New technique to quantify the lipid composition of lipid droplets in porcine oocytes and pre-implantation embryos using Nile Red fluorescent probe

The principal objective of this study was to develop a novel method based on confocal microscopy and a solvatochromic fluorescent dye, Nile red (NR) to quantify the main types of lipids in a single mammalian oocyte and embryo. We hypothesize that NR staining followed by the decomposition of NR-spectra identifies and quantifies the triglycerides, phospholipids, and cholesterol in a single oocyte and embryo. We analyzed the lipid droplets in porcine oocytes and pre-implantation embryos up to the hatched blastocyst stage developed in vivo and in cultured blastocysts. The emission spectrum of NR-stained mixture of different lipid types is a convolution of several component spectra. The principal component analysis (PCA) and a multivariate curve resolution-alternating least squares method (MCR-ALS) allowed to decompose the emission spectrum and quantify the relative amount of each lipid type present in mixture. We reported here that the level of the triglycerides, phospholipids and cholesterol in lipid droplets significantly decreases by 17.7%, 26.4% and 23.9%, respectively, from immature to mature porcine oocytes. The content of triglycerides and phospholipids remains unchanged in droplets of embryos from the zygote up to the morula stage. Then the triglyceride level decreases in the blastocyst by 15.1% and in the hatched blastocyst by 37.3%, whereas the amount of phospholipids decreases by 10.5% and 12.5% at the blastocyst and hatched blastocyst stages, respectively. In contrast, the content of cholesterol in droplets does not change during embryo cleavage. The lipid droplets in the blastocyst produced in vivo contain lower amounts of triglycerides (by 26.1%), phospholipids (by 14.2%) and cholesterol (by 34.8%) than those in the blastocyst cultured in NCSU-23 medium. In conclusion, our new technique is suitable to quantify the content of triglycerides, phospholipids and cholesterol in individual mammalian oocytes and embryos. Our findings indicate an important role for lipids during porcine oocyte maturation and early embryonic development, and suggest an altered lipid metabolism in cultured embryos.     

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 r² = 0.84, and for blastocysts r² = 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.     

Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.     

The storage of nutritional resources during vitellogenesis of Panstrongylus megistus (Hemiptera: Reduviidae): the pathways of lipophorin in lipid delivery to developing oocytes

In this work, we have analyzed the pathways by which lipophorin (Lp) delivers its lipid cargo to developing oocytes of Panstrongylus megistus, a hematophagous vector of Chagas’ disease. Lp, vitellin, total lipids and proteins were measured in ovarian tissues at different stages of the reproductive cycle. Localization of Lp in developing oocytes, mainly at their cortical area, was demonstrated by immunofluorescence assays using an anti-Lp antibody labeled with FITC. In vivo approaches injecting fluorescently labeled Lp to follow the course of the entire particle (Lp-DiI or Lp-Oregon Green) or its lipid cargo (Lp-Bodipy-FA) were monitored by laser scanning confocal microscopy. Significant increases in the amounts of lipids, proteins and vitellin were observed in ovarian tissue with the progress of vitellogenesis. Unexpectedly, an increase in the amount of Lp was also observed. The experiments in vivo demonstrated that the uptake of fluorescent Lp labeled on its protein or lipid moiety by developing oocytes occurred very fast, being impaired at low temperatures. The co-injection of fluorescent Lp and vitellogenin (Vg) showed that both particles co-localized inside yolk bodies, confirming the endocytic pathway for Lp. When the fate of lipids transferred to oocytes was evaluated in vitellogenic females by co-injecting Lp-Bodipy-FA and Lp-DiI, the signal for Bodipy-FA was found in both lipid droplets and yolk bodies. In contrast, in injected females kept at 4 °C the fluorescence was reduced, being observed exclusively in lipid droplets, implying that lipid transfer to the oocyte was diminished but not abolished. Taken together, the results demonstrate that in the hematophagous P. megistus, the storage of lipid resources by developing oocytes occurs by the convergence of different pathways by which Lp maximizes the delivery of its lipid cargo. In addition, it was also shown that, to some extent, lipids stored in the oocyte lipid droplets can also originate from endocytosed Vg. The relevance of these events in the context of the physiology of reproduction in P. megistus is discussed.     

Spectrofluorometric studies of the lipid probe, nile red

We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.     

Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development: Lipid component evolution

The objective of this study was to characterize the morphometry and ultrastructure of porcine preantral and antral follicles, especially the lipid component evolution. Ovarian tissue was processed for light microscopy. Ovarian tissue and dissected antral follicles (< 2, 2–4, and 4–6 mm) were also processed for transmission electron microscopy using routine methods and using an osmium-imidazole method for lipid detection. Primordial follicles (34 ± 5 μm in diameter, mean ± SD) had one layer of flattened-cuboidal granulosa cells around the oocyte, primary follicles (40 ± 7 μm) had a single layer of cuboidal granulosa cells around the oocyte, and secondary follicles (102 ± 58 μm) had two or more layers of cuboidal granulosa cells around the oocyte. Preantral follicle oocytes had many round mitochondria and both rough and smooth endoplasmic reticulum. In oocytes of primordial and primary follicles, lipid droplets were abundant and were mostly located at the cell poles. In secondary and antral follicles, the zona pellucida completely surrounded the oocyte, whereas some microvilli and granulosa cells projected through it. Numerous electron-lucent vesicles and vacuoles were present in the oolemma of secondary and antral follicles. Based on osmium-imidazole staining, most of these structures were shown to be lipid droplets. As the follicle developed, the appearance of the lipid droplets changed from small and black to large and gray, dark or dark with light streaks, suggesting that their nature may change over time. In summary, although porcine follicles and oocytes had many similarities to those of other mammalian species, they were rich in lipids, with lipid droplets with varying morphological patterns as the follicle developed.     

Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development: lipid component evolution

The objective of this study was to characterize the morphometry and ultrastructure of porcine preantral and antral follicles, especially the lipid component evolution. Ovarian tissue was processed for light microscopy. Ovarian tissue and dissected antral follicles (< 2, 2-4, and 4-6 mm) were also processed for transmission electron microscopy using routine methods and using an osmium-imidazole method for lipid detection. Primordial follicles (34 ± 5 μm in diameter, mean ± SD) had one layer of flattened-cuboidal granulosa cells around the oocyte, primary follicles (40 ± 7 μm) had a single layer of cuboidal granulosa cells around the oocyte, and secondary follicles (102 ± 58 μm) had two or more layers of cuboidal granulosa cells around the oocyte. Preantral follicle oocytes had many round mitochondria and both rough and smooth endoplasmic reticulum. In oocytes of primordial and primary follicles, lipid droplets were abundant and were mostly located at the cell poles. In secondary and antral follicles, the zona pellucida completely surrounded the oocyte, whereas some microvilli and granulosa cells projected through it. Numerous electron-lucent vesicles and vacuoles were present in the oolemma of secondary and antral follicles. Based on osmium-imidazole staining, most of these structures were shown to be lipid droplets. As the follicle developed, the appearance of the lipid droplets changed from small and black to large and gray, dark or dark with light streaks, suggesting that their nature may change over time. In summary, although porcine follicles and oocytes had many similarities to those of other mammalian species, they were rich in lipids, with lipid droplets with varying morphological patterns as the follicle developed.     

Application of nile blue and nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle

Using frozen sections from human muscle biopsies, we assessed the value of Nile blue and Nile red, two fluorescent probes, as stains for lipid droplets in normal and pathological skeletal muscle fibers. In normal muscle, lipid storage disorders, and mitochondrial myopathies, Nile blue stained the lipid droplets as yellow-gold fluorescent structures. The lipid droplets were also seen as yellow-gold fluorescent structures in Nile red-stained sections, but the outstanding feature in these preparations was the staining of the membrane network of the muscle fibers and membrane proliferations in pathological muscle as red-orange fluorescent structures. These results suggest that both Nile blue and Nile red stains are useful for visualization of lipid droplets and membrane proliferations in pathological muscle biopsies.     

脂质在卵母细胞发育过程中的双重作用

卵母细胞是雌性动物的生殖细胞,其质量决定雌性动物的繁殖能力。卵母细胞含有丰富的脂质,大部分以甘油三酯的形式储存在脂滴中。脂滴的大小、颜色以及分布模式与卵母细胞的发育能力相关。卵母细胞中甘油三酯可以脂解为脂肪酸,脂肪酸的β-氧化是卵母细胞和早期胚胎发育的重要能量来源。卵母细胞中脂质沉积过多会增加活性氧的含量(reactive oxygen species,ROS),并且破坏线粒体和内质网的功能,最终使卵母细胞的发育受阻。本文通过总结卵母细胞中脂质对卵母细胞发育的积极与消极两个方面的作用,为读者展示脂质在卵母细胞发育过程中的双重作用,让读者辩证地了解脂质对卵母细胞发育的影响。     

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.     

Lipid phase transitions in cat oocytes supplemented with deuterated fatty acids

Cryopreservation of oocytes has already been used to preserve genetic resources, but this technology faces limitations when applied to the species whose oocytes contain large amounts of cytoplasmic lipid droplets. Although cryoinjuries in such oocytes are usually associated with the lipid phase transition in lipid droplets, this phenomenon is still poorly understood. We applied Raman spectroscopy of deuterium-labeled lipids to investigate the freezing of lipid droplets inside cat oocytes. Lipid phase separation was detected in oocytes cryopreserved by slow-freezing protocol. For oocytes supplemented with stearic acid, we found that saturated lipids form the ordered phase being distributed at the periphery of lipid droplets. When an oocyte is warmed to physiological temperatures after cooling, a fraction of saturated lipids may remain in the ordered conformational state. The fractions of monounsaturated and polyunsaturated lipids redistribute to the core of lipid droplets. Monounsaturated lipids undergo the transition to the ordered conformational state below −10°C. Using deuterated fatty acids with a different number of double bonds, we reveal how different lipid fractions are involved in the lipid phase transition of a cytoplasmic lipid droplet and how they can affect cell survival. Raman spectroscopy of deuterated lipids has proven to be a promising tool for studying the lipid phase transitions and lipid redistributions inside single organelles within living cells.     

Nile red: a selective fluorescent stain for intracellular lipid droplets

We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.     

Identification of homologous binding proteins in porcine and bovine gametes

The purpose of this study was to determine if sperm and oocyte proteins that mediate plasma membrane interaction during mammalian fertilization are conserved among porcine and bovine gametes. We examined homologous and heterologous sperm and zona-free oocyte interactions to determine the extent of cross-reactivity between the gametes of these two ungulate species. First, the numbers of ejaculated porcine and bovine sperm bound to the oocyte plasma membrane of intact porcine and bovine oocytes were determined in vitro. There was no significant difference between the number of porcine or bovine sperm that bound to porcine or bovine oocytes (P > 0.25). Second, individual porcine and bovine sperm plasma membrane proteins were identified by binding of homologous or heterologous oocyte plasma membrane to whole sperm plasma membrane on Western ligand blots. The relative amount of labeled oocyte plasma membrane bound to individual sperm plasma membrane proteins was analyzed by laser densitometry. Eight porcine sperm plasma membrane proteins and seven bovine sperm plasma membrane proteins were bound by both porcine and bovine oocyte plasma membrane. A significantly greater relative amount of porcine oocyte plasma membrane than bovine oocyte plasma membrane was bound to the 14- and 10-kD porcine sperm plasma membrane proteins (P < 0.001 and P < 0.01, respectively). A 27-kD bovine sperm plasma membrane protein bound proportionally more bovine oocyte plasma membrane probe than porcine oocyte plasma membrane probe (P < 0.04). These results are consistent with conservation of similar receptor ligand interactions at the gamete plasma membrane among porcine and bovine gametes.     

Application of the standard addition method for the absolute quantification of neutral lipids in microalgae using Nile red

Microalgae are considered one of the best candidates for biofuel production due to their high content in neutral lipids, therefore, an accurate quantification of these lipids in microalgae is fundamental for the identification of the better candidates as biodiesel source.Nile red is a fluorescent dye widely employed for the quantification of neutral lipids in microalgae. Usually, the fluorescence intensity of the stained samples is correlated to the neutral lipid content determined with standard methods, in order to draw a standard curve and deduce the neutral lipids concentration of the unknown samples positioning their fluorescence intensity values on the curve.Standard methods used for the neutral lipids determination are laborious and often implying solvent extraction and/or other transformation (i.e. saponification or transesterification) of the sample. These methods are also time consuming and may give rise to an underestimation of the lipid content due to variable extraction yields.The approach described in this paper combines the standard addition method and the fluorometric staining using Nile red, avoiding the association of traditional neutral lipids quantification methods to the fluorometric determination. After optimization of instrument parameters and staining conditions, a linear correlation between the fluorescence intensity of each sample stained with the Nile red and its neutral lipids content deduced with the standard addition method was identified. The obtained curve allowed the direct determination of neutral lipids content maintaining a linearity range from 0.12 to 12 μg of neutral lipids per ml of sample, without need of pre-concentration. This curve was then used in the quantification of the neutral lipids content in culture of Skeletonema marinoi (Bacillariophyceae) at different days from the inoculum. This method was also successfully applied on Chaetoceros socialis (Bacillariophyceae) and Alexandrium minutum (Dinophyceae).     

Changes of lipid composition in non-cultured and cultured porcine embryos

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.     

Lipid Content and Composition during the Oocyte Development of Two Gorgonian Coral Species in Relation to Low Temperature Preservation

Our previous studies have suggested that chilling sensitivity of coral oocytes may relate to their relatively high lipid intracellular content and lipid composition. The distribution of lipids during the oocyte development was determined here for the first time in two gorgonian species (Junceella juncea and Junceella fragilis). The main lipid classes in the two gorgonian oocytes were total lipid, wax ester, triacylglycerol, total fatty acid, phosphatidylethanolamine and phosphatidylcholine. The results indicated that early stage oocytes of J. juncea and J. fragilis were found to have increased lipid content than late stage oocytes. The content of wax ester was significantly higher in the early stage oocytes of two gorgonian corals (51.0±2.5 and 41.7±2.9 μg/mm(3)/oocyte) than those of late stage oocytes (24.0±1.4 and 30.4±1.2 μg/mm(3)/oocyte, respectively). A substantial amount of phosphatidylethanolamine and total fatty acid was detected at each stage of oocyte development in two gorgonian ranges from 107 to 42 μg/mm(3)/oocyte and 106 to 48 μg/mm(3)/oocyte, whilst low levels of phosphatidylcholine were found in two gorgonian oocytes. The levels of total lipid in the late stage oocytes of J. juncea were significantly higher than those of J. fragilis. The observed differences may partially be related to different habitat preferences as higher lipid levels in J. juncea, a deeper-water coral species exposed to lower temperature seawater, might relate to adjustments of cell membranes in order to increase membrane fluidity.     

Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence

A rapid estimation method of the intracellular lipid content in microorganisms using a fluorescent probe, Nile red, was established by optimization of the Nile red staining and data processing. The protocol was designed to be applicable to a wide range of microorganisms and culture conditions. In the optimized procedure, cells diluted with buffer were stained with 0.24-0.47 microg/ml of Nile red for 5 min, and the fluorescent emission spectra in the wavelength region of 400 to 700 nm excited at 488 nm were acquired before and after the Nile red addition. The fluorescence intensity corresponding to the intracellular lipid amount was determined at the peak of the corrected spectrum. The value showed a linear relation with the lipid content of various oleaginous fungi and yeasts measured by the conventional method. The relative intensities against the unit lipid amounts were almost similar except for one yeast. For the application to mycelia forming various types of pellets, a simple and easy pretreatment of shaking with glass beads for 5-10 min was added to the protocol. The established method was applicable to estimate the lipid content of a wide range of microorganism cultures containing 2-5000 microg-lipid/ml-broth.     

Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes

We determined the incidence of activation, male pronuclear formation, and apposition of pronuclei in porcine oocytes following intracytoplasmic injection of various porcine sperm components and foreign species spermatozoa, such as that of cattle, mouse or human. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. In contrast, injection of either sperm tail or a trypsin- or NaOH-treated sperm head failed to induce oocyte activation. Because injection of mouse, bovine, or human spermatozoon activated porcine oocytes, the sperm-borne activation factor(s) is not strictly species-specific. Male pronuclear formation and pronuclear apposition were observed in porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. Electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation or pronuclear apposition compared with sperm cell injection alone (P > 0.1). Following porcine sperm injection, the microtubular aster was organized from the neck of the spermatozoon, and filled the whole cytoplasm. In contrast, following injection of bovine, mouse, or human spermatozoon, the maternal-derived microtubules were organized from the cortex to the center of the oocytes, which seems to move both pronuclei to the center of oocytes. Cleavage to the two-cell stage was observed at 19-21 hr after injection of porcine spermatozoon. However, none of the oocytes following injection of mouse, bovine, or human spermatozoa developed to the mitotic metaphase or the two-cell stage. These results suggested that the oocyte activating factor(s) is present in the perinuclear material and that it is not species-specific for the porcine oocyte. Self-organized microtubules seemed to move the pronuclei into center of oocytes when foreign species spermatozoa were injected into porcine oocytes.     

Temporal and spatial assembly of lipid droplet-associated proteins in 3T3-L1 preadipocytes

This study aimed to investigate the relationship between newly formed lipid droplets and lipid droplet surface proteins, including perilipin, adipose differentiation related protein (ADRP), and p200 kDa protein (p200) in 3T3-L1 preadipocytes, during lipogenesis. Sterol ester was used to induce nascent lipid droplets in 3T3-L1 preadipocytes and the sequence of lipids and lipid droplet surface proteins was studied using a combination of immunohistochemistry and Nile red staining/Oil red O. We demonstrated that, although most growing lipid droplets appeared to have a lipid core surrounded by a fluorescent rim of ADRP, perilipin, and p200, tiny protein aggregates of ADRP, perilipin, or p200 could also be found to occur in the absence of lipid accumulation. In addition, ADRP associated with nascent lipid droplets prior to that of perilipin or p200. We provide evidence that lipid droplet surface proteins, especially ADRP and perilipin, are important in serving as a nucleation center for the assembly of lipid to form nascent lipid droplets.     

Immunocytochemistry of the Microtubules of fat-laden cells. Brown fat cells and adrenocortical cells in primary monolayer culture

Cytoplasmic microtubules of fat-laden cells containing fine lipid droplets, such as brown fat cells and adrenocortical cells, were studied in relation to the metabolism of intracellular lipid. In these cells, the amount and distribution of lipid droplets reflect the state of inherent cellular function. Materials used were primary monolayer culture of fetal rat brown fat cells and that of bovine adrenocortical cells. The method was the immunocytochemistry with anti-tubulin antibody. When brown fat cells were being lipolyzed or the steroidogenesis of adrenocortical cells were being stimulated, the cytoplasmic microtubules in the cells were organized in a radial pattern in response to the behavior of the lipid droplets. It is assumed that the microtubules were in the regulation of cellular function in terms of the metabolism of lipid droplets in these cells. We have devised, in the course of the current study, a double fluorescence technique as an observational method whereby microtubules were observed immunocytochemically and lipid droplets by a secondary fluorescence with phosphine E staining.