RESUMO
Human embryonic stem (ES) cells have been proposed as a renewable source of pluripotent cells that can be differentiated into various cell types for use in research, drug discovery and in the emerging area of regenerative medicine. Exploitation of this potential will require the development of ES cell culture conditions that promote pluripotency and a normal cell metabolism, and quality control parameters that measure these outcomes. There is, however, relatively little known about the metabolism of pluripotent cells or the impact of culture environment and differentiation on their metabolic pathways. The effect of two commonly used medium supplements and cell differentiation on metabolic indicators in human ES cells were examined. Medium modifications and differentiation were compared in a chemically defined and feeder-independent culture system. Adding serum increased glucose utilisation and altered amino acid turnover by the cells, as well as inducing a small proportion of the cells to differentiate. Cell differentiation could be mitigated by inhibiting p38 mitogen-activated protein kinase (p38 MAPK activity). The addition of Knockout Serum Replacer also increased glucose uptake and changed amino acid turnover by the cells. These changes were distinct from those induced by serum and occurred in the absence of detectable differentiation. Induction of differentiation by bone morphogenetic protein 4 (BMP4), in contrast, did not alter metabolite turnover. Deviations from metabolite turnover by ES cells in fully defined medium demonstrated that culture environment can alter metabolite use. The challenge remains to understand the impact of metabolic changes on long-term cell maintenance and the functionality of derived cell populations.
Assuntos
Aminoácidos/metabolismo , Metabolismo dos Carboidratos/fisiologia , Meios de Cultura , Células-Tronco Embrionárias/metabolismo , Glucose/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , HumanosRESUMO
Oocyte-cumulus cell bidirectional communication is essential for normal development of the oocyte and cumulus cells (CCs) within the follicle. We showed recently that addition of recombinant growth differentiation factor 9 (GDF9), which signals through the SMAD2/3 pathway, during mouse oocyte in vitro maturation (IVM) increased fetal viability. This study thus aimed to observe the effects of disrupting oocyte-CC bidirectional communication during IVM on oocyte developmental competence and fetal outcomes. Cumulus-oocyte complexes (COCs) from equine chorionic gonadotropin-primed prepubertal (CBA/C57BL6) mice were cultured with or without 50 mIU/ml follicle-stimulating hormone (FSH) and 10 ng/ml epidermal growth factor (EGF) or 4 muM SMAD2/3 inhibitor SB-431542. Cumulus expansion and first polar body extrusion were then assessed, or COCs were fertilized and stained to evaluate sperm entry or cultured to the blastocyst stage. Embryo development and blastocyst quality were assessed, and Day 4.5 blastocysts were transferred to pseudopregnant recipients to analyze fetal outcomes. SMAD2/3 inhibition or FSH/EGF absence during IVM resulted in decreased cumulus expansion. First polar body extrusion and sperm entry were decreased in the absence of FSH/EGF, whereas only sperm entry was affected in SB-431542-matured COCs. Embryo development and blastocyst rates were unaffected; however, blastocyst quality was significantly altered, with reduced inner cell mass cell numbers in embryos derived from COCs matured in both treatments. When COCs were matured with SB-431542 in the absence of FSH/EGF, cumulus expansion was reduced, but fertilization, embryo development, and embryo quality were not. Inhibition of SMAD2/3 signaling in the presence of FSH/EGF significantly reduced fetal survival but had no effect on implantation or fetal and placental dimensions and morphology.
Assuntos
Células do Cúmulo/fisiologia , Oócitos/crescimento & desenvolvimento , Oócitos/fisiologia , Comunicação Parácrina , Animais , Benzamidas/farmacologia , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Contagem de Células , Células do Cúmulo/efeitos dos fármacos , Dioxóis/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Hormônio Foliculoestimulante/farmacologia , Fator 9 de Diferenciação de Crescimento/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Oócitos/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Gravidez , Resultado da Gravidez , Proteínas Recombinantes/farmacologia , Proteína Smad2/antagonistas & inibidores , Proteína Smad2/metabolismo , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/metabolismo , Interações Espermatozoide-Óvulo/efeitos dos fármacosRESUMO
BACKGROUND: Successful oocyte in vitro maturation (IVM) would eliminate the need for hormonal stimulation used in assisted reproduction techniques. Unfortunately, oocytes matured in vitro have compromised developmental competence possibly due to disrupted oocyte-cumulus communication resulting from inappropriate levels of oocyte-secreted factors such as growth differentiation factor 9 (GDF9). Hence, the aim of this study was to investigate the effects of exogenous GDF9 during IVM of mouse oocytes on development and subsequent fetal viability. METHODS: Cumulus-oocyte complexes from pregnant mare's serum gonadotrophin primed mice were cultured with or without 200 ng/ml exogenous recombinant GDF9, 50 mIU/ml FSH and 10 ng/ml epidermal growth factor (EGF). After 18 h, cumulus expansion was scored and oocytes were fertilized in vitro. Cleavage, blastocyst development, blastocyst total, inner cell mass (ICM) and trophectoderm cell numbers were assessed. Viability of embryos was assessed by transfer to recipient females and pregnancy outcome determined at day 15. RESULTS: Oocytes matured with exogenous GDF9 in the presence of FSH and EGF had higher rates of development, percentage of hatching blastocyst and blastocyst total and ICM cell numbers (all P < 0.05). Although implantation rate and fetal and placental weights were not affected, the number of viable fetuses at day 15 was increased with exogenous GDF9. CONCLUSIONS: Exogenous GDF9 during IVM improved embryo development and fetal viability and provides a promising approach for human IVM.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Viabilidade Fetal/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Oócitos/fisiologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Massa Celular Interna do Blastocisto/citologia , Proteína Morfogenética Óssea 15 , Contagem de Células , Forma Celular , Células Cultivadas , Meios de Cultura , Células do Cúmulo/citologia , Células do Cúmulo/fisiologia , Sinergismo Farmacológico , Fator de Crescimento Epidérmico/farmacologia , Feminino , Fertilização in vitro , Hormônio Foliculoestimulante/farmacologia , Fator 9 de Diferenciação de Crescimento , Camundongos , Camundongos Endogâmicos , Gravidez , Resultado da Gravidez , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: In vitro maturation (IVM) of mammalian oocytes has potential health benefits for patients undergoing assisted reproduction as an alternative to gonadotrophin treatment. This procedure is also useful for studying the process of oocyte and early embryo development. However, oocytes undergoing IVM have much lower competence than in vivo matured oocytes. Efforts to optimize IVM success have focused on replicating in vivo timing, hormonal milieu and cumulus cell responses associated with maturing oocytes. We have previously identified two extracellular matrix proteins, the protease Adamts1 and hyaluronan-binding proteoglycan Versican, produced by mural granulosa cells that selectively incorporate into the periovulatory cumulus-oocyte complex (COC). METHODS: Murine COC were cultured in the presence of epidermal growth factor and/or FSH. mRNA and protein were measured by real time PCR and Western blot and compared to in vivo derived COC. RESULTS: COCs from mice that underwent IVM for 6 or 20 h in the presence of epidermal growth factor, FSH or in combination had a > 10-fold reduction in mRNA (P < 0.05) for Adamts1 and Vcan when compared with in vivo matured COCs. Hyaluronan synthase 2 expression was up-regulated up to 8-fold (P < 0.05) over the unstimulated control, demonstrating successful induction of cumulus gene expression by the IVM conditions. While in vivo matured COCs showed abundant levels of these proteins, COCs that underwent IVM had neither detectable Adamts1, nor intact or Adamts1-cleaved Vcan. Human cumulus and granulosa cells matured in vivo contained abundant mRNA for Adamts1 and Vcan, demonstrating the potential relevance to human IVM. CONCLUSION: These results indicate that extensively altered COC matrix composition is present during IVM and may contribute to the observed poorer competence of the derived oocytes.