RESUMEN
Non-invasive methods of assessing embryo quality are critical for pregnancy success following IVF or intracytoplasmic sperm injection (ICSI). The addition of new non-invasive morphological and biochemical analyses may further improve pregnancy success, allowing the transfer of a single embryo, thereby reducing the risks involved in multiple births following IVF/ICSI. The presence of a protein, soluble human leukocyte antigen-G (sHLA-G), in embryo cultures has been suggested as a way to non-invasively predict embryo quality and pregnancy success, especially when used in conjunction with current embryo quality assessment methods. Detection of sHLA-G in embryo culture medium has been correlated with pregnancy success in 12 studies, but three studies were not able to detect sHLA-G. This is a review of the literature on sHLA-G detection in IVF/ICSI, and reasons are proposed for the reported discrepancies, as well as guidelines for reporting of data in future studies. Furthermore, it is suggested that the use of an HLA-G transgenic mouse model would advance understanding of the mechanism of action of sHLA-G in preimplantation embryos and its correlation to embryo health and viability. Research on a mouse model, combined with clinical studies, should enable the development of a fast and reliable method for utilizing sHLA-G detection to improve pregnancy success after IVF/ICSI.
Asunto(s)
Viabilidad Fetal , Antígenos HLA/sangre , Antígenos de Histocompatibilidad Clase I/sangre , Índice de Embarazo , Diagnóstico Preimplantación/métodos , Animales , Blastocisto/metabolismo , Femenino , Fertilización In Vitro/métodos , Viabilidad Fetal/fisiología , Regulación del Desarrollo de la Expresión Génica , Antígenos HLA/genética , Antígenos HLA/metabolismo , Antígenos HLA/fisiología , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/fisiología , Humanos , Ratones , Modelos Biológicos , Embarazo , Reproducción/genética , SolubilidadRESUMEN
Since 1978 in vitro fertilization (IVF) procedures have resulted in the birth of over 3 million babies. Yet in 2005, IVF procedures had a live birth rate of only 34%, with 32% of these births resulting in multiple pregnancies. These multiple pregnancies were directly attributed to the transfer of multiple embryos to increase the probability that a single, healthy embryo was included. The predominantly accepted noninvasive viability markers for embryos created by IVF are (1) number of cells at specific time points during development and (2) overall morphology of the embryo. Currently, it is difficult to count the number of cells beyond the eight-cell stage noninvasively. We report a nontoxic cell-counting method capable of counting cell numbers ranging from 8 to 26 in live mouse embryos. This method is derived from the fusion of differential interference contrast and optical quadrature microscopy and is verified by epifluorescence images of Hoechst-stained nuclei. The phase-subtraction cell-counting method is the first accurate, nontoxic technique to count cells through the morula stage in mouse embryos and may enhance the use of cell number as a viability marker if adopted for use with human embryos in the IVF clinic.
Asunto(s)
Blastocisto/citología , Recuento de Células/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Confocal/métodos , Microscopía de Contraste de Fase/métodos , Animales , RatonesRESUMEN
PURPOSE: The mouse preimplantation embryo development (Ped) gene product, Qa-2, influences the rate of preimplantation embryonic development and overall reproductive success. Here we investigated the expression pattern of two microRNAs, miR-125a and miR-125b, known to be involved in development in lower organisms, in preimplantation embryos from the two-cell, four-cell, eight-cell, morula, and blastocyst stages of development from the congenic B6.K1 (Ped negative) and B6.K2 (Ped positive) strains of mice. METHOD: B6.K1 and B6.K2 congenic mice differ only in the absence (B6.K1) or presence (B6.K2) of the genes encoding Qa-2 protein. We analyzed the expression of miR-125a and miR-125b in B6.K1 and B6.K2 preimplantation embryos by using real-time PCR. RESULT: We found no variability in miR-125b expression at any developmental stage in both strains. However, miR-125a expression increased during development in both strains and was ten times higher in Ped negative (B6.K1) embryos than in Ped positive (B6.K2) embryos by the blastocyst stage of development. CONCLUSION: Our results show that the absence of the Ped gene profoundly affects the level of a miRNA (miR-125a) known to regulate early development. The implication is that miR-125a is likely involved in the regulation of timing of early development in mice.
Asunto(s)
Blastocisto/metabolismo , Desarrollo Embrionario/genética , Antígenos de Histocompatibilidad Clase I/genética , MicroARNs/biosíntesis , MicroARNs/genética , Animales , Secuencia de Bases , Células Cultivadas , Femenino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/fisiología , Datos de Secuencia Molecular , EmbarazoRESUMEN
Preimplantation mouse embryos express both classical (class Ia) and nonclassical (class Ib) MHC class I proteins, and yet are not rejected by the maternal immune system. Although the function of the embryonic MHC class Ia proteins is unknown, one MHC class Ib protein, Qa-2, the product of the preimplantation embryo development (Ped) gene, actually enhances reproductive success. Similar in structure to MHC class Ia proteins, Qa-2 protein is a trimer of the alpha (heavy) chain, beta(2) microglobulin and a bound peptide. Studies on the folding, assembly and trafficking of MHC class Ia molecules to the cell surface have revealed this process to be dependent on multiple protein chaperone molecules, but information on the role of chaperone molecules in Qa-2 expression is incomplete. Here, we report the detection of mRNA for four chaperone molecules (TAP1, TAP2, calnexin and tapasin) in preimplantation embryos. We then focused on the role of the MHC-dedicated chaperone, tapasin, on Qa-2 protein expression. First, we demonstrated that tapasin protein is expressed by preimplantation embryos. Then, we used tapasin knockout mice to evaluate the role of tapasin in Qa-2 protein expression on both T cells and preimplantation embryos. We report here that optimal cell surface expression of Qa-2 is dependent on tapasin in both T cells and preimplantation embryos. Identification of the molecules involved in regulation of MHC class I protein expression in early embryos is an important first step in gaining insight into mechanisms of escape of embryos from destruction by the maternal immune system.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Blastocisto/inmunología , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Chaperonas Moleculares/inmunología , Linfocitos T/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Animales , Blastocisto/citología , Femenino , Regulación de la Expresión Génica/genética , Antígenos de Histocompatibilidad Clase I/genética , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Péptidos/inmunología , Embarazo , Pliegue de Proteína , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Linfocitos T/citología , Microglobulina beta-2/inmunologíaRESUMEN
The preimplantation embryo development (Ped) gene regulates the rate of preimplantation embryonic cleavage division and subsequent embryo survival. In the mouse, the Ped gene product is Qa-2 protein, a nonclassical MHC class I molecule encoded by four tandem genes, Q6/Q7/Q8/Q9. Most inbred strains of mice have all four genes on each allelic chromosome, making a total of eight Qa-2 encoding genes, but there are a few strains that are missing all eight genes, defining a null allele. Mouse strains with the presence of the Qa-2 encoding genes express Qa-2 protein and produce embryos with a faster rate of preimplantation embryonic development and a greater chance of embryo survival compared to mouse strains with the null allele. There is extensive evidence that the human homolog of Qa-2 is HLA-G. HLA-G in humans, like Qa-2 in mice, is associated with enhanced reproductive success. The human population is an outbred population. Therefore, for a better comparison to the human population, we undertook an investigation of the presence of the genes encoding Qa-2 in an outbred population of mice. We used Real-Time Quantitative PCR to quantify the number of Qa-2 encoding genes in a population of 32 wild mice identified as Mus musculus domesticus both by morphologic assessment and by PCR analysis of their DNA. We found great variability in the number of Qa-2 encoding genes in the wild mice tested. The wild mouse with the highest number of Qa-2 encoding genes had 85 such genes, whereas we discovered one wild mouse without any Qa-2 encoding genes. Evolutionary implications of a range of Qa-2 encoding gene numbers in the wild mouse population are discussed, as well as the relevance of our findings to humans.
Asunto(s)
Dosificación de Gen , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I/genética , Ratones/genética , Ratones/inmunología , Animales , Animales Salvajes/genética , Animales Salvajes/inmunología , Secuencia de Bases , ADN/genética , Cartilla de ADN/genética , Evolución Molecular , Femenino , Genotipo , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Embarazo , Especificidad de la EspecieRESUMEN
PROBLEM: Qa-2, the product of the Ped (preimplantation development) gene, regulates the rate of cell division of preimplantation mouse embryos by an unknown mechanism. Due to the limited availability of preimplantation embryos, T cells were used as a model system to assess the possible roles of Fyn and Lck, and two downstream effectors, PI-3 kinase and Akt, in Qa-2 induced cell proliferation. METHOD OF STUDY: Resting T cells were stimulated to proliferate by treating with mouse anti-Qa-2 antibody, cross-linking with anti-mouse immunoglobulin, and adding PMA. The effects of kinase inhibitors on this proliferation were studied. Co-immunoprecipitates of T-cell lysates were analyzed for possible associations between Qa-2 and Fyn or Lck. Fyn knockout mice (Fyn-/-) were used to determine whether Fyn is required for T-cell activation induced by cross-linking Qa-2. RESULTS: An inhibitor of Src family kinases and inhibitors of PI-3 kinase and Akt suppressed proliferation of resting T cells induced by cross-linking Qa-2. Fyn, but not Lck, co-immunoprecipitated with Qa-2. Fyn-/- T cells failed to proliferate in response to Qa-2 cross-linking. CONCLUSION: Fyn, PI-3 kinase, and Akt are required for the activation of T cells by cross-linking Qa-2.
Asunto(s)
Proliferación Celular , Antígenos de Histocompatibilidad Clase I/metabolismo , Activación de Linfocitos , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Androstadienos/farmacología , Animales , Anticuerpos , Blastocisto/enzimología , Blastocisto/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Reactivos de Enlaces Cruzados/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulinas/metabolismo , Inmunoprecipitación , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfolinas/farmacología , Nucleósidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-fyn/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fyn/deficiencia , Proteínas Proto-Oncogénicas c-fyn/genética , Piridazinas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacología , WortmaninaRESUMEN
High spatial resolution Raman maps of fixed cells in an aqueous environment are reported. These maps were obtained by collecting individual Raman spectra via a Raman microspectrometer in a raster pattern on a 0.5-microm grid and assembling pseudocolor maps from the spectral hypercubes by multivariate methods. The Raman maps show the nucleus and the nucleoli of cells as well as subcellular organization in the cytoplasm. In particular, the distribution of mitochondria in the perinuclear region could be demonstrated by correlating distinct areas of the Raman maps with corresponding areas of fluorescence maps of the same cells after staining with mitochondria-specific labels. To the best of our knowledge, this is the first report of label-free detection of mitochondria inside a somatic mammalian cell using Raman microspectroscopy.
Asunto(s)
Mitocondrias/fisiología , Mitocondrias/ultraestructura , Aldehídos , Fenómenos Biofísicos , Biofisica , Colorantes Fluorescentes , Células HeLa , Humanos , Microscopía Fluorescente , Mitocondrias/química , Espectrometría RamanRESUMEN
The number of cells in a preimplantation embryo is directly correlated to the health and viability of the embryo. There are currently no methods to count the number of cells in late-stage preimplantation embryos noninvasively. We assessed the ability of optical quadrature microscopy (OQM) to count the number of cells in mouse preimplantation embryos noninvasively. First, to test for possible light toxicity, we exposed two-cell mouse embryos to OQM and differential interference contrast (DIC) microscopy and assessed their ability to develop to the blastocyst stage. We found no inhibition of development from either mode of microscopy for up to 2 h of light exposure. We also imaged eight-cell to morula stage mouse preimplantation embryos by OQM nd developed two methods for counting the number of cells. The contour signature method (CSM) used OQM images alone and the phase subtraction method (PSM) used both OQM and DIC images. We compared both methods to standard cell counting techniques and found that the PSM was superior to all other noninvasive cell counting methods. Our work on mouse embryos should be applicable to human embryos. The ability to correctly count the number of cells in human preimplantation embryos could lead to the transfer of fewer embryos in in vitro fertilization (IVF) clinics and consequently a lower rate of high-risk multiple-infant births.
Asunto(s)
Blastocisto/citología , Microscopía de Interferencia/métodos , Animales , Recuento de Células/métodos , Desarrollo Embrionario/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Óptica y Fotónica , EmbarazoRESUMEN
HLA-G protein is the functional homolog of Qa-2, the product of the mouse preimplantation embryo development (Ped) gene. Embryos expressing Qa-2 on the cell surface exhibit a faster rate of preimplantation cleavage and preferential survival in utero compared with Qa-2-negative embryos. Qa-2 is glycosylphosphatidylinositol (GPI) linked in the cell membrane. As a result, Qa-2 proteins cluster in cholesterol- and sphingolipid-rich lipid raft microdomains in the cell membrane and can signal via raft-associated intracellular signaling molecules. Using T cells as a model system, cross-linking of Qa-2 on the cell membrane has been shown to induce proliferation of resting cells. HLA-G, like Qa-2, lacks a cytoplasmic domain capable of transducing signals from the cell surface to the nucleus, but unlike Qa-2, HLA-G has a short six-amino acid cytoplasmic tail rather than a GPI anchor. To test whether HLA-G, like Qa-2, is located in lipid rafts and can act as a signaling molecule, we used an HLA-G transgenic mouse system. T cells were isolated and tested for HLA-G expression by immunofluorescence and for localization of HLA-G in lipid rafts by immunofluorescence and Western blotting. Next, the T cells were cross-linked with anti-HLA-G antibody to test for induction of proliferation. Our novel results show that HLA-G, like GPI-linked Qa-2, is present in lipid rafts in the cell membrane and can act as a signaling molecule to induce proliferation of resting T cells.
Asunto(s)
Antígenos HLA/fisiología , Antígenos de Histocompatibilidad Clase I/fisiología , Microdominios de Membrana/inmunología , Transducción de Señal/inmunología , Animales , Complejo CD3/metabolismo , Línea Celular Tumoral , Proliferación Celular , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Activación de Linfocitos/genética , Microdominios de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Transducción de Señal/genética , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
We report for the first time the detection of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos. Cholera toxin beta (CTbeta), which binds to the raft-enriched ganglioside GM1, was selected to label rafts. In a novel application a Qdot reagent was used to detect CTbeta labeling. This is the first reported use of nanocrystals in mammalian embryo imaging. Comparative membrane labeling with CTbeta and lipophilic membrane dyes containing saturated or unsaturated aliphatic tails showed that the detection of GM1 in mouse oocytes and embryo membranes was consistent with the identification of cholesterol- and sphingolipid-enriched rafts in the cell membrane. Distribution of the GM1 was compared with the known distribution of non-raft membrane components, and disruption of membrane rafts with detergents confirmed the cholesterol dependence of GM1 on lipid raft labeling. Complementary functional studies showed that cholesterol depletion using methyl-beta-cyclodextrin inhibited preimplantation development in culture. Our results show that the membranes of the mouse oocyte and zygote are rich in lipid rafts, with heterogeneous and stage-dependent distribution. In dividing embryos, the rafts were clearly associated with the cleavage furrow. At the morula stage, rafts were also apically enriched in each blastomere. In blastocysts, rafts were detectable in the trophectoderm layer, but could not be detected in the inner cell mass without prior fixation and permeabilization of the embryo. Lipid rafts and their associated proteins are, therefore, spatio-temporally positioned to a play a critical role in preimplantation developmental events.
Asunto(s)
Fase de Segmentación del Huevo/metabolismo , Microdominios de Membrana/metabolismo , Oocitos/metabolismo , Actinas/metabolismo , Animales , Toxina del Cólera/metabolismo , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Femenino , Gangliósido G(M1)/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Embarazo , Puntos Cuánticos , Coloración y Etiquetado/métodos , Cigoto/metabolismo , beta-CiclodextrinasRESUMEN
PURPOSE: The mouse preimplantation embryo development (Ped) gene product, Qa-2, which is the homolog of human HLA-G, influences the rate of preimplantation embryonic development and overall reproductive success. The sex ratio in preimplantation embryos from Ped gene congenic mice was examined in order to determine whether embryo sex is a confounding factor in the control of the rate of preimplantation development. METHODS: B6.K1 (Ped slow) and B6.K2 (Ped fast) congenic mice differ only in the absence (B6.K1) or presence (B6.K2) of the genes encoding Qa-2 protein. We analyzed the sex of B6.K1 (n=221) and B6.K2 (n=260) preimplantation embryos by using Real-Time PCR with primers specific for the X and Y chromosomes. RESULTS: We found that there was no statistically significant difference in the ratio of male to female preimplantation embryos in either strain. CONCLUSIONS: We conclude that the sex of the embryos is not a confounding factor that affects the Ped gene control of the rate of preimplantation development. Therefore, the Ped gene is entirely responsible for mediating the faster development of B6.K2 embryos compared to B6.K1 embryos.
Asunto(s)
Blastocisto , Desarrollo Embrionario/genética , Ratones Congénicos/genética , Razón de Masculinidad , Animales , Cartilla de ADN , Femenino , Antígenos de Histocompatibilidad/genética , Masculino , Ratones , Reacción en Cadena de la Polimerasa , Procesos de Determinación del Sexo , Factores SexualesRESUMEN
PURPOSE: A major gene responsible for the control of preimplantation cleavage rate is the Ped gene, the product of which is the Qa-2 protein. Fast, but not slow developing mouse embryos express the Qa-2 protein. Platelet-activating factor (PAF) is a novel and potent signaling phospholipid that has unique pleiotropic properties in addition to platelet activation. PAF plays a significant role in virtually every reproductive event, including ovulation, fertilization, implantation, and parturition. The role of the Ped gene in PAF production by preimplantation embryos is yet to be established. The presence of this gene provides embryos with a reproductive advantage over those that are Ped negative, and may also serve as a regulator of PAF synthesis. The study hypothesis is that the amount of PAF produced is dependent upon the presence or absence of the Ped gene. METHODS: B6.K1 (Ped negative) and B6.K2 (Ped positive) mouse embryo-conditioned culture media were assayed for PAF content by a PAF-specific radioimmunoassay. RESULTS: There was a significant (p < 0.001) difference in blastocyst development rates between the Ped+ B6.K2 (61.0%) and the Ped- B6.K1 (25.3%) embryo culture groups. There was a significant difference (p < 0.05) in PAF production between the Ped+ B6.K2 (4.70+/-0.46 pmol per embryo) embryo culture group and the Ped- B6.K1 (10.02+/-3.49 pmol per embryo) embryo group. The B6.K1 (Ped-) embryo group produced >2x more PAF than did the B6.K2 (Ped+) group. CONCLUSIONS: The Ped gene plays a role in PAF production and release in preimplantation stage embryos. The use of two mouse identical strains, except for the Ped gene, show that its presence is associated with an increase in developmental potential. Embryos where the Ped gene was absent produced significantly higher levels of PAF, which may aid in their survival.
Asunto(s)
Blastocisto , Desarrollo Embrionario , Antígenos de Histocompatibilidad Clase I/genética , Factor de Activación Plaquetaria/metabolismo , Animales , Blastocisto/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , EmbarazoRESUMEN
The Ped (preimplantation embryo development) gene, whose product is Qa-2 protein, is correlated with a faster rate of preimplantation development (Ped fast phenotype) in mice that express Qa-2 protein compared with mice with an absence of Qa-2 protein (Ped slow phenotype). In the current study, we have used two congenic mouse strains differentially expressing the Ped gene, strain B6.K1 (Ped slow; Qa-2 negative) and strain B6.K2 (Ped fast; Qa-2 positive), to investigate the effects of Ped gene expression on postnatal growth profiles, systolic blood pressure and adult organ allometry. At birth, B6.K1 mice were moderately lighter than B6.K2 mice. B6.K1 mice became heavier during postnatal life (P < 0.05) and had elevated systolic blood pressure at 21 weeks of age when compared with B6.K2 mice (P = 0.006). B6.K1 mice also demonstrated elevated serum angiotensin-converting enzyme (ACE) activity, a known regulator of blood pressure (P = 0.037). Altered organ:body weight ratios were also observed, with the B6.K1 females having a higher ratio for lungs than B6. K2 females (P = 0.014). These data provide evidence of an association between the rate of preimplantation embryo development, postnatal growth and later cardiovascular function.
Asunto(s)
Presión Sanguínea/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Crecimiento/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/fisiología , Animales , Peso al Nacer/genética , Fenómenos Fisiológicos Cardiovasculares , Desarrollo Embrionario/fisiología , Femenino , Pulmón/anatomía & histología , Pulmón/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos , Tamaño de los Órganos/genética , Peptidil-Dipeptidasa A/sangre , Caracteres SexualesRESUMEN
Memory lymphocytes play a central role in the secondary immune response. The concentration of memory lymphocytes increases with age. A high level of cell surface CD44 is a marker of memory lymphocytes compared to naïve lymphocytes which express a low level of CD44. Major histocompatibility complex (MHC) class I protein expression also increases with age. To explore a possible correlation between the expression of CD44 and MHC class I protein (Kb), peripheral blood lymphocytes from 27 C57BL/6 mice ranging in age from 3 months to 33 months were isolated by Ficoll-Hypaque gradient centrifugation. Single and double indirect immunofluorescence assays were then performed with rat IgG anti-CD44 and/or mouse IgG anti-Kb as first antibodies, and phycoerythrin (PE) labeled goat anti-rat IgG and/or fluorescein (FITC) labeled goat anti-mouse IgG as second antibodies. Cells were then analyzed by using a FACScan flow cytometer. As expected, the percentage of lymphocytes expressing high levels of CD44 (memory cells) increased significantly with age and the expression of Kb increased significantly with age. Interestingly, the expression of Kb in lymphocytes expressing high levels of CD44 (memory cells) was 72% more than in cells expressing low levels of CD44 (naive cells) regardless of age.
Asunto(s)
Envejecimiento/inmunología , Antígenos H-2/metabolismo , Receptores de Hialuranos/metabolismo , Memoria Inmunológica/fisiología , Subgrupos Linfocitarios/metabolismo , Animales , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Masculino , Ratones , Ratones Endogámicos C57BLAsunto(s)
Biomarcadores/análisis , Fertilización In Vitro , Antígenos HLA/sangre , Antígenos de Histocompatibilidad Clase I/sangre , Resultado del Embarazo , Adulto , Medios de Cultivo , Desarrollo Embrionario , Femenino , Antígenos HLA-G , Humanos , Valor Predictivo de las Pruebas , Embarazo , Embarazo MúltipleRESUMEN
The transporter associated with antigen processing 1 and 2 (TAP1 and TAP2) genes belong to the ATP-binding cassette family of transporter genes. They provide peptides necessary for the assembly of major histocompatibility complex (MHC) class I molecules by transporting these peptides into the endoplasmic reticulum. As MHC class I protein expression increases with age, we have explored the effect of age on the transcription of MHC class I genes (Kb) and TAP1 and TAP2 genes in C57BL/6 mice. Blood and spleen lymphocytes were isolated from mice aged from 3 months to over 24 months. RNA was extracted and mRNA for Kb, TAP1, TAP2 was quantified using slot-blot hybridization followed by densitometry. There was a parallel age-related increase (1.5-fold) in blood lymphocyte mRNA of these genes from 3 months to 21 months. In mice over 24 months old there was a decrease in Kb and TAP1 mRNA, but an increase in TAP2 mRNA. In spleen lymphocytes an age-related increase in all three mRNA species occurred throughout life. While MHC class I and Tap genes underwent very similar age-related changes, MHC class I mRNA was about 50 times more abundant than either TAP1 or TAP2 mRNA.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Envejecimiento/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Transcripción Genética/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Bazo/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
The current consensus in the literature is that ovulated oocytes that are not fertilized die by apoptosis, but the details of the proteins involved in the apoptotic pathways have not been elucidated. In this paper we confirm that caspase-3, the executioner of apoptosis, is expressed in mouse oocytes, and show that two initiators of apoptosis, caspase-8 and caspase-9, are expressed in mouse oocytes. Comparisons were made of caspase-3, -8, and -9 activities in superovulated oocytes that were freshly collected or allowed to age in vivo or in vitro. We found that caspase-3 activity significantly increased in aged oocytes compared with young oocytes (p < 0.001), and that both caspase-8 activity and caspase-9 activity decreased in aged oocytes compared with young oocytes (p < 0.001 for caspase-8 and p < 0.05 for caspase-9 activity). A comparison of superovulated with naturally ovulated oocytes showed the same amount of caspase-8 activity in each, but a significant (p < 0.001) decrease in caspase-9 activity in naturally ovulated compared with superovulated oocytes. There was no difference in caspase-3, -8, or -9 activity in oocytes compared with zygotes. Finally, we showed that culture of oocytes in staurosporine increased the activity of caspase-8 and caspase-9. In conclusion, the finding of both caspase-8 and caspase-9 activity in oocytes shows that unfertilized oocytes have the machinery to undergo apoptosis by using either the extrinsic (caspase-8 dependent) or intrinsic (caspase-9 dependent) pathways.
Asunto(s)
Caspasas/metabolismo , Oocitos/enzimología , Cigoto/enzimología , Animales , Caspasa 3 , Caspasa 8 , Caspasa 9 , Ratones , Microscopía Fluorescente , Oocitos/citología , Estaurosporina/farmacología , Superovulación/fisiología , Cigoto/citologíaRESUMEN
Qa-2, a murine class Ib major histocompatibility complex (MHC) molecule, is a possible functional homolog of human leukocyte antigen G (HLA-G). Both molecules have been implicated in immunoregulation and embryonic development and both occur in membrane-bound and soluble isoforms that arise by alternative splicing. Soluble splice variants have been implicated in the reproductive functions of HLA-G. While soluble variants of Qa-2 have been previously detected in T lymphocytes, we now demonstrate the presence of mRNA for one of the two known soluble forms of Qa-2 in eight-cell embryos and in blastocysts. Qa-2 is glycosylphosphatidylinositol (GPI) linked in the outer leaflet of the cell membrane and is found in lipid raft microdomains where other raft-associated proteins transduce signals into the cell. In contrast, HLA-G has a truncated six amino acid cytoplasmic tail. By fluorescence co-localization in JEG-3 cells, using fluorescent cholera toxin beta subunit (a lipid raft marker) and anti-HLA-G antibody, we have demonstrated that membrane-bound HLA-G also localizes to lipid rafts, consistent with functional homology between the two molecules. Finally, our experiments in which we have purified Qa-2 and transferred it via a process known as protein painting to Qa-2 negative cells represent a model for potential therapy involving HLA-G.
Asunto(s)
Blastocisto/inmunología , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/genética , Animales , Línea Celular , Membrana Celular/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Antígenos HLA/análisis , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Microdominios de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Embarazo , ARN/aislamiento & purificación , Empalme del ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
The human major histocompatibility complex (MHC), in addition to its role in the regulation of cell-cell interactions in the immune response, also influences reproductive success. Human leukocyte antigen-G (HLA-G) is an MHC class I gene of particular interest in reproductive biology because of its specific expression on fetal cytotrophoblast cells, and its reported involvement both in protection of the developing fetus from destruction by the maternal immune response and in the prevention of maternal pre-eclampsia. HLA-G has 15 known alleles at the DNA level, and allelic frequency varies among ethnic groups. This study describes the results of an inaugural attempt to correlate an HLA-G genetic polymorphism with pregnancy outcome in a patient population undergoing IVF. The study group was composed of 102 Caucasian women. A maternal HLA-G genetic polymorphism was investigated by polymerase chain reaction (PCR) analysis of DNA collected from granulosa cells surrounding oocytes harvested for the IVF procedure. While no statistically significant correlation was identified in this initial study, larger studies examining DNA from trios of mother, father and offspring are planned.
Asunto(s)
Embrión de Mamíferos/fisiología , Genotipo , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/genética , Resultado del Embarazo , Adulto , Alelos , Secuencia de Bases , ADN/análisis , Desarrollo Embrionario y Fetal , Femenino , Fertilización In Vitro , Frecuencia de los Genes , Células de la Granulosa/química , Antígenos HLA-G , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Embarazo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The Ped gene influences the rate of cleavage of preimplantation embryos and their subsequent survival. Embryos that express the product of the Ped gene, Qa-2 protein, cleave at a faster rate than embryos with an absence of Qa-2 protein. In addition, the Ped gene has pleiotropic effects on reproduction. Thus, there is a reproductive advantage to those mouse strains that are Qa-2 positive. The presence or absence of Qa-2 is reflected at the DNA level by the presence or absence (deletion polymorphism) of the gene(s) encoding Qa-2 protein. Many inbred and wild-derived mouse strains have been characterized as Qa-2 positive or negative, but no previous studies have looked at the distribution of the Ped gene in a population of free-living wild mice. The purpose of this study was to determine the Ped gene deletion polymorphism frequency in a sample of free-living wild mice. Twenty-nine mice were collected and identified as Mus musculus. Genomic DNA extraction was performed on tail tips, and PCR was used to amplify a region from the Ped gene. Known Qa-2 positive and negative mice were used as controls. Results showed that all 29 wild mice were positive for the Ped gene. Since the Ped gene is dominant and provides a reproductive advantage, it is not surprising that all of the wild mice were Qa-2 positive. However, our assay could not distinguish homozygous from heterozygous mice. It is possible that the Qa-2 deletion polymorphism is segregating in the population, and a larger sample size would identify some Qa-2 negative mice.