Distribution Of Filipin-sterol Complexes Research Articles

Distribution of filipin-sterol complexes in sperm membranes from hypercholesterolaemic rabbits.

The distribution of membrane filipin-sterol complexes (FSC) was examined ultrastructurally in cauda epididymal sperm from normal and hypercholesterolaemic rabbits. Membrane FSC were quantitatively analysed on replicas of filipin-treated cells. We determined a significant difference in FSC concentration in the plasma membrane of the acrosome region (PMAR) of hypercholesterolaemic animals compared to normal rabbits. Hypercholesterolaemic animals had 0.56 +/- 0.05 FSC complex per micron 2 (enriched Cholesterol diet: Diet 2) in the marginal segment of PMAR; 0.62 +/- 0.05 FSC complex per micron 2 (enriched Cholesterol and fish oil diet: Diet 3) and only 0.28 +/- 0.01 FSC complex per micron 2 for normal animals (Control Diet 1). In the principal (anterior) segment we found 0.54 +/- 0.10 FSC complex per micron 2 (Diet 2), 0.56 +/- 0.03 FSC complex per micron 2 (Diet 3) and 0.30 +/- 0.04 FSC complex per micron 2 (Control Diet 1). We also counted 0.47 +/- 0.1 FSC complex per micron 2 in the equatorial segment of PMAR for Diet 2, 0.27 +/- 0.05 and 0.28 +/- 0.04 FSC complex per micron 2 in Diet 1 and Diet 3 respectively. Diet 4 (fish oil) did not differ from the control. An increase in the Cholesterol (Chol) level in biological membranes or a difference in the Chol membrane domains could cause a variation in the membrane rigidity that could modify the sperm membrane fusion capacity and functionality. The results presented in this paper are in agreement and could explain the decrease in the kinetic of the sperm acrosome reaction that we have observed in experimentally hypercholesterolaemic rabbits (Díaz-Fontdevila & Bustos-Obregón, 1992).

Changes in the distribution of filipin-sterol complexes in the boar sperm head plasma membrane during epididymal maturation and in the uterus.

The distribution of filipin-sterol complexes (FSCs) and intramembranous particles (IMPs) in the plasma membrane of the late spermatid of the boar and of the sperm obtained from the epididymides, ejaculates, and uterus 2 hours after mating was examined by a freeze-fracture replica technique. In the late spermatid, the FSC density was found to be very low. A majority of the FSCs in the acrosomal plasma membrane (APM) appeared as protuberances on the E face in the epididymal, ejaculate, and uterine sperm. The density of the FSCs in the principal segment (PS) of the APM was 291 +/- 44 FSC/microns2 (mean +/- standard deviation, S.D.), 322 +/- 41 FSC/microns2 and 355 +/- 31 FSC/microns2 in the caput, corpus, and cauda epididymidis, respectively. In comparison with the cauda epididymal sperm, the FSC density gradually decreased in the PS of the ejaculated (277 +/- 39 FSC/microns2) and uterine sperm (243 +/- 50 FSC/microns2). The reduction was especially remarkable in the equatorial segment (ES), where the density of FSCs in ejaculated and uterine sperm decreased to about half and less than half of that in the cauda epididymal sperm, respectively. Large (13 nm) and small (8 nm) IMPs were distributed evenly and densely in the P face of the APM in the late spermatid, epididymal, and ejaculated sperm. In the uterine sperm, IMP-free areas were observed in the P face of the plasma membrane, a feature thought to represent one of the capacitation changes of the boar sperm.

Freeze-fracture observations on changes in the distribution of Filipin-sterol complexes during epididymal maturation and capacitation of boar spermatozoa

It is believed that i ntramembra.nous sterols play an essential role in membrane stability and permeability. To investigate the distribution changes of sterols in sperm membrane during epididymal maturation and capacitation, filipin has been used as a cytochemical probe for the detection for membrane sterols. Using this technique in combination with freeze fracturing, we examined the boar spermatozoa under various physiological conditions.The spermatozoa were collected from: 1) caput, corpus and cauda epididymides, 2) sperm rich fraction of ejaculates, and 3)the uterus 2hr after natural coition. They were fixed with 2.5% glutaraldehyde in 0.05M cacodylate buffer (pH 7.4), and treated with the filipin solution (final concentration : 0.02.0.05%) for 24hr at 4°C with constant agitation. After the filipin treatment, replicas were made by conventional freeze-fracture technique. The density of filipin-sterol complexes (FSCs) was determined in the E face of the plasma membrane of head regions.

Distribution of filipin-sterol complexes in the unmyelinated nerve fibre

The filipin-sterol technique was employed, together with freeze-fracture, to investigate the fine structure of the unmyelinated nerve fibre in the peripheral nerve. No heterogeneity was observed in the distribution of filipin labelling either in the Schwann cell plasma membrane or along the axolemma. The distribution of labelling is contrasted with that in the myelinated nerve fibre and related to the relative morphology and electrophysiology of the two types of fibre.

Freeze-fracture localization of filipin-sterol complexes in plasma- and cyto-membranes of Pneumocystis carinii.

The polyene antibiotic, filipin, was used as the probe for demonstrating sterols in the freeze-fractured plasma- and cytomembranes of Pneumocystis carinii. The distribution of filipin-sterol complexes was homogeneous on the plasma membrane throughout all developmental stages from trophozoite to cyst; however, the density of the complexes gradually decreased with the progress of development. In the trophozoite, the density of the complexes was 485 +/- 42/micron2 on the P face and 341 +/- 27/micron2 on the E face. It was 249 +/- 50 on the P face and 132 +/- 48 on the E face in the precyst and 138 +/- 24 and 59 +/- 20, respectively, in the cyst. The membranes of nucleus, mitochondria, and small round bodies showed more or fewer complexes while no complexes were found in the membranes of one endoplasmic reticulum. In nuclear and mitochondrial membranes, some small scattered clusters of complexes were observed. Two types of vacuoles were distinguished: one having many complexes in its membrane and the other having none at all.

Filipin-sterol complexes at Schmidt-Lanterman incisures.

Employing the freeze-fracture technique, the distribution of filipin-sterol complexes was determined for membranes of peripheral nerve myelin. A heterogeneous distribution of complexes was observed with the greatest abundance on membranes associated with the cytoplasmic channels of Schmidt-Lanterman and longitudinal incisures. In addition there was an irregular network of well-labelled membrane bands in compact myelin. The results are related to a possible role for these channels and bands in the biochemical turnover of cholesterol in myelin.

Distribution of filipin-sterol complexes in the myelinated nerve fiber

Using filipin as a cytochemical probe to reveal the distribution of cholesterol, myelinated peripheral nerve fibers were examined in freeze-fracture replicas. Filipin-sterol complexes were most abundant in the Schwann cell and axonal plasma membranes. In the Schwann cell plasma membrane there was no heterogeneity in complex distribution in relation to the subjacent cytoplasmic network. In myelin lamellae there was a decrease in complexes from outer to inner lamellae and some aggregation of complexes in individual lamellae. The density of complexes in cytoplasmic organelles varied from absent in mitochondria to high in lysosome-like bodies. The results are interpreted in terms of the related biochemical composition and biophysical properties of cell membranes, with particular reference to the myelinated nerve fiber. The influence of diffusion barriers and gradients on the formation of complexes by filipin is considered.

Distribution of filipin-sterol complexes on cultured muscle cells: cell-substratum contact areas associated with acetylcholine receptor clusters.

Specialized areas within broad, close, cell-substratum contacts seen with reflection interference contrast microscopy in cultures of Xenopus embryonic muscle cells were studied. These areas usually contained a distinct pattern of light and dark spots suggesting that the closeness of apposition between the membrane and the substratum was irregular. They coincided with areas containing acetylcholine receptor clusters identified by fluorescence labeled alpha-bungarotoxin. Freeze-fracture of the cells confirmed these observations. The membrane in these areas was highly convoluted and contained aggregates of large P-face intramembrane particles (probably representing acetylcholine receptors). If cells were fixed and then treated with the sterol-specific antibiotic filipin before fracturing, the pattern of filipin-sterol complex distribution closely followed the pattern of cell-substratum contact. Filipin-sterol complexes were in low density in the regions where the membrane contained clustered intramembrane particles. These membrane regions were away from the substratum (bright white areas in reflection interference contrast; depressions of the P-face in freeze-fracture). Filipin-sterol complexes were also in reduced density where the membrane was very close to the substratum (dark areas in reflection interference contrast; bulges of the P-face in freeze-fracture). These areas were not associated with clustered acetylcholine receptors (aggregated particles). This result suggests that filipin treatment causes little or no artefact in either acetylcholine receptor distribution or membrane topography of fixed cells and that the distribution of filipin-sterol complexes may closely parallel the microheterogeneity of membranes that exist in living cells.

Occurrence and distribution of filipin-sterol complexes in chloroplast envelope membranes of algae and higher plants as visualized by freeze-fracture

The occurrence and planar distribution of 3-β-hydroxysterols in chloroplast envelope membranes of different algae and higher plants has been studied with the freeze-fracture technique using the polyene antibiotic filipin as cytochemical marker. The inner chloroplast envelope membrane in all organisms studied is devoid of filipin-sterol complexes. The outer chloroplast envelope membranes of isolated higher plant chloroplasts (spinach, pea) and of chloroplasts of the mossPolytrichum piliferum are lacking filipin-sterol complexes, thus indicating a very low concentration of 3-β-hydroxysterols in chloroplast envelope membranes of higher plants. In contrast filipin-sterol complexes are abundant in the outer chloroplast envelope membrane of the flagellatesChlamydomonas reinhardii, Cryptomonas erosa, andEuglena gracilis. The chloroplast-ER surrounding the plastid ofCryptomonas erosa also exhibits filipin-sterol complexes. Functional and phylogenetic aspects of these observations are discussed.