RESUMO
OBJECTIVE: A number of studies to improve in vitro culture conditions have been tried over past ten years by using co-culture system with helper somatic cells. However, the mechanism of coculture is poorly understood. This study was designed to understand the mechanism for the mode of actual action of co-culture system of ICR strain's 1-cell embryos with human oviduct epithelial cells by examining the effect of conditioned medium and contactless coculture using a cell culture insert on the embryo development and by measuring the level of superoxide anion from conditioned medium after co-culture. METHODS: ICR strain's zygote embryos were cultured in medium alone (control), coculture, conditioned medium, or contactless coculture system for 6 days. Conditioned media (CM) were prepared as following 5 groups. All CM were collected after culturing oviduct cells for 2 days. CM-1 was stored at -20degrees C until use, and CM-2 was prepared just before use as a culture medium. CM-3 was cocultured with embryos and retrieved just before use. CM-4 and CM-5 were derives from the microfilteration of CM-2 and CM-3, respectively, using Microcon-10 (10 kDa molecular weight cut-off). The percentage of the embryos developed to hatched blastocyst stage and the level of superoxide anion in supernatant from medium alone culture (control), coculture, and contactless coculture were measured. RESULTS: The rates of embryo development to the hatched blastocyst stage were significantly higher in coculture (43%) than in control (0%) (p<0.05). The CM-1 group had no embryo development since 2-cell embryonic stage, whereas the CM-2, CM-3, CM-4 and CM-5 groups had the improved development to 4 or 8 cell embryo stage, but the similar rate of development to hatched blastocyst compared to control. The effect of coculture on embryo develpment was disappeared in the contactless coculture group. The level of superoxide anion was significantly reduced in coculture group compared to control. CONCLUSION: It is concluded that the present coculture system overcomes the 2-cell block in vitro and improves the embryo development. This beneficial effect may be due to the direct cell-cell contact between embryo and helper cells or the removal of deleterious components from medium rather than the embryotrophic factors.
Assuntos
Animais , Feminino , Humanos , Gravidez , Blastocisto , Técnicas de Cultura de Células , Técnicas de Cocultura , Meios de Cultivo Condicionados , Desenvolvimento Embrionário , Estruturas Embrionárias , Células Epiteliais , Peso Molecular , Oviductos , Superóxidos , Linfócitos T Auxiliares-Indutores , ZigotoRESUMO
No abstract available.
Assuntos
Feminino , Gravidez , Desenvolvimento Embrionário , Estruturas Embrionárias , Óxido NítricoRESUMO
No abstract available.
Assuntos
Macrófagos Peritoneais , Óxido Nítrico , Óxido Nítrico Sintase , Choque , Fator de Necrose Tumoral alfaRESUMO
This study was performed to determine the effects of nitric oxide on human sperm cell function. Semen samples were obtained from normal healthy volunteers. Motile spermatozoas collected by swim-up method were incubated up to 24 hours in Ham's F-10 medium supplemented with a various concentration of sodium nitroprusside (nitric oxide releasing agent). Sperm motility, hyperactivation, acrosome reaction rate, and acrosin activity were determined. The results are as follows; 1. 1mM of SNP resulted in a significant decrease in sperm motility (44.8%+/- 8.9%:78.1%+/-6.3%, and hyperactivation (10.4%+/-6.4%:477%+/-9.5%) after incubation for 3 hours compared with the control group (Ham's F-10 alone), but had no effect on acrosome reaction. 2. At 100muM SNP, sperm motility was reduced after incubation for 6 hours (54.8%+/- 3.2%) compared with that of the control group (82.7% +/- 8.9%), but hyperactivation and acrosome reaction were not affected. 3. However, a lower concentration (less than 101M) of SNP had no effect on sperm motility and hyperactivation for 8 hours of incubation but significantly decreased them when incubation periods were increased up to 24 hours compared with the control group. On the other hand, 1muM and l0muM SNP significantly increased the acrosome reaction rate in both acrosomal status (17.3%+/-5.2%,23.5%+/-4.7%, respectively) and acrosin activity (34.3muIU+/- 10.5muIU, 45.6muIU+/-5.6muIU, respectively) as compared with the control group (7.0%+/-4.0%, 9.5muIU+/-3.4muIU). These results indicate that SNP, NO releasing agent, has a dose-dependent effects on the sperm cell function. Therefore it may positively affect the fertilization by promoting acrosomal reaction at a lower concentration (less than 101M).