Lipid Droplets Of Various Sizes Research Articles

Morphological Analysis of Human Milk Membrane Enclosed Structures Reveals Diverse Cells and Cell-like Milk Fat Globules

Over the past decade, the cellular content of human milk has been a focus in lactation research due to the benefit a potential non-invasive stem cell compartment could provide either to the infant or for therapeutic applications. Despite an increase in the number of studies in this field, fundamental knowledge in regard to milk cell identification and characterisation is still lacking. In this project, we investigated the nature, morphology and content of membrane enclosed structures (MESs) and explored different methods to enrich human milk cells (HMCs) whilst reducing milk fat globule (MFG) content. Using both flow cytometry and immunofluorescence imaging, we confirmed previous reports and showed that nucleated HMCs make up a minority of milk-isolated MESs and are indistinguishable from MFGs without the use of a nuclear stain. HMC heterogeneity was demonstrated by differential uptake of nuclear stains Hoechst 33258 and DRAQ5™ using a novel technique of imaging milk MESs (by embedding them in agar), that enabled examination of both extracellular and intracellular markers. We found that MESs often contain multiple lipid droplets of various sizes and for the first time report that late post-partum human milk contains secretory luminal binucleated cells found across a number of participants. After investigation of different techniques, we found that viably freezing milk cells is an easy and effective method to substantially reduce MFG content of samples. Alternatively, milk MESs can be filtered using a MACS® filter and return a highly viable, though reduced population of milk cells. Using the techniques and findings we’ve developed in this study; future research may focus on further characterising HMCs and the functional secretory mammary epithelium during lactation.

How great white sharks nourish their embryos to a large size: evidence of lipid histotrophy in lamnoid shark reproduction.

ABSTRACTThe great white shark (Carcharodon carcharias) exhibits viviparous and oophagous reproduction. A 4950 mm total length (TL) gravid female accidentally caught by fishermen in the Okinawa Prefecture, Southern Japan carried six embryos (543-624 mm TL, three in each uterus). Both uteri contained copious amounts of yellowish viscous uterine fluid (over 79.2 litres in the left uterus), nutrient eggs and broken egg cases. The embryos had yolk stomachs that had ruptured, the mean volume of which was approximately 197.9 ml. Embryos had about 20 rows of potentially functional teeth in the upper and lower jaws. Periodic acid Schiff (PAS)-positive substances were observed on the surface and in the cytoplasm of the epithelial cells, and large, secretory, OsO4-oxidized lipid droplets of various sizes were distributed on the surface of the villous string epithelium on the uterine wall. Histological examination of the uterine wall showed it to consist of villi, similar to the trophonemata of Dasyatidae rays, suggesting that the large amount of fluid found in the uterus of the white shark was likely required for embryo nutrition. We conclude that: (1) the lipid-rich fluid is secreted from the uterine epithelium only in early gestation before the onset of oophagy, (2) the embryos probably use the abundant uterine fluid and encased nutrient eggs for nutrition at this stage of their development, and (3) the uterine fluid is the major source of embryonic nutrition before oophagy onset. This is the first record of the lipid histotrophy of reproduction among all shark species.

Cross-validation of techniques for measuring lipid content of bovine oocytes and blastocysts

The main objective was to test and validate a fluorescence approach to quantify lipid content of individual bovine oocytes and blastocysts. For Experiment 1, denuded oocytes were evaluated, as well as in vitro-produced blastocysts in a factorial design: cows versus feedlot heifers; three additives during Days 2.5–7.5 of culture (Control; 10% FCS; 0.3 μM phenazine ethosulfate (PES), an electron acceptor that oxidizes NADPH); and two blastocyst stages (early versus expanded). All blastocysts were graded subjectively for darkness (1 = clear … 4 = dark). In Experiment 2, denuded oocytes were used to measure lipid content in a factorial design of: cows versus heifers and four subjective darkness grades (1 = clear … 4 = dark). To quantify lipids, oocytes and 7.5 d blastocysts were fixed and then stained with 1 μg/mL Nile Red dye in mPBS overnight. A digital photograph of the equatorial part of the oocyte and embryo was taken at 200×, and fluorescence intensity (Arbitrary Fluorescence Units, AFU) was measured with Image Pro software. Reverse images of the same photographs were used to count numbers of cytoplasmic lipid droplets of various sizes (LC). The linear regression equation of LC with AFU in oocytes had an r 2 = 0.84, and for blastocysts r 2 = 0.91. The LC and AFU also had similar coefficients of variation from the ANOVA for blastocysts (38 vs 44%, respectively). Treatment differences were of similar magnitude with both procedures: lipid content in oocytes and blastocysts from heifers and cows was similar (P > 0.1); PES reduced lipid accumulation, and FCS increased it relative to the Control for AFU (18.6 vs 46.6 vs 36.9 units, respectively), and LC (1763 vs 4081 vs 3310, respectively; all, P < 0.01). Early blastocysts resulted in more lipid accumulation per unit area than expanded ones based on AFU (41.5 vs 26.6) and LC (3519 vs 2583; both P <0.01). There was a strong relationship (P < 0.01) between subjective oocyte and blastocyst darkness and lipid content. The less labor intensive fluorescence staining was a reliable technique for quantifying lipid droplets in oocytes and blastocysts.

Epithelioid Variant of Pleomorphic Liposarcoma: A Comparative Immunohistochemical and Ultrastructural Analysis of Six Cases with Emphasis on Overlapping Features with Epithelial Malignancies

Pleomorphic liposarcoma (PL) is the least common subtype of liposarcoma, displaying a lipoblastic, malignant fibrous histiocytoma (MFH)-like and, less frequently, an epithelioid growth pattern. The epithelioid morphology in PL is still underrecognized and may closely simulate other epithelial neoplasms, mainly adrenal cortical carcinoma (ACC). No electron microscopic (EM) studies of the epithelioid variant of PL have been previously described, nor have there been studies of its immunoreactivity with A103 or f -inhibin. The purpose of this study is to analyze the histological, immunohistochemical, and EM features of epithelioid PL in an attempt to better explore the distinction from their epithelial mimickers, such as ACC. A panel of 5 antibodies was studied, including A103, f -inhibin, smooth muscle actin (SMA), AE1/AE3, and Cam 5.2. Out of 22 cases of PLs, 6 cases characterized by the presence of both epithelioid phenotype and pleomorphic lipoblasts were identified from the EM archives. There were 4 females and 2 males, with a mean age of 58 (range, 39-78). Two lesions arose in the thigh and 1 each in the abdominal wall, chest wall, anterior mediastinum, and retroperitoneum, with tumor size ranging from 7 to 17 cm (mean, 13 cm). Histologically, 2 PLs were pure epithelioid, whereas the other 4 had a mixed epithelioid and MFH-like appearance. Immunohistochemically, A103 (4/6), SMA (4/6), and AE1/AE3 (1/6) revealed a various degree of positive reactions. No immunolabeling for f -inhibin or Cam5.2 was detected in any case. By EM, the epithelioid areas revealed round or polyhedral cells with lipid droplets of various sizes and number, intimately apposed cell surfaces, occasional junction-like structures (4/6), and micropinocytotic vesicles (4/6). Interestingly, the ribosome-lamellar complexes, once thought to be characteristic of hairy cell leukemia but rarely seen in solid tumors, were noted in one pure epithelioid PL. When compared to the MFH-like area, rough endoplasmic reticula (RER) were less well developed, but mitochondria were more prominent in the epithelioid components. Neither mitochondria with tubulovesicular cristae nor prominent smooth endoplasmic reticula indicative of ACC were seen. Well-formed external lamina was not present. Other features to support a higher level of epithelial differentiation, such as lumen formation, microvilli, and tonofilaments, were not found. In conclusion, focal A103 reactivity in epithelioid undifferentiated tumors should be interpreted with caution before rendering the diagnosis of a primary or metastatic ACC, especially when examining biopsy specimens. The possibility of an epithelioid variant of PL must be excluded; f -inhibin can serve as a useful adjunct in this regard. In addition to variable intracytoplasmic fat droplets, the distinctive ultrastructural features of epithelioid variant of PL include numerous mitochondria, pinocytotic vesicles, junction-like structures, and, rarely, ribosome-lamellar complex. Despite some overlapping features, electron microscopy remains a useful tool to distinguish between epithelioid PL and ACC.

Ultrastructural Studies on the Uropygial Glands of Quail.

To investigate the processes of lipid synthesis and secretion of the uropygial gland of the adult male quail, glandular cells of the shallow and deep portions of the secretory tubule were observed ultrastructurally. All glandular cells in the shallow and deep portions displayed a lamellar structure, which was divided into basal, transitional and degenerating layers. In the shallow portion, some glandular cells in the transitional layer contained small lipid droplets, and others conspicuously large lipid droplets. Both cell types had a well-developed lamellar smooth endoplasmic reticulum (sER), and a well-developed Golgi apparatus. The former cells also contained sER with a lattice structure in the middle. In the degenerating layer, the glandular cells had many lipid droplets of moderate size and organellar structure had been lost due to autolysis. In the deep portion of the secretory tubule, the glandular cells of the transitional layer were filled with lipid droplets of various sizes which were surrounded by well-developed follicular or rod sER in which organella such as Golgi apparatus were also well developed. Rhombic crystalline structures were also observed. The glandular cells of the degenerating layer displayed a tendency to assemble lipid droplets of various sizes which compressed the nuclei to a stellate appearance. The structures of the sER and Golgi apparatus were well maintained, while the mitochondria were degenerated.These results suggest that lipid in the quail uropygial gland is synthesized in the sER and Golgi apparatus of glandular cells of the transitional layer, and particularly in the deep rather than the shallow portions.

Cyclic AMP-Receptor Protein Activity in Rat Preputial Cells

Cells from the rat preputial gland--a type of sebaceous gland--exhibited specific responsiveness of cyclic 3',5'-adenosine monophosphate (AMP) dependent protein kinase to stimulation by agents that elevate intracellular cyclic AMP. Electron microscopy shows that the rat preputial gland resembles the human sebaceous gland, not only in terms of containing a sebocyte-like population of cells in an acinar arrangement at different maturational stages, but also in the morphology of its organelles such as abundant and sometimes atypical mitochondria, many perinuclear lysosomes with crystalline inclusions, lipid droplets of various sizes, and peroxisomes. Other cell types, among them duct and inflammatory cells, were evident in the tissue sections, but constituted a minor component. Responses to stimulation of the adenylate cyclase-protein kinase pathways were determined using preputial cells that had been both freshly dispersed and grown in monolayer culture. Stimulation with isoproterenol (IPR) or forskolin (FS) resulted in both cases in an increase of cyclic AMP binding of the regulatory (R) subunits of cyclic AMP-dependent protein kinase, as determined by photoaffinity labeling of R subunits with an azido analog of cyclic AMP ([32P]-8-azido cyclic AMP). Cells from the epidermis under comparable conditions responded to a lesser degree and with a different distribution of R subunit isoforms. There are, therefore, differences in receptor activity as well as in the transduction pathways between the two types of epithelial cell populations. These results indicate that the preputial gland contains precursor cells that differentiate in culture to retain specific molecular mechanisms of action mediated via cyclic AMP.

Pathogenesis of Fatty Liver in Rats Fed A High Protein Diet Without Pyridoxine

Fatty liver induced in rats by a high protein diet without pyridoxine was studied morphologically. The microscopic change was characterized by accumulation of fat in hepatocytes of the centrolobular and midzonal areas. Electron microscopic examination at an early stage showed marked accumulation of small osmiophilic particles in the granular endoplasmic reticulum and vesicles throughout the cytoplasm with similar particles in the spaces of Disse. After 4 weeks, numerous lipid droplets of various sizes were seen in pericanalicular lysosomes in hepatocytes with concomitant increase in the triglyceride level. The droplets gradually formed larger droplets in the cytoplasm. After 8 weeks, myelin figures together with fat droplets were seen in continuity with the endoplasmic reticulum and occasionally crystal clefts were observed within lysosomes of hepatocytes. These findings suggest that development of fatty liver results from impaired lysosomal degradation of lipid.pyridoxine deficiency fatty liver myelin figure lysosome

Fine structural changes in the liver of young and old rats as influenced by microsomal enzyme inducers.

When given orally to young and old rats, pregnenolone-16 alpha-carbonitrile, spironolactone, or phenobarbital, known microsomal enzyme inducers, caused an increase in smooth endoplasmic reticulum. Dexamethasone, while a potent microsomal enzyme inducer, did not cause smooth endoplasmic reticulum increase. In both untreated and treated old rats, there was dilatation and vesiculation of rough endoplasmic reticulum with occasional granular material present in vesicles. Cytoplasmic lipid droplets of various sizes were frequent. Some mitochondria exhibited polymorphism and a variation in matrical density. Lysosomes and autophagic vacuoles as well as lipid droplets of various sizes were frequent in all groups. These results show that microsomal enzyme inducers influence the subcellular structure of hepatocytes in old rats.

セボレヤ症状ラットの肝臓および皮膚に関する組織学的観察

1. Degenerative changes occurred in the liver of rats administered with sperm oil, and the hepatic cells of the central part showed atrophy and necrosis. The blood corpuscles in capillaries around the hepatic central vein increased in number, indicating the symptoms of an early stage of congestive liver.2. In the rats with seborrhea induced by cotton seed oil, the symptoms of fatty liver were observed and numerous lipid droplets of various sizes were detected in the cytoplasm of hepatic cells around Glisson's sheath.3. The sebaceous glands were markedly hypertrophied and functionally stimulated. The lipid granules in the cells of these glands increased in size and number.

Development of the cytoplasmic membranous organelle in the endodermal cells of the yolk sac of the bat Tadarida brasiliensis cynocephala

The “membranous organelle” in the cytoplasm of the endodermal cells of the yolk sac develops directly from the rough endoplasmic reticulum (ER). The organelle is very small, but recognizable prior to mid-gestation when the endodermal cells first hypertrophy, and at this time it consists of only a few hexagonal channels. The membranes forming the walls of the channels are continuous with the rough ER, and mitochondria are usually in close proximity to the developing structure. Glycogen is observed within the channels of the organelle even at this early stage, though none may be present in the remainder of the cytoplasm. The structure remains quite inconspicuous until about a month before birth when it increases in size, becoming as large as the nucleus, and is the dominant structure in the cytoplasm. At the height of its development it surrounds numerous lipid droplets of various sizes, and glycogen is at all times present within the membranous channels.

ELECTRON MICROSCOPIC STUDIES ON CHOLESTEROL-INDUCED CEREBRAL ATHEROSCLEROSIS IN COCKERELS.

Cerebral atherosclerotic lesions in cockerels were studied with aid of light and electron microscopes. Twelve birds were given cholesterol in addition to the standard broiler feed. Twelve other birds serving as controls received the feed alone. The cholesterol-fed birds exhibited alterations in the endothelial cells of the leptomeningeal and brain tissue vessels. The endothelial cells showed vacuoles containing crescent-shaped lipid bodies and the subendothelial space contained a granular-fibrillary substance and lipid-filled macrophages. The contours of the basement membranes were somewhat granular. The internal elastic lamina occasionally seemed split and fragmentary and had lost its normal fibrillar structure. The collagen fibrils were more evenly distributed than in the vessel walls of the control group and followed a more irregular course, but the periodicity was normal. Vacuolation of the perinuclear parts of smooth muscle cells was rare. Especially in the hypothalamic region, small lipid-containing vacuoles were observed within the cytoplasm of the cells of the brain tissue proper. From the fact that some of the vacuoles were completely translucent and others contained electron-dense lipid droplets of various sizes, it was inferred that these may represent different stages of transformation of the chemical state of the lipids.

The fine structure of mesenchymatous cells in the regenerating forelimb of the adult newt Triturus

The mesenchymatous cells in the regenerating forelimb of the adult newt were examined with the electron microscope. Most of the cells have an obvious and voluminous rough-surfaced endoplasmic reticulum occurring as numerous elongated strands, as small and large vesicles, or as irregularly shaped dilations; in a small number of cells the reticulum is sparse. In general, the cells have large ovoid nuclei, occasional prominent nucleoli, and distinct double nuclear envelopes with pores. The outer membrane of the envelope makes frequent incursions into the cytoplasm to merge with elements of the endoplasmic reticulum. There are numerous lipid droplets of various sizes within the cytoplasm. The fine structure of the cells of the adult regenerate is compared with that of the larva, and the possible significance of the differences in terms of the developmental capacities of the cells is discussed.