Projects George Posthuma

Technical Research

1) Preparation of non-embedded sections from High Pressure Frozen (HPF) tissues or cells for immunocytochemistry.
(E van Donselaar; G. Posthuma; Bruno Humbel (external advisor); J.W. Slot, (supervisor)

We follow two approaches:

The rehydration approach.
Samples are cryofixed by HPF, dehydrated during cryosubstitution in acetone in the presence of chemical fixatives. The fixed samples are then rehydrated and, after sucrose infusion, frozen again and cryosectioned as in our standard procedure. These sections are finally used for immunogold labeling.

Mouse chondrocyte. Cryosection prepared after the rehydration approach.

The section fixation approach.
Samples are cryofixed by HPF and cryosectioned at –150°C. The frozen sections are picked up and thawed in drops containing fixatives. After the sections are fixed, they are transferred to appropriate buffers and used for immunogold labeling as in our standard procedure.
This approach circumvents any dehydration step.

Mouse chondrocyte. Cryosectioning followed by section fixation.

2) Calibration of correlative live cell imaging and immune-EM.
(Oorschot et al, 2002. J. Histochem & Cytochem, 50, 1067-1080)

The method will been made applicable to various cells, antigens and fluorescent probes, and will be used to detect proteins at subcellular level at any given time during their transport itinerary in live cells.

Cell Biological Research

1) Quality of the oocyte in the ovarian follicle pool.
(Collaboration with Maarten Vinken, Dept. of Obstetrics & Gynaecology, University Medical Centre Utrecht, 3584 CX Utrecht, The Netherlands, and Prof E.te Velde
Dept. of Obstetrics & Gynaecology, Diakonessenhuis Utrecht, 3582 KE Utrecht, The Netherlands)

In western society the age at which women give birth to their first child has gradually increased. At the same time the chance of getting pregnant decreases after the age of 29. In this multidisciplinary research project, initiated by the Dept of Gynecology of the UMCU, an attempt is made to find parameters that describe the quality of resting and developing follicles. Both physiological parameters, like the vascularisation and hormone concentrations, as well as morphological parameters are taken in consideration. First we have investigated whether on the basis of morphological parameters a difference could be observed in biopsies from healthy women aged from 23 to 45. They were divided into two groups (23-32 and 38-45) and the oocytes were screened using conventional electon microscopical techniques for age related morphological changes. In elder women stereological analysis pointed out that the ooplasmic fraction of vacuoles was significantly increased whereas the number of mitochondria decreased. Furthermore, the mitochondrial matrix was less dense and the SER and Golgi complex were more dilated. We want to extend this study to immuno-electron microscopy and investigate whether oxygen radical scavengers like superoxide dismutase play a role in the ageing of ovarian follicles and their surrounding granulosa cells.

Ultrastructural differences found in the resting ovarian follicle pool in healthy young women.
1A) Primary follicle of good quality. The oocyte nucleus is large with a regular membrane; the mitochondria are clustered around the nucleus; and multivesicular bodies, lipid droplets, and vacuoles are absent. The granulosa cells have large nuclei with uncondensed chromatin and cytoplasm of low electron density, in which many mitochondria are present. Bar= 5 µm. 1B) Detail of 1A; mitochondria with intact membranes,transverse cristae, and a matrix of moderate electron density. Bar=300 nm. 1C) Detail of 1A; SER in flat tubular and vesicular form. Bar=200 nm. 2A) Intermediary follicle of poor quality. The membranes of the oocyte nucleus and the mitochondria are ruptured; numerous small vacuoles and a number of lipid droplets are present in the ooplasm. One granulosa cell shows condensation of chromatin on the nuclear membrane. Bar=5 µm. 2B) Detail of 2A; mitochondria with ruptured outer membranes, and the cristae are largely destroyed. Bar=250 nm. 2C) Detail of 2A; SER in swollen tubular and vesicular form. Bar=250 nm.

2) Amyloid formation in blood.
(Collaboration with Barend Bouma,Department of Medical Oncology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands)

In a number of diseases the normal way a protein is folded has changed. Due to the formation of so called cross-ß structures, the properties of such a protein changes and it is less susceptible to degradation and it is more likely to form larger protein aggregates called amyloid fibrils, which in their turn end up in plaques, which we know from Alzheimers disease and transmissible spongiform encephalopathies. This misfolding can be the result from a mutation in the DNA, or due to changes in the molecule later on. One of the alterations is the way a protein is folded after the addition of sugar moieties to an already finished protein (advanced glycation endproduct, AGE). The clearance of both amyloid proteins and protein-AGE adducts is performed by multiligand receptors, such as receptor for AGE, CD36, and Scavenger receptors A and B type I, and the serine protease tissue-type plasminogen activator (tPA).
When albumin, a major blood component, is exposed to relatively high sugar concentrations during its lifespan, glycation can alter its structure and the formation of fibrils is a fact. Using transmission electron microscopy we were able to demonstrate fibres consisting of glycated albumin. Further more circular dichroism spectropolarimetry confirmed the presence ß-sheets in these fibrils, which also reacted with Congo red (an amyloid specific dye). This would explain how an initially globular protein can bind to the same multiligand ‘cross-ß structure’ receptors and to tPA as earlier described for glycated ligands and amyloid ligands.

Albumin-carbohydrate adducts formed after incubation with glyceraldehyde/NaCNBH3. The majority of the albumin aggregates were globular, occasionally linear rods (arrowheads), ranging from 250-400 nm in length were observed. (Bar = 100 nm).