RESUMEN
BACKGROUND: Recently we discovered that the human oviduct synthesizes abundant prostacyclin (PGI(2)). Gene knock-out studies suggest that PGI(2) is essential to endometrial decidualization, but the effects of PGI(2) on sperm and embryos have not been reported. METHODS: The effects of PGI(2) on human sperm were analysed by a computer-assisted semen analysis system. The effects of PGI(2) on mouse embryos were examined based on the rates of complete hatching. The expression of PGI(2) receptor (IP) was evaluated by Western blot analysis and immunohistochemistry. The binding of PGI(2) to embryos was confirmed by radioligand binding assay. Finally, cAMP levels were assessed in PGI(2)-challenged embryos. RESULTS: Iloprost (a stable PGI(2) analogue) did not affect the motility or the overnight survivability of human sperm. Western blot analysis did not detect IP in the sperm plasma membrane. In contrast, the hatching of mouse embryos was enhanced by iloprost (ED(50) 6.7 nmol/l). Exposure to iloprost during 8-cell to morulae or morulae to early blastocyst stages was critical to enhanced hatching. This coincided with the developmental stage-specific expression of IP. Although iloprost bound to blastocysts, it did not significantly increase cAMP. CONCLUSION: PGI(2) enhanced the hatching of mouse embryos but not the motility of human sperm.
Asunto(s)
Desarrollo Embrionario y Fetal/efectos de los fármacos , Epoprostenol/farmacología , Motilidad Espermática/efectos de los fármacos , Animales , Blastocisto/efectos de los fármacos , Western Blotting , Membrana Celular/química , Supervivencia Celular/efectos de los fármacos , AMP Cíclico/análisis , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/fisiología , Epoprostenol/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Iloprost/análisis , Iloprost/farmacología , Inmunohistoquímica , Masculino , Ratones , Mórula/efectos de los fármacos , Ensayo de Unión Radioligante , Receptores de Epoprostenol/análisis , Espermatozoides/ultraestructuraRESUMEN
A high-performance liquid chromatographic (HPLC) method utilizing ultraviolet absorbance coupled with radioisotove detection was developed for the precise and simultaneous determination of iloprost and misoprostol. This assay allows complete resolution of iloprost diastereoisomers and has a total run time of approximately twenty minutes. Samples were prepared for chromatographic analysis by extracting a mixture of tritiated drugs from rat plasma with acetonitrile. The resulting solutions were chromatographed on a reversed phase Zorbax Rx-C8 column using 0.02M potassium phosphate (pH 3.0), acetonitrile, and methanol (46:30:24, v/v) at a flow rate of 1.7 mL/min. 2-Naphthoic acid was employed as an internal standard. The correlation coefficient for varying concentrations of tritiated iloprost (12.7 Ci/mmol specific activity) from 2.18 ng/mL to 21.8 ng/mL was 0.995, and the correlation coefficient for concentrations of tritiated misoprostol (50 Ci/mmol specific activity) from 0.617 ng/mL to 6.17 ng/mL was 0.993. The high selectivity and sensitivity of this assay make it useful for the simultaneous quantitation of iloprost and misoprostol.
Asunto(s)
Iloprost/análisis , Misoprostol/análisis , Animales , Cromatografía Líquida de Alta Presión , Iloprost/sangre , Hígado/química , Ratones , Misoprostol/sangre , Estructura Molecular , Prostaglandinas/análisis , Prostaglandinas/farmacología , Ratas , Estándares de Referencia , EstereoisomerismoRESUMEN
A high-performance liquid chromatographic (HPLC) method utilizing absorbance detection was developed for the rapid and precise determination of the stable prostacyclin analogue iloprost in 5% dextrose in water solution (D5W). Samples were prepared for chromatographic analysis by extracting the drug into chloroform, evaporating the solvent, and solubilizing the residue in methanol. The resulting solutions were chromatographed on a Shandon ODS Hypersil column using 0.02 M potassium phosphate (pH 3.0), methanol, and acetonitrile (456:144:400, v/v) at a flow rate of 1.8 mL/min. 2-Naphthoic acid was employed as an internal standard. The detector response at 207 nm was linear (correlation coefficient greater than 0.9998) for concentrations of iloprost from 10 ng on-column, the lower limit of quantitation, up to 7 micrograms on-column. The precision of the method was approximately 5% based on peak area, with a recovery of iloprost from D5W of 87%. Liquid-liquid extraction was found to be superior to solid-phase sorbent extraction as a sample preparation method due to incompatibility of dextrose with the sorbent bed.