RESUMEN
This chapter provides an ImageJ/Fiji automated macro approach to remove the vitelline membrane autofluorescence in live Drosophila embryo movies acquired in a 4D (3D plus time) fashion. The procedure consists in a segmentation pipeline that can cope with different relative intensities of the vitelline membrane autofluorescence, followed by a developed algorithm that adjusts the extracted outline selection to the shape deformations that naturally occur during Drosophila embryo development. Finally, the fitted selection is used to clear the external glowing halo that, otherwise, would obscure the visualization of the internal embryo labeling upon projection or 3D rendering.
Asunto(s)
Embrión no Mamífero/diagnóstico por imagen , Imagenología Tridimensional/métodos , Microscopía Intravital/métodos , Membrana Vitelina/diagnóstico por imagen , Animales , Animales Modificados Genéticamente , Artefactos , Drosophila/embriología , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Desarrollo Embrionario , Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Imagenología Tridimensional/instrumentación , Microscopía Intravital/instrumentación , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Grabación en Video/instrumentación , Grabación en Video/métodos , Membrana Vitelina/química , Membrana Vitelina/embriologíaRESUMEN
In human pregnancy, abnormal placental hemodynamics likely contribute to the etiology of early-onset preeclampsia and fetal intrauterine growth restriction. The mouse is increasingly being deployed to study normal and abnormal mammalian placental development, yet the placental hemodynamics in normal pregnancy in mice is currently unknown. We used ultrasound biomicroscopy to noninvasively image and record Doppler blood velocity waveforms from the maternal and embryonic placental circulations in mice throughout gestation. In the uterine artery, peak systolic velocity (PSV) increased significantly from 23+/-2 (SE) to 59+/-3 cm/s, and end-diastolic velocity (EDV) increased from 7+/-1 to 28+/-2 cm/s in nonpregnant versus full-term females so that the uterine arterial resistance index (RI) decreased from 0.70+/-0.02 to 0.53+/-0.02. Velocities in the maternal arterial canal in the placenta were low and nearly steady and increased from 0.9+/-0.03 cm/s at embryonic day 10.5 (E10.5) to 2.4+/-0.07 cm/s at E18.5. PSV in the umbilical artery increased steadily from 0.8+/-0.1 cm/s at E8.5 to 15+/-0.6 cm/s at E18.5, whereas PSV in the vitelline artery increased from 0.6+/-0.1 cm/s at E8.5 to 4+/-0.2 cm/s at E13.5 and then remained stable to term. In the umbilical artery, the EDV detection rate was 0% at Asunto(s)
Circulación Sanguínea/fisiología
, Hemodinámica/fisiología
, Placenta/irrigación sanguínea
, Preñez/fisiología
, Cordón Umbilical/irrigación sanguínea
, Útero/irrigación sanguínea
, Membrana Vitelina/irrigación sanguínea
, Animales
, Arterias/diagnóstico por imagen
, Arterias/fisiología
, Velocidad del Flujo Sanguíneo
, Desarrollo Embrionario/fisiología
, Femenino
, Retardo del Crecimiento Fetal/etiología
, Retardo del Crecimiento Fetal/fisiopatología
, Ratones
, Microscopía Acústica
, Placenta/diagnóstico por imagen
, Placenta/fisiología
, Preeclampsia/etiología
, Preeclampsia/fisiopatología
, Embarazo
, Arterias Umbilicales/diagnóstico por imagen
, Arterias Umbilicales/fisiología
, Cordón Umbilical/diagnóstico por imagen
, Cordón Umbilical/fisiología
, Útero/diagnóstico por imagen
, Útero/fisiología
, Membrana Vitelina/diagnóstico por imagen
, Membrana Vitelina/fisiología
RESUMEN
Fractionation of vitelline envelope (VE) extracts from carp eggs made possible the efficient polymerization of a VE-like structure. The structure corresponded to the fourth layer of the VE or fertilization envelope (FE), and its organization was achieved by reassembly in vitro after solubilization of the sheets composed of filamentous substances or network-like aggregates which were induced by a cortical alveolus sialoglycoprotein or thrombin. The sialoglycoprotein was a serine proteinase and immunolocalized only in the structure at the periphery of cortical alveoli, not in the VE and yolk granules. Ultrastructural features of the VE-like structure suggested that reassembly in vitro occurred via several intermediates in the process of polymerization. A polyclonal antibody produced against one of the assembled VE components, a 64 kDa protein, more intensely immunostained the outer periphery of the VEs than other areas, and immunoelectron microscopy showed that immunogold particles specifically labeled reassembled VE-like structures and major skeletons of the networks or network-like sheets. The protein with a molecular weight of 64 kDa was found to be a DNase. Thus, these results suggest a new approach to investigating not only the FE assembly process in vitro but also the organizing relationship between the major skeleton of the VE or FE and other additional constituents.