RESUMO
AIMS: To investigate experiences and expectations of parents/siblings of adults with Down Syndrome (DS) regarding oral healthcare, and explore factors impacting on access and experience of dental care for this group. DESIGN: A two phase qualitative and quantitative study using in-depth interviews with a convenience sample of six parents/siblings, and a postal questionnaire of 200 parents/siblings of adults with DS who are members of the Down Syndrome Association. RESULTS: The main themes elicited from the qualitative interviews related to concern, experiences, parents'/siblings' attitudes, preferences and information. The response rate from the postal questionnaire was 63.5%. Adults with DS attended the dentist regularly but received little restorative treatment. Experience of oral healthcare was influenced by the attitudes and skills of dental health professionals; stigma; and relatives' expectations of dentists, their oral health beliefs, information and support received, knowledge and priorities. Parents/siblings wanted dentists to be proactive in providing more information on oral health issues in collaboration with other health and social care professionals. CONCLUSIONS: Whilst most adults with DS visited the dentist regularly, relatively little treatment had been provided. Parents highlighted a need for appropriate and timely oral health information early in their child's life, and access to dentists who were sympathetic, good communicators and well-informed about DS.
Assuntos
Assistência Odontológica para Doentes Crônicos/psicologia , Síndrome de Down/psicologia , Educação em Saúde Bucal , Pais/psicologia , Relações Profissional-Família , Irmãos/psicologia , Adulto , Atitude do Pessoal de Saúde , Interpretação Estatística de Dados , Desumanização , Assistência Odontológica para Doentes Crônicos/estatística & dados numéricos , Pesquisa em Odontologia/métodos , Feminino , Acessibilidade aos Serviços de Saúde , Humanos , Entrevistas como Assunto , Masculino , Inquéritos e QuestionáriosRESUMO
Insulin-like growth factor II (IGF-II) and its receptor, the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor, are first expressed from the zygotic genome at the two-cell stage of mouse development. However, their role is not clearly defined. Insulin-like growth factor II is believed to mediate growth through the heterologous type 1 IGF and insulin receptors, whereas the IGF-II/M6P receptor is believed to act as a negative regulator of somatic growth by limiting the availability of excess levels of IGF-II. These studies demonstrate that IGF-II does have a role in growth regulation in the early embryo through the IGF-II/M6P receptor. Insulin-like growth factor II stimulated cleavage rate in two-cell embryos in vitro. Moreover, this receptor is required for the glycaemic response of two-cell embryos to IGF-II and for normal progression of early embryos to the blastocyst stage. Improved development of embryos in crowded culture supports the concept of an endogenous embryonic paracrine activity that enhances cell proliferation. These responses indicate that the IGF-II/M6P receptor is functional and likely to participate in such a regulatory circuit. The functional role of IGF-II and its receptor is discussed with reference to regulation of early development.
Assuntos
Desenvolvimento Embrionário/fisiologia , Fator de Crescimento Insulin-Like II/fisiologia , Receptor IGF Tipo 2/fisiologia , Animais , Transporte Biológico , Blastocisto/fisiologia , Feminino , Imunofluorescência , Glucose/metabolismo , Fator de Crescimento Insulin-Like II/farmacologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Gravidez , Receptor IGF Tipo 2/deficiência , Receptor IGF Tipo 2/genética , Técnicas de Cultura de TecidosRESUMO
The role of growth hormone (GH) in embryonic growth is controversial, yet preimplantation embryos express GH, insulin-like growth factor I (IGF-I) and their receptors. In this study, addition of bovine GH doubled the proportion of two-cell embryos forming blastocysts and increased by about 25% the number of cells in those blastocysts with a concentration-response curve showing maximal activity at 1 pg bovine GH ml(-1), with decreasing activity at higher and lower concentrations. GH increased the number of cells in the trophectoderm by 25%, but did not affect the inner cell mass of blastocysts. Inhibition of cell proliferation by anti-GH antiserum indicated that GH is a potent autocrine or paracrine regulator of the number of trophectoderm cells in vivo. Type 1 IGF receptors (IGF1R) were localized to cytoplasmic vesicles and plasma membrane in the apical domains of uncompacted and compacted eight-cell embryos, but were predominantly apparent in cytoplasmic vesicles of the trophectoderm cells of the blastocyst, similar to GH receptors. Studies using alpha IR3 antiserum which blocks ligand activation of IGF1R, showed that IGF1R participate in the autocrine or paracrine regulation of the number of cells in the inner cell mass by an endogenous IGF-I-IGF1R pathway. However, alpha IR3 did not affect GH stimulation of the number of trophectoderm cells. Therefore, GH does not use secondary actions via embryonic IGF-I to modify the number of blastocyst cells. This result indicates that GH and IGF-I act independently. GH may selectively regulate the number of trophectoderm cells and thus implantation and placental growth. Embryonic GH may act in concert with IGF-I, which stimulates proliferation in the inner cell mass, to optimize blastocyst development.
Assuntos
Blastocisto/efeitos dos fármacos , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Animais , Comunicação Autócrina , Blastocisto/citologia , Blastocisto/metabolismo , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Hormônio do Crescimento/imunologia , Soros Imunes/farmacologia , Camundongos , Camundongos Endogâmicos , Comunicação Parácrina , Receptor IGF Tipo 1/análise , Receptor IGF Tipo 1/imunologia , Receptor IGF Tipo 1/metabolismo , Estimulação QuímicaRESUMO
Studies across several species, particularly the mouse, show that growth hormone (GH, somatotrophin) is an important determinant of litter size, and to a lesser extent, of birth length. GH acts at all stages of development, from ovulation through preimplantation development to the late fetus, with actions on both embryo/fetus and mother contributing to successful fetal development. The fact that these are not more obvious in vivo is likely a result of redundancy of cytokine hormone action, particularly in relation to prolactin, which shares common actions and receptor locations with GH.
Assuntos
Desenvolvimento Embrionário e Fetal/fisiologia , Hormônio do Crescimento/fisiologia , Animais , Desenvolvimento Embrionário/fisiologia , Feminino , Hormônio do Crescimento/antagonistas & inibidores , Humanos , Troca Materno-Fetal/fisiologia , Camundongos , Camundongos Transgênicos , Ovulação/fisiologia , Gravidez , Prolactina/fisiologia , Ratos , Receptores da Somatotropina/genética , Receptores da Somatotropina/fisiologiaRESUMO
FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta-catenin and AF-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta-catenin such that very little of either protein remained following 72h culture. The residual beta-catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta-catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta-catenin and AF-6 in vivo.
Assuntos
Embrião de Mamíferos/fisiologia , Endopeptidases/metabolismo , Endopeptidases/fisiologia , Transativadores , Animais , Blastocisto/metabolismo , Western Blotting , Adesão Celular , Comunicação Celular , Divisão Celular , Células Cultivadas , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cinesinas/metabolismo , Camundongos , Microscopia de Fluorescência , Miosinas/metabolismo , Oligonucleotídeos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Ligação Proteica , Fatores de Tempo , Ubiquitina Tiolesterase , beta CateninaRESUMO
Although mouse oocytes and cleavage-stage embryos prefer pyruvate and lactate for metabolic fuels, they do take up and metabolize glucose. Indeed, presentation of glucose during the cleavage stages is required for subsequent blastocyst formation, which normally relies on uptake and metabolism of large amounts of glucose. Expression of the facilitative glucose transporter GLUT1 was examined using immunohistochemistry and Western blotting, and in polyspermic oocytes, metabolism of glucose was measured and compared with that of pyruvate and glutamine. GLUT1 was observed in all oocytes and embryos, and membrane and vesicular staining was present. Additionally, however, in polyspermic oocytes, the most intense staining was in the pronuclei, and this nuclear staining persisted in cleaving normal embryos. Furthermore, GLUT1 expression appeared to be up-regulated both in nuclei and plasma membranes following culture of oocytes in the absence of glucose. In polyspermic oocytes, the metabolism of glucose, but not of pyruvate or glutamine, was directly proportional to the number of pronuclei formed. After compaction, nuclear staining diminished, and GLUT1 localized to basolateral membranes of the outer cells and trophectoderm. In blastocysts, a weak but uniform staining of inner-cell-mass plasma membranes was apparent. The results are discussed in terms of potential roles for GLUT1 in pronuclei of oocytes and zygotes, nuclei of cleavage-stage embryos, and a transepithelial transport function for GLUT1, probably coupled with GLUT3, in compacted embryos and blastocysts.
Assuntos
Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Desenvolvimento Embrionário , Proteínas de Transporte de Monossacarídeos/análise , Oócitos/metabolismo , Oócitos/ultraestrutura , Animais , Blastocisto/metabolismo , Blastocisto/ultraestrutura , Western Blotting , Membrana Celular/química , Núcleo Celular/química , Células Cultivadas , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Glutamina/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Gravidez , Ácido Pirúvico/metabolismo , Zigoto/metabolismo , Zigoto/ultraestruturaRESUMO
PROBLEM: The importance of early pregnancy factor (EPF) at the pre-implantation stage of development (days 1-3 post-coitum [p.c.]) has been previously established in this laboratory. However, the role of EPF at the implantation stage (days 4.5-5 p.c.) has not been determined. This present study therefore investigates the role of EPF at this important developmental stage, both in vivo and in vitro. METHOD OF STUDY: Mated mice were passively immunized with anti-EPF antibodies at the peri-implantation stage (days 3.5-4 p.c.) and embryo implantation recorded. Parallel studies were conducted in vitro, where the effect of anti-EPF antibodies on trophoblast outgrowth of blastocysts was determined. RESULTS: Administration of anti-EPF antibodies in vivo at the peri-implantation stage of development resulted in failure of embryos to implant. Similarly, trophoblastic outgrowth of blastocysts was adversely affected in the presence of anti-EPF antibodies. CONCLUSIONS: These results, together with previous findings that anti-EPF antibodies retard embryonic development when administered at the early pre-implantation stage, clearly demonstrate that EPF is required by the embryo at two important developmental stages- the one-two-cell stage and the peri-implantation stage.
Assuntos
Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Camundongos/embriologia , Peptídeos/fisiologia , Proteínas da Gravidez , Fatores Supressores Imunológicos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/toxicidade , Blastocisto/efeitos dos fármacos , Chaperonina 10 , Implantação do Embrião/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Idade Gestacional , Imunização Passiva , Imunoglobulina M/imunologia , Imunoglobulina M/farmacologia , Imunoglobulina M/toxicidade , Mórula/efeitos dos fármacos , Peptídeos/antagonistas & inibidores , Peptídeos/imunologia , GravidezRESUMO
Provision of the maternal factors, albumin and/or insulin to embryos in vitro restores preimplantation morphological development and cell proliferation to that seen in vivo. The hypothesis that the preimplantation effects of insulin or albumin would be reflected in increased fetal growth rate was examined. Two-cell embryos were cultured 48-50 h in medium supplemented with 0.17 micromol/l, 15 micromol/l albumin or 0.17 micromol/l insulin and the resultant blastocysts transferred to pseudopregnant recipients. Fetal and placental mass and skeletal development were determined at E19 or E20 (day 19 or 20 of embryonic development). Preimplantation access to insulin or albumin increased fetal growth by 4-6%. Combining insulin and albumin did not produce a further increment in fetal growth. The fetal growth achieved by providing preimplantation access to insulin, albumin or both was equivalent to that of in-vivo developed blastocysts. The conclusions are that: (i) preimplantation access to maternal insulin and albumin is required for normal fetal growth rates in the mouse and (ii) the increments in inner cell mass cell number and metabolic rates induced by insulin (and possibly albumin) reflect a requirement for maternal growth factors during preimplantation stages to optimize fetal development.
Assuntos
Desenvolvimento Embrionário , Feto/fisiologia , Insulina/fisiologia , Prenhez/metabolismo , Albumina Sérica/fisiologia , Animais , Osso e Ossos/embriologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Feminino , Peso Fetal , Feto/anatomia & histologia , Feto/efeitos dos fármacos , Idade Gestacional , Insulina/metabolismo , Insulina/farmacologia , Camundongos , Camundongos Endogâmicos , Gravidez , Albumina Sérica/metabolismo , Albumina Sérica/farmacologiaRESUMO
The inability of the embryo to utilize glucose as a fuel before compaction has been an area of much speculation. It is suggested that limitations in glucose transporter processes are the prime reasons for this. The recent identification of GLUT3 as the transporter responsible for the uptake of maternal glucose after compaction may provide the missing link in this puzzle. Furthermore, the coincidence of its expression with the onset of embryonic glucose utilization suggests that GLUT3 may be involved in the determination of metabolic priorities of the embryo. A model for the uptake of glucose by the blastocyst based on the function of two facilitative glucose transporters, GLUT3 and GLUT1, is proposed which can accommodate growth factor regulation of embryonic processes and is consistent with both the well established biochemical characteristics of GLUT proteins and the physiology of the embryo.
Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário e Fetal , Proteínas de Transporte de Monossacarídeos/fisiologia , Proteínas do Tecido Nervoso , Animais , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 2 , Transportador de Glucose Tipo 3 , GravidezRESUMO
The results of this study challenge the widely held view that growth hormone (GH) acts only during the postnatal period. RNA phenotyping shows transcripts for the GH receptor and GH-binding protein in mouse preimplantation embryos of all stages from fertilized eggs (day 1) to blastocysts (day 4). An antibody specific to the cytoplasmic region of the GH receptor revealed receptor protein expression, first in two-cell embryos, the stage of activation of the embryonic genome (day 2), and in all subsequent stages. In cleavage-stage embryos this immunoreactivity was localized mainly to the nucleus, but clear evidence of membrane labeling was apparent in blastocysts. GH receptor immunoreactivity was also observed in cumulus cells associated with unfertilized oocytes but not in the unfertilized oocytes. The blastocyst receptor was demonstrated to be functional, exhibiting the classic bell-shaped dose-response curves for GH stimulation of both 3-O-methyl glucose transport and protein synthesis. Maximal stimulation of 40-50% was seen for both responses at less than 1 ng/ml recombinant GH, suggesting a role for maternal GH. However mRNA transcripts for GH were also detected from the morula stage (day 3) by using reverse transcription-PCR, and GH immunoreactivity was seen in blastocysts. These observations raise the possibility of a paracrine/autocrine GH loop regulating embryonic development in its earliest stages.
Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Hormônio do Crescimento/biossíntese , Hormônio do Crescimento Humano/farmacologia , Receptores da Somatotropina/biossíntese , 3-O-Metilglucose/farmacocinética , Análise de Variância , Animais , Transporte Biológico/efeitos dos fármacos , Blastocisto/citologia , Proteínas de Transporte/biossíntese , Feminino , Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/fisiologia , Camundongos , Camundongos Endogâmicos , Mórula/citologia , Mórula/fisiologia , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Transcrição Gênica , Zigoto/citologia , Zigoto/fisiologiaRESUMO
The robust independence of preimplantation embryo development in vitro suggests that the developmental programme is autonomous. The rapid accumulation of evidence during the last decade for participation of many hormones, growth factors and their receptors in these early stages of embryogenesis has challenged this conclusion. In this review, the insulin and epidermal growth factor families, which have been best studied in mice, are used to illustrate the different roles growth factors may play in preimplantation physiology and the circuits that possibly mediate their participation. Tumour necrosis factor alpha, an inhibitory factor and growth hormone previously considered to be restricted to orchestrating postnatal growth and development, is also discussed. In the absence of results indicating the existence of a master regulatory factor, the data support the hypothesis that the redundancy of growth factor actions may provide fail-safe protection to the preimplantation developmental programme.
Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário/fisiologia , Substâncias de Crescimento/fisiologia , Animais , Feminino , Fertilização in vitro/métodos , Substâncias de Crescimento/genética , Humanos , Camundongos , Fenótipo , Gravidez , RNA/genética , RNA/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/fisiologia , Zigoto/crescimento & desenvolvimentoRESUMO
The first differentiative event in mammalian development is segregation of the inner cell mass and trophectoderm (TE) lineages. The epithelial TE cells pump fluid into the spherical blastocyst to form the blastocyst cavity. This activity is fuelled by glucose supplied through facilitative glucose transporters. However, the reported kinetic characteristics of blastocyst glucose transport are inconsistent with those of the previously identified transporters and suggested the presence of a high-affinity glucose carrier. We identified and localized the primary transporter in TE cells. It is glucose transporter GLUT3, previously described in the mouse as neuron-specific. In the differentiating embryo, GLUT3 is targeted to the apical membranes of the polarized cells of the compacted morula and then to the apical membranes of TE cells where it has access to maternal glucose. In contrast, GLUT1 was restricted to basolateral membranes of the outer TE cells in both compacted morulae and blastocysts. Using antisense oligodeoxynucleotides to specifically block protein expression, we confirmed that GLUT3 and not GLUT1 is the functional transporter for maternal glucose on the apical TE. More importantly, however, GLUT3 expression is required for blastocyst formation and hence this primary differentiation in mammalian development. This requirement is independent of its function as a glucose transporter because blastocysts will form in the absence of glucose. Thus the vectorial expression of GLUT3 into the apical membrane domains of the outer cells of the morula, which in turn form the TE cells of the blastocyst, is required for blastocyst formation.
Assuntos
Blastocisto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Transporte de Monossacarídeos/fisiologia , Proteínas do Tecido Nervoso , Animais , Feminino , Imunofluorescência , Transportador de Glucose Tipo 3 , Camundongos , Microscopia Confocal , Oligonucleotídeos Antissenso , GravidezRESUMO
The roles of glucose deprivation, insulin, and insulin-like growth factor I (IGF-I) in the regulation of glucose transport in the mouse blastocyst were examined. Glucose transport, measured by uptake of 3-0-methyl glucose (3-OMG), was increased by 19% (P < 0.01) in response to glucose deprivation. Both IGF-I and insulin stimulated uptake, but IGF-I was 1,000-fold more potent than insulin, increasing uptake by 51% at 1.7 pM (P < 0.001). These effects began to appear after 20 min of incubation with growth factors, and required the simultaneous presence of glucose. The relative potencies of insulin and IGF-I suggest that the actions of IGF-I and insulin were both mediated via the IGF-I receptor. The inactivity of a specific agonistic insulin receptor antibody (B10) confirms this and suggests that this action may be independent of signalling through IRS-1. Cycloheximide decreased growth factor-stimulated transport by about 40%, indicating that both protein synthesis and transporter recruitment from cytoplasmic stores are responsible for maximal stimulation. These characteristics are consistent with GLUT1-facilitated glucose uptake and suggest that GLUT1 is the regulatable transporter in mouse blastocysts. Stimulation of GLUT1 may be a ubiquitous feature of the autocrine/ paracrine activity of IGF-I in cell growth and proliferation.
Assuntos
3-O-Metilglucose/metabolismo , Blastocisto/metabolismo , Fator de Crescimento Insulin-Like I/fisiologia , Insulina/fisiologia , Receptor IGF Tipo 1/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Cicloeximida/farmacologia , Feminino , Masculino , Camundongos , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
The effects of insulin on the endocytic activity of mouse blastocysts in vitro were investigated using confocal laser scanning microscopy, quantitative image analysis and electron microscopy. Confocal studies showed that fluorescein isothiocyanate-labelled markers, dextran (fluid phase) and albumin (combined membrane and fluid phase), were endocytosed by blastocysts and localized within vesicles (about 2.5 microns in diameter) in the outer trophectoderm cells. No labelling was detected in the inner cell mass cells or the blastocoel cavity. Treatment with 170 nmol insulin l-1 stimulated the endocytosis of fluorescently labelled dextran in freshly collected blastocysts, increasing mean vesicle diameter per embryo by 15% (P < 0.05) after incubation with insulin for 2.5 h and mean vesicle number per embryo by 56% (P < 0.01) after 6 h. Both effects were also evident in blastocysts that had been cultured from the late eight-cell stage. Blastocysts incubated for 6 h with insulin displayed increased convolutions in the trophectoderm apical membrane compared with controls, indicating increased membrane activity and suggesting macropinosome formation. Collectively, these results suggest that insulin enhances endocytosis in the trophectoderm by stimulating uptake at the apical membrane into larger and more numerous endocytic vesicles and with some evidence of vesicle fusion. This mechanism may provide a metabolic basis for the stimulation by insulin of biosynthesis, proliferation and morphological development in early embryos.
Assuntos
Blastocisto/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Insulina/farmacologia , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Células Cultivadas , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Endogâmicos , Microscopia Confocal , Microscopia EletrônicaRESUMO
Fluid phase endocytosis in mouse blastocysts was characterized using the fluid phase marker, 3H-dextran, which did not bind to the membrane. This nonsaturable uptake occurred via an energy-requiring process, with only 20% accountable by diffusion as indicated by analysis at 4 degrees C. Insulin stimulated uptake of 3H-dextran by 30% (P < 0.05) over the first hr. The rate of uptake then decreased in both control and insulin-treated blastocysts. However, by 2 hr, insulin-treated blastocysts contained 38% more 3H-dextran (38%; P < 0.01) than control blastocysts. Incubation of blastocysts in protein-free medium increased 3H-dextran uptake to a rate equivalent to 12% of the blastocyst volume/min (1,500 +/- 240 pliter/hr), compared to 4.5% and 1.5% of the blastocyst volume/min for uptake in the presence of 0.1 g BSA/l and 10 g BSA/l, respectively. Confocal microscopic studies of fluorescently labelled dextran uptake in blastocysts, cultured in the absence of BSA, showed an increase in weak fluorescence labelling in the trophectoderm cells of blastocysts, compared to blastocysts cultured in the presence of BSA. There was no diffusion of fluorescence label into the blastocoel cavity. This is consistent with fluid being endocytosed, possibly by a large number of small pinocytic vesicles. Thus fluid-phase endocytosis in blastocysts is stimulated by insulin, increasing the delivery of nutrient-containing fluid into blastocysts. In the absence of protein, embryos also increase fluid uptake, possibly in an attempt to maintain the rate of supply of protein nutrient to trophectoderm cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Blastocisto/citologia , Endocitose , Animais , Blastocisto/fisiologia , Membrana Celular/fisiologia , Dextranos/metabolismo , Feminino , Inulina/metabolismo , Camundongos , Microscopia Confocal , GravidezRESUMO
Mouse blastocysts take up glutamine by specific transport systems. Glutamine is an important precursor for macromolecular synthesis and a potential alternative fuel to glucose. This study compared glutamine uptake in blastocysts and isolated inner cell masses and characterized the major participating systems in the latter. Inner cell masses take up glutamine by facilitated transport systems. The identity of these was investigated using substrate competition and kinetic studies. Na(+)-dependent uptake of 13 mumol glutamine l-1 was inhibited by 60% by 1 mmol tryptophan l-1, 25% by 1 mmol 2-amino-2-norbornanecarboxylic acid l-1 and 50% by 1 mmol lysine l-1. Furthermore, 1 mmol 2-methyl(amino)isobutyric acid (MeAIB) l-1 inhibited uptake by 29%. Kinetic analysis of MeAIB-resistant uptake revealed a predominant Na(+)-dependent facilitated uptake system with Km and Vmax values of 434 +/- 72 mumol l-1 and 237 +/- 38 fmol per inner cell mass per 10 min, respectively. The inhibition of Na(+)-dependent uptake by tryptophan, lysine and the analogue 2-amino-2-norbornanecarboxylic acid suggests that most uptake of glutamine by inner cell masses occurs via the same system that predominates in whole blastocysts, Bo,+. The period of assay was so brief that significant participation of the inner cell mass in whole blastocyst uptake was precluded showing that system Bo,+ is expressed by both the trophectoderm and inner cell mass components of the blastocyst. However, MeAIB inhibited uptake by inner cell masses but not by blastocysts. This MeAIB-sensitive uptake had a Km value of 4.3 +/- 1.7 mmol l-1 and a Vmax value of 451 +/- 119 fmol per inner cell mass per 10 min. These characteristics suggest the first embryonic appearance of system A, which is a common Na(+)-dependent transporter in many somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Blastocisto/metabolismo , Glutamina/farmacocinética , Aminoácidos/metabolismo , Animais , Transporte Biológico , Camundongos , Camundongos Endogâmicos , Sódio/metabolismoRESUMO
Membrane transport of glucose divorced from metabolism, was analysed in 2-cell embryos, morulae and blastocysts in the preimplantation mouse. A non-metabolizable radiolabelled analogue, 3,0 methyl D-glucose (3OMG) was used, and glucose was used as well in morulae and blastocysts; incubation times were < or = 5 min. Uptake occurred by combination of a non-saturable process, resistant to cytochalasin-B, and a facilitated process exhibiting classic Michaelis-Menten kinetics. The rate constant for the non-saturable component increased from 1.22 +/- 0.12 pL embryo-1 min-1 in 2-cell embryos to 2.08 +/- 0.44 pL embryo-1 min-1 in blastocysts, determined using 3OMG. The Km values of the saturable component for 3OMG at 22 degrees C were relatively constant at about 6.5 mM in 2-cell embryos, morulae and blastocysts. At 37 degrees C, the Km increased from 6 mM in 2-cell embryos to 17 mM in blastocysts. Vmax increased about five-fold during development from the 2-cell stage to the morula stage and about three-fold during development to the blastocyst. The Km values for glucose in morulae and blastocysts were constant at about 1.3 mM at 37 degrees C. Uptake of 3OMG in blastocysts was inhibited by glucose and stimulated by incubation in glucose-free medium. There was no kinetic evidence for the participation of multiple saturable components in uptake by blastocysts or morulae. This supports the observation that the glucose transporter GLUT2, which is first expressed at the 8-cell stage to supplement GLUT1 expressed in the oocyte, does not contribute to the uptake of environmental glucose and is, therefore, probably restricted in expression to abcoelic membrane areas of the trophectoderm. Together with the known values of glucose in uterine fluid, the kinetic data indicate that most glucose enters the trophectoderm by this GLUT1 at a rate which directly reflects the external glucose concentrations. The activity increased on a cellular basis as development proceeded, suggesting increased activity to meet the increasing metabolic requirements of the blastocyst for glucose.
Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Metilglucosídeos/farmacocinética , 3-O-Metilglucose , Animais , Transporte Biológico , Blastocisto/metabolismo , Técnicas de Cultura , Feminino , Glucose/farmacocinética , Masculino , Camundongos , Gravidez , TemperaturaRESUMO
It has become clear that the mammalian embryo participates in a complex dialogue with the maternal physiology. The language of the dialogue is growth factor signalling. The embryo expresses receptors for insulin, IGFs, GH, EGF and cytokines including LIF, and CSFs; whilst ligands are secreted by the supporting tissues of the oviduct and uterus, and in some cases, the embryo itself. In the preimplantation period when the embryo is travelling to the uterus and passing through its first differentiation, these ligands affect embryonic physiology, apparently in ways that optimise developmental potential and synchronise embryonic and maternal physiologies. It is not yet clear in most cases whether this is by autocrine, paracrine or endocrine mode. In the crucial peri-implantation phase the embryo is preparing to invade the maternal system for which extensive uterine remodelling is necessary. A model is proposed in which a cascade of growth factor activities, orchestrated by the ovarian steroid patterns, choreographs the biochemical players (ECM proteinases and their inhibitors) which initiate this activity.
Assuntos
Blastocisto/fisiologia , Substâncias de Crescimento/fisiologia , Animais , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Feminino , Humanos , Insulina/fisiologia , Gravidez , Receptor de Insulina/fisiologia , Receptores de Somatomedina/fisiologia , Somatomedinas/fisiologiaRESUMO
Mouse blastocysts, in vitro, endocytosed 100 micrograms/ml 125I-labelled bovine serum albumin (BSA) at a rate equivalent to 192 +/- 27 microliters/hr/mg embryonic protein over the first 20 min. Insulin stimulated this initial uptake by 30% (P < 0.05). After this time, accumulation of 125I-labelled BSA began to plateau as the endocytosed 125I-labelled BSA was catabolized and 125I was released from the cells. Insulin caused an approximately 72% (P < 0.05) increase in the amount of uncatabolized 125I-labelled BSA remaining in insulin-treated blastocysts after 2 hr as compared to control blastocysts. Insulin partially inhibited catabolism of endocytosed 125I-labelled BSA during the first 2 hr following transfer to nonradioactive medium. After this time, degradation ceased in both control and insulin-treated blastocysts, leaving a small, uncatabolized protein pool remaining in the embryos; however, as a result of insulin's inhibitory effects on the initial catabolic rate, the uncatabolized protein pool was 30% (P < 0.05) larger in insulin-treated blastocysts after the 4 hr chase. Insulin inhibited endogenous protein degradation in blastocysts by 37% (P < 0.05). Combined with previous studies showing a 90% increase in endogenous protein synthesis in blastocysts following short-term stimulation with insulin (Harvey and Kaye, 1988), these results suggest that insulin acts to increase the endogenous protein reserves in the embryo. Dose-response studies indicated an EC50 of 0.5 pM for insulin's stimulation of 125I-labelled BSA accumulation, consistent with action via its own receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Blastocisto/efeitos dos fármacos , Insulina/farmacologia , Soroalbumina Bovina/metabolismo , Animais , Blastocisto/metabolismo , Endocitose/efeitos dos fármacos , Técnicas In Vitro , Fator de Crescimento Insulin-Like I/farmacologia , Cinética , Leucina/metabolismo , Camundongos , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/metabolismoRESUMO
Previous studies showed that both insulin and insulin-like growth factor-1 (IGF-1) stimulate metabolism and growth of preimplantation embryos. Because the effects of insulin occur with very low doses, it was suggested that its effects were mediated by its own receptors. However, the effects of IGF-1 occurred at higher doses, suggestive of cross reaction with the insulin receptor but still in the range for mediation via its own receptor. The aim of this study was to investigate the mediation of the metabolic and growth effects of insulin and IGF-1 using a specific insulin receptor antagonist. The antagonistic B-10 Fab fragment (B-10f) completely blocked stimulation of protein synthesis by both insulin and IGF-1, indicating that the insulin receptor mediates this action of both hormones. Alternately, only insulin's stimulation of inner cell mass mitogenesis and morphological development was inhibited by the B-10 Fab fragment. This showed that growth stimulation by insulin and IGF-1 was mediated via different receptors, insulin through its own receptor and IGF-1 through some other receptor. However, mediation via the IGF-2 receptor is not excluded since IGF-1 stimulates compaction when there is evidence for only the presence of the IGF-2 receptor. In summary, insulin or IGF-1 at physiological concentrations stimulates preimplantation mouse embryos, suggesting an important role for both these growth factors in early development.