIVE: Analyse relationship between abortion and exposition of polluted air in early gestation DESIGN: After created chambers of exposure, we share our total group of 60 animals in 2 sub groups.One of them was exposed and other one no exposed. After the end of gestation our rst intention was analyse fetal weight, but we were surprised with high number of early abortion in ambient chamber. MATERIALS AND METHODS: Chambers of exposition was criated for simulating ambiental polluted air and clean air. The exposure chambers are assembled side by side in the garden of Medical School with heavy trafc. Daily concentrations of following pollutans were performed.A total of 60 Swiss mice, were divided in 2 groups, in the clean chamber or ambient chamber. Initially female and male were in the same cage in ratio 1/1. After check a ejaculatory plug, female and male are separeted and females were alone in their respectives cages. After rst day of pregnancy they were located in clean chamber or ambient chamber, and stayed there for 3 next weeks. Cesarean section was performed after 19 days of gestation, and abort check was performed at time of delivery. Imediatelly before cesarean section mothers were sacried following internacional norms, by cervical dislocation. RESULTS: A total of 60 Swiss mice females began our data.18 animals were excluded because they didn t become pregnant. A total of 387 fetuses were alive at birth time. 60 early abortions were detectated. From these 48 came from ambient chambers, or 80% of total abortion. After realized analyses of variance a signicant relantionship between polluted air and abortion was found (p 0.012). CONCLUSION: A positive relantionship between air pollution and abortion were found in our study.We found only early abortions that mean a importance of exposition in rst week of gestation.

P-691 Using Real-Time PCR to Study Hormonal Regulation of ImplantationRelated Gene Expression. K. Dragisic, H. C. Liu, Z. Y. He, Z. Rosenwaks. Cornell University Medical Center, New York, NY. OBJECTIVE: Endometrial cytokines and adhesion molecules involved in implantation may be hormonally regulated. The purpose of our study was to use real-time PCR to quantify steroid inuence on endometrial gene expression. DESIGN: Mouse endometrial tissue was cultured with hormonal media. Gene expression patterns were studied by real time PCR. MATERIALS AND METHODS: Endometrial cells were cultured from 3-week old BDF1 mice (Charles River Co.) The enzymatically digested glands and stroma (2x105 cell density) were seeded in 4 groups and incubated for 10 days with different hormonal media: Control, Estradiol (E2, 1x10-8 M), Progesterone (P, 1x10-8M) and E2 P. RNA was extracted from endometrial cells on day 10 of incubation using an Rneasy Mini Kit (Qiagen) and quantitated. Reverse transcription was performed. Relative quantitation of mRNA was performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystems) using TaqMan real-time technology. Cytokine primers and probes were designed using Applied Biosystems inventoried probes. Quantitation was normalized to endogenous control mouse -actin. Decidualization was conrmed by measuring decidual/ trophoblastic prolactin-related protein (d/tPRP) mRNA in P and E2 P treated cells (Gynecol Endocrinol 2001;15: 426-432). Multiplex PCR was performed with 6-FAM as the reporter dye for each protein and VIC for -actin. RESULTS: 30mcl of total RNA was obtained from each group: Control concentration, 240.4mcg/mL; E2, 296.4mcg/mL; P, 460.9 mcg/mL; E2 P, 432.5 mcg/mL. All samples were detected twice, and mean results are reported. The threshold cycle (Ct) was calculated by determining the point at which uorescence emission by degradation of the hybridization probe exceeded a threshold limit (Genome Research 1996;6: 986-994). Ct values decrease linearly with increasing target quantity and reect input sample concentration. The Ct for blank sample cycles was 40, and protein mRNA levels were stratied by hormonal treatment.

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Abstracts

Vol. 84, Suppl 1, September 2005