RESUMEN
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2014 there were six themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of animal models, xenobiotics, pathological biomarkers, genetics and epigenetics, and stillbirth and fetal growth restriction.
Asunto(s)
Biomarcadores/análisis , Modelos Animales de Enfermedad , Placenta/efectos de los fármacos , Placenta/metabolismo , Complicaciones del Embarazo/patología , Xenobióticos/toxicidad , Animales , Epigénesis Genética/fisiología , Femenino , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/patología , Humanos , Exposición Materna/efectos adversos , Enfermedades Placentarias/inducido químicamente , Enfermedades Placentarias/genética , Enfermedades Placentarias/metabolismo , Embarazo , Complicaciones del Embarazo/diagnóstico , MortinatoRESUMEN
The primitive cardiac tube starts beating 6-8 weeks post fertilization in the developing embryo. In order to describe normal cardiac development during late first and early second trimester in human fetuses this study used microarray and pathways analysis and created a corresponding 'normal' database. Fourteen fetal hearts from human fetuses between 10 and 18 weeks of gestational age (GA) were prospectively collected at the time of elective termination of pregnancy. RNA from recovered tissues was used for transcriptome analysis with Affymetrix 1.0 ST microarray chip. From the amassed data we investigated differences in cardiac development within the 10-18 GA period dividing the sample by GA in three groups: 10-12 (H1), 13-15 (H2) and 16-18 (H3) weeks. A fold change of 2 or above adjusted for a false discovery rate of 5% was used as initial cutoff to determine differential gene expression for individual genes. Test for enrichment to identify functional groups was carried out using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Array analysis correctly identified the cardiac specific genes, and transcripts reported to be differentially expressed were confirmed by qRT-PCR. Single transcript and Ontology analysis showed first trimester heart expression of myosin-related genes to be up-regulated >5-fold compared with second trimester heart. In contrast the second trimester hearts showed further gestation-related increases in many genes involved in energy production and cardiac remodeling. In conclusion, fetal heart development during the first trimester was dominated by heart-specific genes coding for myocardial development and differentiation. During the second trimester, transcripts related to energy generation and cardiomyocyte communication for contractile coordination/proliferation were more dominant. Transcripts related to fatty acid metabolism can be seen as early as 10 weeks and clearly increase as the heart matures. Retinol receptor and gamma-aminobutyric acid (GABA) receptor transcripts were detected, and have not been described previously in human fetal heart during this period. For the first time global gene expression of heart has been described in human samples to create a database of normal development to understand and compare with known abnormal fetal heart development.
Asunto(s)
Desarrollo Fetal , Corazón Fetal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Adulto , Femenino , Corazón Fetal/embriología , Humanos , Análisis de Matrices Tisulares , TranscriptomaRESUMEN
OBJECTIVE: This study aimed to describe brain development during the first (B1) and second trimester (B3) in human fetuses. DESIGN: Ten brains from 10 to 18 weeks of gestational age (GA) were collected, and the RNA was used for transcriptome analysis (Affymetrix 1.0 ST microarray chip). Differences in brain development within 10 to 18 GA were investigated by dividing the sample into 10 to 12 (B1), 13 to 15(B2) and 16 to 18(B3) weeks. A fold change of 2 or above, with a false discovery rate of 5%, was used as cut-off to determine differential gene expression for individual genes. Quantitative real-time PCR was used to confirm differences. Tests for enrichment procedures (using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) were then used to identify functional groups of mRNA. RESULTS: At 10 to 12 weeks, brains showed neuronal migration to be upregulated. From 10 to 18 weeks, brains showed genes coding for neuronal migration, differentiation and connectivity upregulated. ALDH1A1 and NPY genes, marker of spinal cord and striatum, were upregulated in B1 and B3 brains, respectively. Also, SLITRK6-HAS2 and CRYAB-PCDH18 genes for ear and eye sensory input were upregulated in B1. CONCLUSIONS: For the first time, brain global gene expression was described in human samples. Period B1 was dominated by genes coding for neuronal migration, differentiation, programmed cell death and sensory organs. B3 was dominated by neuronal proliferation, branching and myelination. Creating such a database will allow comparison with abnormals in future studies.
Asunto(s)
Encéfalo/metabolismo , Feto/metabolismo , Expresión Génica , Proteínas del Tejido Nervioso/genética , Primer Trimestre del Embarazo , Segundo Trimestre del Embarazo , Femenino , Expresión Génica/fisiología , Perfilación de la Expresión Génica , Edad Gestacional , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The development of the placenta is imperative for successful pregnancy establishment, yet the earliest differentiation events of the blastocyst-derived trophectoderm that forms the placenta remain difficult to study in humans. Human embryonic stem cells (hESC) display a unique ability to form trophoblast cells when induced to differentiate either by the addition of exogenous BMP4 or by the formation of cellular aggregates called embryoid bodies. While mouse trophoblast stem cells (TSC) have been isolated from blastocyst outgrowths, mouse ESC do not spontaneously differentiate into trophoblast cells. In this review, we focus on addressing the similarities and differences between mouse TSC differentiation and hESC-derived trophoblast differentiation. We discuss the functional and mechanistic diversity that is found in different species models. Of central importance are the unique signaling events that trigger downstream gene expression that create specific cellular fate decisions. We support the idea that we must understand the nuances that hESC differentiation models display so that investigators can choose the appropriate model system to fit experimental needs.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Transducción de Señal/fisiología , Trofoblastos/citología , Animales , Células Madre Embrionarias/fisiología , Femenino , Humanos , Ratones , Placenta/citología , Placenta/fisiología , Embarazo , Trofoblastos/fisiologíaRESUMEN
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of models and technical issues involved in placenta research: 1) comparative placentation and animal models; 2) advanced techniques in placental histopathology; 3) human pluripotent stem cells as a model for trophoblast differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Modelos Animales , Placenta/patología , Placentación/fisiología , Células Madre Pluripotentes/fisiología , Trofoblastos/fisiología , Animales , Femenino , Humanos , Placenta/citología , EmbarazoAsunto(s)
Experimentación Animal/ética , Experimentación Animal/normas , Personal de Laboratorio Clínico/tendencias , Relaciones Públicas/tendencias , Medicina Reproductiva , Experimentación Animal/legislación & jurisprudencia , Bienestar del Animal , Animales , Comunicación , Unión Europea , Regulación Gubernamental , Humanos , Estados Unidos , Organización Mundial de la SaludRESUMEN
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology: 1) epigenetics and imprinting in the placenta; 2) growth factors and villous trophoblast differentiation; 3) role of the placenta in regulating fetal exposure to xenobiotics during pregnancy; 4) infection and the placenta.
Asunto(s)
Epigénesis Genética/fisiología , Impresión Genómica/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Placenta/fisiología , Complicaciones Infecciosas del Embarazo/fisiopatología , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Trofoblastos/fisiología , Xenobióticos/efectos adversos , Diferenciación Celular/fisiología , Femenino , Humanos , Placenta/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/fisiopatologíaRESUMEN
The human embryo is not a feasible experimental system for the detailed study of implantation and early placentation, so surrogate systems have been sought for investigating the determination of the trophectoderm lineage, its differentiation into trophoblasts of the early implantation site, and subsequently the morphogenesis of the definitive placenta. An alternative to the use of embryos for studying early placental development was revealed by work with human embryonic stem cells (hESC), demonstrating BMP2/4-stimulated trophoblast differentiation, and spontaneous formation from embryoid bodies (EBs). These cells display a trophoblastic transcriptome, as well as a placental protein and steroid hormone secretory profile, and invasive and chemotactic behavior resembling human placental trophoblasts. With EB-derived trophoblasts, two-dimensional and three-dimensional paradigms and other modifications of the culture environment, including extracellular matrix and aggregation with placental fibroblasts, impact on trophoblast differentiation. Recent studies have questioned the identity of the trophoblasts directed by BMP treatment of hESC, and careful attention to culture conditions is needed to interpret different results among research groups. Although the precise placental counterpart of the hESC-derived trophoblast remains unclear, hESC-derived trophoblasts remain an intriguing platform for modeling early implantation.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Trofoblastos/citología , Animales , Cuerpos Embrioides/fisiología , Femenino , Humanos , Embarazo , PrimatesRESUMEN
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.
Asunto(s)
Estado de Salud , Placenta/fisiología , Animales , Investigación Biomédica/tendencias , Diferenciación Celular , Epigénesis Genética , Femenino , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunomodulación , Masculino , MicroARNs/fisiología , Fisiología Comparada/tendencias , Placenta/citología , Placenta/inmunología , Placentación , Embarazo , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Trasplante de Células Madre/tendencias , Células Madre/citología , Células Madre/inmunología , Trofoblastos/citología , Trofoblastos/inmunologíaRESUMEN
The aim of this study was to characterize the expression of the rhesus HLA-E ortholog Mamu-E, particularly at the maternal-fetal interface. Mamu-E expression was confirmed by locus-specific RT-PCR in the placenta as well as in peripheral blood mononuclear cells (PBMC) and other organs. We evaluated the utility of antibodies recognizing HLA-E (MEM-E/06 against native HLA-E, MEM-E/02 against denatured HLA-E) to detect Mamu-E by flow cytometry/immunofluorescence, Western blot, and immunohistochemistry (IHC). Western blot analysis of cells and selected transfectants confirmed the recognition of Mamu-E but not Mamu-AG by antibodies MEM-E/06 and HC10 but not MEM-E/02. Immunohistochemical staining of frozen sections of rhesus placenta with the MEM-E/06 antibody demonstrated expression in most populations of rhesus monkey trophoblast cells, including villous cytotrophoblasts (strong positive staining), apical membrane of syncytiotrophoblasts (light to moderate staining) and extravillous cytotrophoblasts (moderate to strong staining, especially endovascular trophoblasts in early pregnancy). Expression was not trophoblast cell-specific, especially at term, when endothelial cells in both the chorionic plate and placental villi showed strong staining for Mamu-E. Staining of rhesus extravillous trophoblast cells suggested the co-expression of Mamu-E and Mamu-AG (the rhesus HLA-G homolog) on these cells. MEM-E/06 was shown also to react with differentiating rhesus placental syncytiotrophoblasts in primary culture, detecting intracellular and weak surface expression of Mamu-E. We conclude that the gestation-dependent co-expression of Mamu-E with Mamu-AG in villous and extravillous trophoblast cells suggests important and perhaps complementary but distinct roles of these two non-classical MHC class I loci in pregnancy at the maternal-fetal interface. In addition, the MEM-E/06 antibody will be useful for the detection of Mamu-E at the maternal-fetal interface in the rhesus monkey.
Asunto(s)
Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Placenta/inmunología , Animales , Femenino , Antígenos HLA/análisis , Antígenos de Histocompatibilidad Clase I/análisis , Macaca mulatta/inmunología , Modelos Animales , Embarazo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Antígenos HLA-ERESUMEN
Trophoblast cell lines are important research tools used as a surrogate for primary trophoblast cells in the study of placental function. Because the cellular origins of transformed trophoblasts are likely to be diverse, it would be of value to understand the unique and shared phenotypes of the cells on a global scale. We have compared two widely used cell lines, BeWo and JEG3, by microarray analysis in order to identify differentially expressed genes. Results indicated that approximately 2700 genes were differentially expressed between the cell lines, with principal differences observed in the biological processes of response to stress, cell adhesion, signal transduction, and protein and nucleobase metabolisms. These data suggest that BeWo and JEG3 cell lines, and perhaps other trophoblast cell lines, are sufficiently dissimilar from each other such that they will be differentially suited for specific experimental paradigms.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Trofoblastos/citología , Trofoblastos/fisiología , Línea Celular , Cartilla de ADN , Femenino , Humanos , Integrinas/genética , Placenta/citología , Placenta/fisiología , Reacción en Cadena de la Polimerasa , Embarazo , Proteínas/genética , Transcripción GenéticaRESUMEN
Leukocyte Ig-like receptors (LILRs) are a family of receptors that have inhibitory and activating functions and widely expressed by lymphoid and myeloid cells. Here we report the identification of the rhesus monkey LILRs by screening of rhesus spleen and decidua cDNA libraries and RT-PCR cloning. We obtained eight different full-length clones with structural and functional diversity similar to human LILRs, including LILRs with immunoreceptor tyrosine-based inhibitory motifs, LILRs with truncated cytoplasmic tails containing positively charged arginine residues in the transmembrane domain, and putative soluble receptors lacking transmembrane or cytoplasmic domains. Characterization of rhesus LILRs will facilitate use of this non-human primate model for the study of the functional role(s) of LILRs, including immune regulation through interaction with non-classical MHC class I molecules.
Asunto(s)
Macaca mulatta/genética , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Decidua/metabolismo , Femenino , Biblioteca de Genes , Leucocitos/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Bazo/metabolismoRESUMEN
We performed immunohistochemical analysis of DC-SIGN expression at the maternal-fetal interface at different stages of pregnancy in the rhesus monkey. Natural killer cells, monocytes and macrophages were observed in the nonpregnant endometrium, particularly in the luteal phase, and were increased in pregnant endometrium. No DC-SIGN+ cells were observed in the nonpregnant uterus. We observed decidual DC-SIGN+ cells within 1 week of implantation, and they increased in number during the first 5 weeks of gestation. DC-SIGN+ cells showed a clear differential distribution in the decidua in the first 2 weeks of pregnancy, being found only adjacent to the implantation site, in marked contrast to the widespread distribution of CD68+ macrophages and CD56+ NK cells throughout the endometrium. DC-SIGN+ cells also showed a more dendritic morphology than the general CD68+ cell population, and analysis of serial sections indicated an overlapping but not identical localization of these markers. Mature dendritic cells could not be detected as judged by total absence of immunostaining for CD83, CD86, DEC-205, or CD1a. DC-SIGN+ cells were defined as MHC class II+ and CD14+ by flow cytometry. We conclude that DC-SIGN expression is an early response by the primate maternal immune system to the implanting embryo. The selectively distributed population of DC-SIGN+ decidual leukocytes may represent a morphologically and phenotypically distinct subpopulation of decidual macrophages of early pregnancy that could contribute to the establishment of maternal-fetal immune tolerance.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Regulación de la Expresión Génica , Lectinas Tipo C/metabolismo , Macaca mulatta/fisiología , Intercambio Materno-Fetal/fisiología , Embarazo/fisiología , Receptores de Superficie Celular/metabolismo , Animales , Antígenos CD/metabolismo , Biomarcadores/análisis , Femenino , Feto/fisiología , InmunohistoquímicaRESUMEN
A novel cDNA was cloned from human endometrium, matching a human gene with the interim name KIAA1463. An mRNA identified by 5'-rapid amplification of cDNA ends was found to be 3349 nt in length. PCR analysis also identified another transcript of 6626 nt, with an open reading frame encoding a 900 amino acid protein. A fold recognition program identified similarity to firefly luciferase containing an AMP-binding motif; hence, we refer to the predicted protein as the AMP binding/luciferase-like protein (ALLP). ALLP mRNA and protein were expressed throughout the female reproductive tract with the highest levels found in the ovary and uterus. In situ hybridization and immunohistochemistry showed predominant localization of the ALLP mRNA/protein in endometrial glandular epithelium and within the theca and granulosa cells in the ovary. In the endometrium expression of ALLP, mRNA and protein were higher during d 16-21 of the secretory phase of the cycle. Western blot analysis showed decreased expression of ALLP in the postmenopausal endometrium, and hormone replacement therapy increased the expression of ALLP. Endometrial adenocarcinoma cell lines expressed more ALLP, compared with cultured primary endometrial cells or normal endometrial tissue. The ubiquitous expression of ALLP in reproductive and nonreproductive tissues suggests that this protein, which is probably regulated by ovarian steroids, plays an important metabolic role and may be involved in such processes as implantation and tumorigenesis.
Asunto(s)
Adenosina Monofosfato/metabolismo , Proteínas Portadoras/análisis , Genitales Femeninos/química , Luciferasas/análisis , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Endometrio/química , Femenino , Humanos , Luciferasas/química , Ciclo Menstrual , Datos de Secuencia Molecular , ARN Mensajero/análisisRESUMEN
The distribution of uterine leukocytes during the periimplantation period cannot be readily evaluated in human pregnancy. Using immunohistochemistry we examined the distribution of macrophages, natural killer (NK) cells, and T cells in the non-pregnant endometrium and in the decidua at early stages of implantation and pregnancy in the rhesus monkey. CD68+ macrophages, CD56+ lymphocytes and CD3+ T cells were present in the proliferative and secretory endometrium. The number of macrophages and CD56+ lymphocytes dramatically increased at implantation (day 14-15 of pregnancy) and continued to be high in early pregnancy decidua. Macrophages were conspicuously more numerous in proximity to implantation site (decidua basalis) as compared to sites peripheral to the developing placenta (decidua parietalis), and were found in close association with cytotrophoblasts adjacent to the decidua, as well as around arteries invaded by extravillous cytotrophoblasts. In contrast to macrophages, CD56+ lymphocytes were more evenly distributed throughout the decidua. Few CD3+ T cells were seen in pregnancy, being scattered in the endometrial stroma with occasional aggregate formation. The distribution of uterine leukocytes vis-à-vis trophoblasts at the rhesus monkey implantation site and in early pregnancy suggests different roles for macrophages and uterine NK cells in the response to trophoblast invasion.
Asunto(s)
Endometrio/citología , Células Asesinas Naturales/citología , Macaca mulatta/fisiología , Macrófagos/citología , Animales , Antígenos CD/metabolismo , Endometrio/metabolismo , Femenino , Técnicas para Inmunoenzimas , Células Asesinas Naturales/metabolismo , Macrófagos/metabolismo , Embarazo , Linfocitos T/citología , Linfocitos T/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismoRESUMEN
OBJECTIVE: Type I diabetes mellitus during pregnancy is associated with dysregulation of the oxygen and glucose metabolic pathways, both of which affect placental villous growth and function. Alteration of placental development in women with diabetes may contribute to the increased risk of preeclampsia, macrosomia, or fetal growth restriction. METHODS: To evaluate placental growth in the setting of maternal diabetes, immunohistochemical techniques were used to examine fibroblast growth factor-2 (FGF-2) expression, cell proliferation (Ki67), and apoptosis (Apo-Tag) in placentas from diabetic and nondiabetic patients. RESULTS: Immunostaining for FGF-2 in placentas from diabetic women demonstrated an increase in intensity within the villous stroma and syncytiotrophoblast (P<.05). Associated with these changes in FGF-2 expression, placentas from diabetic women showed no change in villous mitotic activity but did show decreased stromal compartment apoptosis. When expressed as a ratio of Ki67-positive:Apo-Tag-positive nuclei as an index of relative cell turnover, the stromal compartment showed a significant trend towards decreased nuclei turnover (P<.05), suggesting relative tissue growth in diabetic patients. CONCLUSION: Increased FGF-2 expression and decreased stromal cell compartment turnover in the diabetic placenta might be a compensatory mechanism in response to the altered physiologic milieu of maternal diabetes on placental function.
Asunto(s)
Apoptosis , División Celular , Diabetes Mellitus Tipo 1/metabolismo , Factor 2 de Crecimiento de Fibroblastos/análisis , Placenta/química , Embarazo en Diabéticas , Diabetes Mellitus Tipo 1/patología , Femenino , Edad Gestacional , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Antígeno Ki-67/análisis , Placenta/citología , EmbarazoRESUMEN
To reduce the number of animals required for controlled studies of marmoset oocytes and early embryos, a superovulation protocol was developed for the common marmoset. Females were given up to 50 i.u./day recombinant human follicle stimulating hormone (FSH)--(r-hFSH) for 6 days. Ovaries were visualized by a modified laparoscopic technique and follicular aspiration was performed using a needle and suction apparatus inserted directly through an otoscope speculum. The number of follicles + ovulation points (+/- S.E.) was 2.9 (+/- 0.2) in controls and 14.1 (+/- 1.6; P < or = 0.001) in the 50 i.u. r-hFSH per day animals. Oocytes, typically at the germinal vesicle stage at collection, extruded a first polar body within 26 hours. In vitro fertilization was performed and embryos developed to the hatched blastocyst stage (34%). With many high quality oocytes and the ability to synchronize cycles, the marmoset is a valuable primate model for examining nuclear reprograming and early embryonic events.
Asunto(s)
Callithrix/fisiología , Hormona Folículo Estimulante/farmacología , Ovario/efectos de los fármacos , Animales , Blastocisto/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Femenino , Fertilización In Vitro , Humanos , Masculino , Oocitos/efectos de los fármacos , Proteínas Recombinantes/farmacología , Superovulación/efectos de los fármacosRESUMEN
The transcriptional regulators aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) modulate the transcription of genes involved in cellular differentiation and proliferation. In this study, we investigated the expression of these transcriptional regulators in the female reproductive tract. AHR and ARNT mRNA transcripts were readily detected by a ribonuclease protection assay in all reproductive tissues examined. The expression of these factors in the endometrium and myometrium did not vary during the menstrual cycle, and was not different in pre- versus post-menopausal women. However, post-menopausal women on continuous hormone replacement therapy had greater expression of AHR but not of ARNT in the endometrium and myometrium when compared with women not taking hormones. Leiomyomas expressed significantly less AHR and ARNT mRNA compared with normal myometrium. The ovaries expressed both AHR and ARNT mRNA, and expression was unaffected by age. Endometriotic ovarian cysts expressed more AHR but not more ARNT mRNA compared with healthy ovarian tissue. However, there were no changes in the expression of AHR or ARNT mRNA in ovarian cancer. In conclusion, the female reproductive tract expresses mRNA for the transcription factors AHR and ARNT, and changes in their expression at select target sites in specific pathological conditions such as endometriosis and uterine leiomyomas suggest a potential role for these factors in the pathogenesis of these conditions.
Asunto(s)
Proteínas de Unión al ADN , Neoplasias de los Genitales Femeninos/metabolismo , Genitales Femeninos/metabolismo , Ovario/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Factores de Transcripción/metabolismo , Útero/metabolismo , Adulto , Anciano , Translocador Nuclear del Receptor de Aril Hidrocarburo , Endometrio/metabolismo , Trompas Uterinas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leiomioma/metabolismo , Ciclo Menstrual/fisiología , Persona de Mediana Edad , Miometrio/metabolismo , Neoplasias Ováricas/metabolismo , Ovario/fisiopatología , Placenta/metabolismo , Embarazo , Receptores de Hidrocarburo de Aril/genética , Distribución Tisular , Factores de Transcripción/genética , Neoplasias Uterinas/metabolismo , Útero/fisiopatologíaRESUMEN
Transgenic mice have provided invaluable information about gene function and regulation. However, because of marked differences between rodents and primates, some areas of human biology such as early embryonic development, aging, and maternal-fetal interactions would be best studied in a nonhuman primate model. Here, we report that gene transfer into rhesus monkey (Macaca mulatta) preimplantation embryos gives rise to transgenic placentas that express a reporter transgene (eGFP). Blastocysts resulting from culture of in vitro fertilized ova were transduced with a self-inactivating lentiviral vector and transferred into recipient females. One twin and one singleton pregnancy were produced from a single stimulation cycle, and one live rhesus monkey was born from each pregnancy. Placentas from all conceptuses showed expression of the transgene as detected by reverse transcription-PCR, ribonuclease protection assay, direct epifluorescence, immunohistochemistry, and Western blot analysis. Integration in somatic tissues of the offspring was not detected. A maternal immune response to the xenogeneic placental antigen was shown by the presence of anti-GFP antibodies in peripheral blood of the recipient females by day 99 of gestation (term = 165 days). These results demonstrate that transgene expression during gestation is compatible with successful pregnancy in nonhuman primates and provides an approach that could be broadly applicable to the development of novel models for primate biomedical research.
Asunto(s)
Desarrollo Embrionario/fisiología , Técnicas de Transferencia de Gen , Vectores Genéticos , Lentivirus , Placenta/metabolismo , Animales , Línea Celular Transformada , Femenino , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Macaca mulatta , Embarazo , Transgenes , Células Tumorales CultivadasRESUMEN
Embryo transfer in the rhesus monkey has been historically limited to transfer of cleavage stage embryos. In order to allow genetic manipulation of rhesus embryos in vitro, without using invasive surgical techniques, it is important to explore the transfer of morula and blastocyst stage embryos. Embryos were produced by in vitro fertilization from gonadotropin-stimulated monkeys, or were obtained by nonsurgical uterine flushing of naturally mated or artificially inseminated females. Nonsurgical transfer was accomplished by inserting a metal guide through the cervix into the uterus, after which a hollow cell sampler was inserted over the guide. The guide was removed and a catheter was inserted containing one to five embryos. Several pregnancies resulted from in vitro- and in vivo-derived blastocysts, and two pregnancies were carried to term resulting in one live birth. Blood samples were collected regularly to monitor plasma levels of chorionic gonadotropin, luteinizing hormone, and progesterone. The recipients received progesterone as a subcutaneous implant or daily injections from the day of transfer. The approach described in this study provides the opportunity to explore transgenic and chimeric models in the monkey by the development of noninvasive methods to transfer late-stage embryos that have been manipulated in vitro.