RESUMEN
Parthenogenetic activation of oocytes is a helpful tool to obtain blastocysts, of which the inner cell mass may be used for derivation of embryonic stem cells. In order to improve activation and embryonic development after parthenogenesis, we tried to use sperm injection and subsequent removal of the sperm head to mimic the natural Ca2+ increases by release of the oocyte activating factor. Visualization of the sperm could be accomplished by Hoechst staining and ultraviolet (UV) light irradiation. To exclude negative effects of this treatment, we examined toxicity on activated mouse oocytes. After activation, oocytes were incubated in Hoechst 33342 or 33258 stain and exposed to UV irradiation. The effects on embryonic development were evaluated. Our results showed that both types of Hoechst combined with UV irradiation have toxic effects on parthenogenetically activated mouse oocytes. Although activation and cleavage rate were not affected, blastocyst formation was significantly reduced. Secondly, we used MitoTracker staining for removal of the sperm. Sperm heads were stained before injection and removed again after 1 h. However, staining was not visible anymore in all oocytes after intracytoplasmic sperm injection. In case the sperm could be removed, most oocytes died after 1 day. As MitoTracker was also not successful, alternative methods for sperm identification should be investigated.
Asunto(s)
Bencimidazoles/toxicidad , Blastocisto/citología , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/efectos de la radiación , Oocitos/citología , Partenogénesis/fisiología , Rayos Ultravioleta/efectos adversos , Animales , Blastocisto/efectos de los fármacos , Blastocisto/efectos de la radiación , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos C57BL , Oocitos/efectos de los fármacos , Oocitos/efectos de la radiación , Partenogénesis/efectos de los fármacos , Partenogénesis/efectos de la radiación , Fármacos Sensibilizantes a Radiaciones/toxicidad , Recuperación de la Esperma , Coloración y Etiquetado/métodosRESUMEN
In embryonic stem cell culture, small molecules can be used to alter key signaling pathways to promote self-renewal and inhibit differentiation. In mice, small-molecule inhibition of both the FGF/MEK/Erk and the GSK3ß pathways during preimplantation development suppresses hypoblast formation, and this results in more pluripotent cells of the inner cell mass (ICM). In this study, we evaluated the effects of different small-molecule inhibitors of the FGF/MEK/Erk and GSK3ß pathway on embryo preimplantation development, early lineage segregation, and subsequent embryonic stem cell derivation in the humans. We did not observe any effect on blastocyst formation, but small-molecule inhibition did affect the number of OCT3/4- and NANOG-positive cells in the human ICM. We found that combined inhibition of the FGF/MEK/Erk and GSK3ß pathways by PD0325901 and CHIR99021, respectively, resulted in ICMs containing significantly more OCT3/4-positive cells. Inhibition of FGF/MEK/Erk alone as well as in combination with inhibition of GSK3ß significantly increased the number of NANOG-positive cells in blastocysts possessing good-quality ICMs. Secondly, we verified the influence of this increased pluripotency after 2i culture on the efficiency of stem cell derivation. Similar human embryonic stem cell (hESC) derivation rates were observed after 2i compared to control conditions, resulting in 2 control hESC lines and 1 hESC line from an embryo cultured in 2i conditions. In conclusion, we demonstrated that FGF/MEK/Erk and GSK3ß signaling increases the number of OCT3/4- and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.
Asunto(s)
Masa Celular Interna del Blastocisto/metabolismo , Células Madre Embrionarias/citología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Proteínas de Homeodominio/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Transducción de Señal , Benzamidas/farmacología , Masa Celular Interna del Blastocisto/citología , Masa Celular Interna del Blastocisto/efectos de los fármacos , Recuento de Células , Técnicas de Cultivo de Célula , Linaje de la Célula , Difenilamina/análogos & derivados , Difenilamina/farmacología , Técnicas de Cultivo de Embriones , Implantación del Embrión/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Proteínas de Homeodominio/genética , Humanos , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Piridinas/farmacología , Pirimidinas/farmacología , Factores de TiempoRESUMEN
OBJECTIVE: To create a pool of frozen donated human oocytes and find the optimal stage for slow controlled-rate freezing of human in vitro matured oocytes. DESIGN: Oocytes at different developmental stages of maturation (germinal vesicle, metaphase I, or metaphase II) and oocytes that failed to fertilize after IVF or intracytoplasmic sperm injection (ICSI) were frozen using a slow controlled-rate freezing protocol. Frozen/thawed oocytes were artificially activated to verify activation potential and compared with oocytes that were not frozen. SETTING: University hospital-based fertility center. PATIENT(S): Stimulated patients undergoing IVF/ICSI treatment donated oocytes left over during their infertility treatment. INTERVENTION(S): Human oocytes were frozen at different stages of maturation. Fresh and frozen/thawed oocytes were activated by electrical pulses followed by incubation in 6-dimethylaminopurine. MAIN OUTCOME MEASURE(S): Survival rate, maturation rate and kinetics, and activation potential. RESULT(S): Human oocytes at all developmental stages have high survival rates after slow controlled-rate freezing. Frozen/thawed germinal vesicle oocytes showed decreased and delayed maturation after thawing. Activation potential was not affected. CONCLUSION(S): A pool of donated human oocytes could be established using slow controlled-rate freezing. Immature oocytes should be frozen after in vitro maturation.
Asunto(s)
Criopreservación/métodos , Fertilización In Vitro/métodos , Oocitos/citología , Oocitos/fisiología , Partenogénesis/fisiología , Adenina/análogos & derivados , Adenina/farmacología , Supervivencia Celular/fisiología , Estimulación Eléctrica , Femenino , Humanos , Técnicas In Vitro , Donación de Oocito , Oocitos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Estimulación QuímicaRESUMEN
Parthenogenetic reconstruction is one major strategy to create patient-specific stem cells. The aim of this study was to find the best artificial activation protocol for parthenogenetic activation of mouse and human oocytes comparing different methods. In a first set of experiments, in-vivo matured mouse oocytes and human failed-fertilized, in-vitro and in-vivo matured oocytes were artificially activated by a chemical (ionomycin) or electrical stimulus. In a second set of experiments, a combination of activating agents (electrical pulses followed by ionomycin or SrCl(2)) was applied in an aim to improve developmental competence. All embryos were evaluated daily until day 6 after activation. Mouse blastocysts were differentially stained to evaluate blastocyst quality. For mouse oocytes and human failed-fertilized oocytes, blastocyst development was significantly higher after electrical activation (P<0.05). For human in-vitro and in-vivo matured oocytes, blastocyst formation was only obtained after electrical activation of in-vitro matured oocytes. After combining activating agents, no differences in development could be observed. In conclusion, this study revealed that for both mouse and human oocytes development to the blastocyst stage was significantly better after electrical activation compared with chemical activation. Combining activating agents had no further positive effect on developmental potential.