Background: Embryonic development proceeds through finely tuned reprogramming of the parental genomes to form a totipotent embryo. Cells within this embryo will then differentiate and give rise to ...all the tissues of a new individual. Early embryonic development thus offers a particularly interesting system in which to analyze functional nuclear organization. When the organization of higher-order chromatin structures, such as pericentromeric heterochromatin, was first analyzed in mouse embryos, specific nuclear rearrangements were observed that correlated with embryonic genome activation at the 2-cell stage. However, most existing analyses have been conducted by visual observation of fluorescent images, in two dimensions or on z-stack sections/projections, but only rarely in three dimensions (3D). Results: In the present study, we used DNA fluorescent in situ hybridization (FISH) to localize centromeric (minor satellites), pericentromeric (major satellites), and telomeric genomic sequences throughout the preimplantation period in naturally fertilized mouse embryos (from the 1-cell to blastocyst stage). Their distribution was then analyzed in 3D on confocal image stacks, focusing on the nucleolar precursor bodies and nucleoli known to evolve rapidly throughout the first developmental stages. We used computational imaging to quantify various nuclear parameters in the 3D-FISH images, to analyze the organization of compartments of interest, and to measure physical distances between these compartments. Conclusions: The results highlight differences in nuclear organization between the two parental inherited genomes at the 1-cell stage, i.e. just after fertilization. We also found that the reprogramming of the embryonic genome, which starts at the 2-cell stage, undergoes other remarkable changes during preimplantation development, particularly at the 4-cell stage.
The solubilization of somatostatin receptors from guinea‐pig pancreas by different non‐denaturing detergents was investigated after stabilization of the receptors by prior binding of ...125I‐Tyr11somatostatin or its analogue 125I‐Leu8, DTrp22, Tyr25somatostatin 28, to pancreatic plasma membranes.
The somatostatin‐receptor complexes were solubilized in a high yield by Zwittergent 3–14 (3‐tetradecyldimethylammonio‐1‐propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X‐100, Chaps (3‐cholamidopropyldimethylammonio‐1‐propanesulfonate) and octyl β‐D‐glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol.
In order to characterize solubilized somatostatin‐receptor comples, membranes receptors were covalently labelled using N‐5‐azido‐2‐nitrobenzoyloxysuccinimide as cross‐linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr= 93000 which interacted with the two radioligands.
In addition, a similar component of Mr= 88000 was characterized after analysis by SDS‐PAGE of membrane receptors covalently cross‐linked with 125I‐Leu8, DTrp22, Tyr25somatostatin 28 by different heterobifunctional reagents: N‐5‐azido‐2‐nitrobenzoyloxysuccinimide, N‐hydroxysuccinimidyl 4‐azidobenzoate, N‐succinimidyl 6‐(4′‐azido‐2′‐nitrophenylamino)hexanoate. Optimal cross‐linking results were obtained with N‐5‐azido‐2‐nitrobenzoloxysuccinimide.
The solubilized somatostatin‐receptor complex was adsorbed to wheat‐germ agglutinin‐agarose column and eluted by specific sugars.
We concluded that the guinea‐pig pancreatic somatostatin receptor in the membrane and in the non‐denaturing detergent solution behaves as a protein monomer of apparent Mr∼ 85000–90000. The somatostatin receptor is a glycoprotein which contains complex‐type carbohydrate chains.
PDF