Distribution Of Lipid Droplets Research Articles

Selection of <i>In Vitro</i>-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.

Induction of the fatty acid 2-hydroxylase (FA2H) gene by Δ(9)-tetrahydrocannabinol in human breast cancer cells.

To investigate gene(s) being regulated by ∆(9)-tetrahydrocannabinol (∆(9)-THC), we performed DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are poorly differentiated breast cancer cells, treated with ∆(9)-THC for 48 hr at an IC50 concentration of approximately 25 µM. Among the highly up-regulated genes (> 10-fold) observed, fatty acid 2-hydroxylase (FA2H) was significantly induced (17.8-fold). Although the physiological role of FA2H has not yet been fully understood, FA2H has been shown to modulate cell differentiation. The results of Oil Red O staining after ∆(9)-THC exposure showed the distribution of lipid droplets (a sign of the differentiated phenotype) in cells. Taken together, the results obtained here indicate that FA2H is a novel ∆(9)-THC-regulated gene, and that ∆(9)-THC induces differentiation signal(s) in poorly differentiated MDA-MB-231 cells.

Induction of 11β-HSD 1 and Activation of Distinct Mineralocorticoid Receptor- and Glucocorticoid Receptor-Dependent Gene Networks in Decidualizing Human Endometrial Stromal Cells

The actions of glucocorticoids at the feto-maternal interface are not well understood. Here, we show that decidualization of human endometrial stromal cells (HESCs) in response to progesterone and cAMP signaling is associated with a strong induction of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) expression and enzyme activity. Decidualization also triggered a gradual decrease in glucocorticoid receptor (GR) expression and reciprocal increase in mineralocorticoid receptor (MR) levels. Gene expression profiling of differentiating HESCs after small interfering RNA (siRNA)-mediated knockdown of either GR or MR identified 239 and 167 significantly regulated genes, respectively. Interestingly, GR-repressed genes were enriched for Krüppel-associated box domain containing zinc-finger proteins, transcriptional repressors involved in heterochromatin formation. In agreement, GR knockdown was sufficient to enhance trimethylated H3K9 levels in decidualizing cells. Conversely, we identified several MR-dependent genes implicated in lipid droplet biogenesis and retinoid metabolism. For example, the induction in differentiating HESCs of DHRS3, encoding a highly conserved enzyme that catalyzes the oxidation/reduction of retinoids and steroids, was enhanced by aldosterone, attenuated in response to MR knockdown, and abolished upon treatment with the MR antagonist RU26752. Furthermore, we demonstrate that decidualization is associated with dynamic changes in the abundance and distribution of cytoplasmic lipid droplets, the formation of which was blocked by RU26752. In summary, progesterone drives local cortisol biosynthesis by decidual cells through induction of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), leading to transcriptional regulation of distinct GR and MR gene networks involved in epigenetic programming and lipid and retinoid metabolism, respectively.

Accumulation of squalene is associated with the clustering of lipid droplets

The isoprenoid squalene is an important precursor for the biosynthesis of sterols. The cellular storage of squalene and its impact on membrane properties have been the subject of recent investigations. In a screen for abnormal lipid droplet morphology and distribution in the yeast Saccharomyces cerevisiae, we found significant lipid droplet clustering (arbitrarily defined as an aggregation of six or more lipid droplets) in a number of mutants (e.g. erg1) that are defective in sterol biosynthesis. Interestingly, these mutants are also characterized by accumulation of large amounts of squalene. Reducing the level of squalene in these mutants restored normal lipid droplet distribution. Moreover, inhibition of squalene monooxygenase in two mammalian cell lines (CHO-K1 and 3T3-L1) by terbinafine also resulted in lipid droplet clustering. These results indicate that the level of squalene may affect the growth and distribution of lipid droplets.

Coherent anti-Stokes Raman scattering and spontaneous Raman spectroscopy and microscopy of microalgae with nitrogen depletion

Microalgae are extensively researched as potential feedstocks for biofuel production. Energy-rich compounds in microalgae, such as lipids, require efficient characterization techniques to investigate the metabolic pathways and the environmental factors influencing their accumulation. The model green alga Coccomyxa accumulates significant amounts of triacylglycerols (TAGs) under nitrogen depletion (N-depletion). To monitor the growth of TAGs (lipid) in microalgal cells, a study of microalgal cells (Coccomyxa sp. C169) using both spontaneous Raman and coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy were carried out. Spontaneous Raman spectroscopy was conducted to analyze the components in the algal cells, while CARS was carried out to monitor the distribution of lipid droplets in the cells. Raman signals of carotenoid are greater in control microalgae compared to N-depleted cells. Raman signals of lipid droplets appear after N-depletion and its distribution can be clearly observed in the CARS microscopy. Both spontaneous Raman spectroscopy and CARS microscopy were found to be suitable analysis tools for microalgae.

Effect of Follicular Aging on the Nuclear Maturation and Distribution of Lipid Droplets in Bovine Oocytes.

Effect of Follicular Aging on the Nuclear Maturation and Distribution of Lipid Droplets in Bovine Oocytes.

Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets

Macroautophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by the autophagosome and delivered to the lysosome. Autophagosome formation is considered to take place on the endoplasmic reticulum and involves functions of autophagy-related (Atg) proteins. Here, we report the identification and characterization of mammalian Atg2 homologues Atg2A and Atg2B. Simultaneous silencing of Atg2A and Atg2B causes a block in autophagic flux and accumulation of unclosed autophagic structures containing most Atg proteins. Atg2A localizes on the autophagic membrane, as well as on the surface of lipid droplets. The Atg2A region containing amino acids 1723-1829, which shows relatively high conservation among species, is required for localization to both the autophagic membrane and lipid droplet and is also essential for autophagy. Depletion of both Atg2A and Atg2B causes clustering of enlarged lipid droplets in an autophagy-independent manner. These data suggest that mammalian Atg2 proteins function both in autophagosome formation and regulation of lipid droplet morphology and dispersion.

Lipid-rich blastomeres in the two-cell stage of porcine parthenotes show bias toward contributing to the embryonic part

This study was designed to determine the fate of the blastomeres in two-cell porcine parthenotes that display uneven size (larger vs. smaller) or cytoplasmic brightness (darker vs. brighter) during development to the blastocyst stage. For the non-invasive tracing of cell lineage, lipophilic fluorescence dye DiI (red) and DiD (blue) was randomly microinjected into each of two different blastomeres in each embryo. In blastocysts derived from the two-cell parthenotes with unevenly-sized blastomeres, no biased contribution was found in the progeny of either blastomere. However, in the blastocysts derived from the two-cell parthenote having different cytoplasmic brightnesses, the progeny of darker (more lipid-rich cytoplasm) blastomeres were more than two-fold more likely to form the embryonic part (43.6%; 17/39) than they were to form the abembryonic part (17.9%; 7/39), while the contribution of brighter blastomeres (less lipid) was just the opposite. The expressions of four marker genes involved in lineage allocation (Cdx2, Tead4, Oct4 and Carm1) were also analyzed in darker and brighter blastomeres of two-cell parthenotes using quantitative RT-PCR. The expression of Carm1 that encodes arginine methyltransferase 1 and that promotes inner cell mass (ICM) differentiation was significantly higher (P<0.05) in darker blastomeres. The ICM marker Oct4 also tended to be more highly expressed in the darker blastomeres, while Cdx2 and the TE marker Tead4 showed comparably higher expressions in the brighter blastomeres. However, in all cases, the marginal differences in the expression levels of Oct4, Cdx2 and Tead4 were not statistically significant (P>0.05). Our findings indicate that expression of genes related to early differentiation, especially Carm1, are partially associated with lipid droplet distribution in the two-cell porcine parthenote and may lead to biased embryonal axis formation.

Lipid droplets as fat storage organelles in Caenorhabditis elegans: Thematic Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man

Lipid droplets are evolutionarily conserved organelles where cellular fat storage and mobilization are exquisitely regulated. Recent studies have defined lipid droplets in C. elegans and explored how they are regulated by genetic and dietary factors. C. elegans offers unique opportunities to visualize lipid droplets at single-cell resolution in live animals. The development of novel microscopy techniques and protein markers for lipid droplets will accelerate studies on how nutritional states and subcellular organization are linked in vivo. Together with powerful tools for genetic and biochemical analysis of metabolic pathways, alteration in lipid droplet abundance, size, and distribution in C. elegans can be readily connected to whole-animal energy homeostasis, behavior, and life span. Therefore, further studies on lipid droplets in C. elegans promise to yield valuable insights that complement our knowledge gained from yeast, Drosophila, and mammalian systems on cellular and organismal fat storage.

Preliminary morphological and biochemical changes in rat liver following postnatal exposure to sodium arsenite

The effects of sodium arsenite exposure on the hepatic maturation period of cellular and functional reorganization in developing rat livers were evaluated. Animals received intraperitoneal injections of sodium arsenite (1.5 mg/kg body weight) or distilled water on days 9 to 28 after birth. On day 29, the animals were sacrificed either by cervical dislocation or by perfusion fixation. The perfusion fixed liver tissue was processed for paraffin embedding, sectioning and hematoxylin and eosin staining. The fresh liver tissue was processed for cryo-sectioning followed by Sudan Black B staining and for biochemical estimation of reduced glutathione. Microscopic observation revealed comparable preserved hepatic lobular patterns and distributions of uninucleate and binucleate hepatocytes in the control and the experimental groups. The mean nuclear area and diameter of the hepatocytes was increased in the experimental group. Lipid droplet distribution pattern in Sudan Black B stained sections revealed higher staining intensity towards the centrilobular area in both groups. Semiquantitative estimation of staining intensity showed lower mean gray values in zone 3 than in zones 2 and 1 (suggestive of the setting in of the adult pattern) in both groups. The reduced glutathione levels in the liver tissue and the altered nuclear size of the hepatocytes in the experimental group suggested the impairment of morphological and biochemical processes induced by arsenic exposure during the postnatal period.

Subcellular lipid droplet distribution in red and white muscles in the obese Zucker rat

Little is known about the subcellular distribution of lipids in insulin-resistant skeletal muscle. However, it has recently been suggested that lipid accumulation in the subsarcolemmal region directly contributes to insulin resistance. Therefore we hypothesised that regional differences in lipid distribution in insulin-resistant muscle may be mediated by: (1) a reduction in fatty acid trafficking into mitochondria; and/or (2) a regional increase in the enzymes regulating lipid synthesis. Transmission electron microscopy was used to quantify lipid droplet and mitochondrial abundance in the subsarcolemmal and intermyofibrillar compartments in red and white muscles from lean and obese Zucker rats. To estimate rates of lipid trafficking into mitochondria, the metabolic fate of radiolabelled palmitate was determined. Key enzymes of triacylglycerol synthesis were also determined in each subcellular region. Subsarcolemmal-compartmentalised lipids represented a small absolute fraction of the overall lipid content in muscle, as regardless of fibre composition (red/white) or phenotype (lean/obese), lipid droplets were more prevalent in the intermyofibrillar region, whereas insulin-resistant white muscles were devoid of subsarcolemmal-compartmentalised lipid droplets. While, in obese animals, lipid droplets accumulated in both subcellular regions, in red muscle of these animals lipids only appeared to be trafficked away from intermyofibrillar mitochondria, a process that cannot be explained by regional differences in the abundance of triacylglycerol esterification enzymes. Lipid accumulation in the subsarcolemmal region is not necessary for insulin resistance. In the intermyofibrillar compartment, the diversion of lipids away from mitochondria in insulin-resistant animals probably contributes to lipid accumulation in this subcellular area.

A role for seipin in lipid droplet dynamics and inheritance in yeast

Malfunctions of processes involved in cellular lipid storage and mobilization induce the pathogenesis of prevalent human diseases such as obesity, type 2 diabetes and atherosclerosis. Lipid droplets are the main lipid storage depots for neutral lipids in eukaryotic cells, and as such fulfil an essential function to balance cellular lipid metabolism and energy homeostasis. Despite significant progress in identifying key metabolic enzymes involved in lipid storage and their regulation in various model organisms, some fundamental questions as to the biogenesis, subcellular distribution and inheritance of lipid droplets are as yet unsolved. In this study, we applied a set of imaging techniques such as high-resolution four-dimensional (4D) live-cell imaging, quantitative microscopy, transmission electron microscopy and electron tomography to gain insight into the spatio-temporal organization of lipid droplets during cellular growth in the yeast Saccharomyces cerevisiae. This analysis revealed a high level of organization of the subcellular positioning of lipid droplets in individual cells, their directed migration towards the cellular periphery and a coordinated transfer of a subpopulation of lipid droplets into daughter cells during cell division. Lipid droplets appear to remain associated with ER membranes during cellular growth independently of their size and subcellular localization. Deletion of FLD1, the functional orthologue of the human BSCL2 gene encoding seipin, leads to impaired dynamics of yeast lipid droplets and defective lipolysis, which might be due to aberrant ER structures in these mutants. Our data suggest a role for yeast seipin as a scaffolding protein that is required for the dynamics of a specific subdomain of the ER, and provide a new aspect for the interpretation of abnormal lipid droplets phenotypes in yeast mutants lacking seipin.

Subcellular localization of skeletal muscle lipid droplets and PLIN family proteins OXPAT and ADRP at rest and following contraction in rat soleus muscle

Skeletal muscle lipid droplet-associated proteins (PLINs) are thought to regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN2 [adipocyte differentiation-related protein (ADRP)] is found only on lipid droplets, while PLIN5 (OXPAT, expressed only in oxidative tissues) is found both on and off the lipid droplet and may be recruited to lipid droplet membranes when needed. Our purpose was to determine whether PLIN5 is recruited to lipid droplets with contraction and to investigate the myocellular location and colocalization of lipid droplets, PLIN2, and PLIN5. Rat solei were isolated, and following a 30-min equilibration period, they were assigned to one of two groups: 1) 30 min of resting incubation and 2) 30 min of stimulation (n = 10 each). Immunofluorescence microscopy was used to determine subcellular content, distribution, and colocalization of lipid droplets, PLIN2, and PLIN5. There was a main effect for lower lipid and PLIN2 content in stimulated compared with rested muscles (P < 0.05). Lipid droplet distribution declined exponentially from the sarcolemma to the fiber center in the rested muscles (P = 0.001, r(2) = 0.99) and linearly in stimulated muscles (slope = -0.0023 ± 0.0006, P < 0.001, r(2) = 0.93). PLIN2 distribution declined exponentially from the sarcolemma to the fiber center in both rested and stimulated muscles (P < 0.0001, r(2) = 0.99 rest; P = 0.0004, r(2) = 0.98 stimulated), while PLIN5 distribution declined linearly (slope = -0.0085 ± 0.0009, P < 0.0001, r(2) = 0.94 rest; slope=-0.0078 ± 0.0010, P = 0.0003, r(2) = 0.91 stimulated). PLIN5-lipid droplets colocalized at rest with no difference poststimulation (P = 0.47; rest r(2) = 0.55 ± 0.02, stimulated r(2) = 0.58 ± 0.03). PLIN2-lipid droplets colocalized at rest with no difference poststimulation (P = 0.48; rest r(2) = 0.66 ± 0.02, stimulated r(2) = 0.65 ± 0.02). Contrary to our hypothesis, these results show that PLIN5 is not recruited to lipid droplets with contraction in isolated skeletal muscle.

Octylphenol affects morphology and steroidogenesis in mouse tumor Leydig cells

Exposure to xenoestrogens has been shown to cause adverse effects on reproductive function of various animal species. However, direct effects of xenoestrogens on Leydig cell function remain scarcely known. The aim of our study was to demonstrate the effects of 4- tert-Octylphenol (OP) on the expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and androgen receptor (AR) in MA-10 Leydig cells. Cells were treated for 3 and 12 h with OP (10 −4–10 −8 M). In cell cultures treated with high OP concentrations for 3 and 12 h morphological alterations were observed. Oil Red O staining revealed differences in lipid droplet size and distribution when compared to controls. Immunoreactive 3β-HSD was found in the cytoplasm, whereas immunoreactive AR was detected in the nuclei of both control and OP-exposed Leydig cells. A dose-related decrease in the expression of both proteins in OP-exposed cells was found. Qualitative results by immunocytochemistry and Western blot were confirmed by further quantitative analyses. Radioimmunological analysis revealed time-dependent secretion of progesterone (P 4) by control and OP-exposed Leydig cells. In 3 and 12 h OP-treated Leydig cells a significant decrease in P 4 level was found. Our study demonstrated a dose- and time-dependent action of OP on both morphology and steroidogenic function of MA-10 cells. It seems likely that OP besides acting via ER may mediate its action through AR modulating Leydig cell function.

Hypolipidemic Effect of n‐Butanol Extract from Asparagus officinalis L. in Mice fed a High‐fat Diet

During industrial processing of Asparagus (Asparagus officinalis L.), around half of each spear is discarded. However, these discarded asparagus (by-products) might be used as food supplements for their potential therapeutic effects. This study evaluated the hypolipidemic effect of n-butanol extract (BEA) from asparagus by-products in mice fed a high-fat diet (HFD). Continuous HFD feeding caused hyperlipidemia, oxidative stress and liver damage in mice. Interestingly, while BEA significantly decreased the levels of body weight gain, serum total cholesterol and low density lipoprotein cholesterol, it dramatically increased the high density lipoprotein level when administered at three different doses (40, 80 or 160 mg/kg body weight) for 8 weeks in hyperlipidemic mice. In addition, BEA decreased the levels of alanine transaminase, aspartate transaminase and alkaline phosphatase in serum. Finally, superoxide dismutase activity and the total antioxidation capacity were evidently increased, while the malondialdehyde level and the distribution of lipid droplets were reduced in liver cells of BEA-treated mice. Taken together, the findings of this study suggested that BEA had a strong hypolipidemic function and could be used as a supplement in healthcare foods and drugs or in combination with other hypolipidemic drugs.

Abstract 50: Comparison of NMR lipid profiles in mitotic arrest and apoptosis as indicators of drug resistance

Abstract Aim: To compare NMR-visible lipids in paclitaxel exposed cells undergoing apoptosis or mitotic arrest in order to explore their utility as a biomarker of drug resistance. Methods: Cultured cervical cell lines (HeLa, C33A and Me180, ATCC, USA) were exposed to 1 µM paclitaxel (Sigma, UK) for 8, 16, 24, and 48 hours (n=3). Diffusion-weighted (DW) spectra were acquired using a 11.74T spectrometer (Avance Bruker BioSpin, Germany) using a stimulated echo sequence with bipolar gradients. Cellular morphology was assessed with transmission electron microscopy (TEM) using uranyl acetate followed by lead citrate staining. Following TOPRO-3 and nile red co-staining, flow cytometry and confocal microscopy verified cell size and viability, visualized cell cycle phase distribution, apoptotic features, and accumulation of cytoplasmic lipid droplets. Western blots were used to assess activation (by phosphorylation) of cytoplasmic phospholipase A2 (P-cPLA2) and expression of fatty acid synthase (FAS) at each stage. Results: 24 h after exposure to paclitaxel, all lines showed &amp;gt;65% mitotic arrest, and HeLa showed apoptosis as well. At 48 h HeLa cells progressed to apoptosis (due to mitotic catastrophe) while C33A and Me180 cells progressed beyond mitotic arrest to normal morphology or multinucleation indicating resistance. There was significant increase in saturated and unsaturated lipids at 24 h (mitotic arrest) followed by a further striking increase at 48 h in all lines, especially in the 5.3ppm peak (unsaturated) in HeLa at 48 h. Unsaturated lipids increased more than saturated lipids in all lines, triglycerides increase the most in HeLa cells. Resistant lines showed lower increased methylene / methyl (1.3 ppm / 0.9 ppm) ratio (from 1.3 to 2.8, 1.7 to 2.8 compared with 1.5 to 3.8 in HeLa cells at 48 h). The levels of P-cPLA2 and FAS were unchanged at 24 h followed by a drop at 48 h in HeLa cells. The percentage of cells displaying lipid droplets increased significantly at 24 and 48 h in all lines with increase in droplet size only in HeLa cells at 24 and 48 h. This implies that the larger droplets are associated with apoptosis but that smaller ones are less specific. Apoptotic cells showed 3-4 x the number of droplets compared to cells arrested in mitosis. Conclusion: After exposure of cells to paclitaxel, increase in lipids on NMR results from increased visibility not synthesis (more droplets, FAS not increased). In apoptosis predominant increases are in unsaturated fatty acids in larger droplets, whereas paclitaxel resistant lines accumulate smaller droplets with significantly less triglycerides. Acknowledgements: This work was funded by the EC FP6 Marie Curie Action: Early Stage Training (contact No. 020718). We also acknowledge the support received for the CRUK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) (grant C1060/ A10334) and NHS Funding to the NIHR Biomedical Research Centre. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 50.

Hypolipidaemic and hepatoprotective effects of ethanolic and aqueous extracts from Asparagus officinalis L. by-products in mice fed a high-fat diet

Asparagus (Asparagus officinalis L.) by-products, i.e. the parts of the spears discarded during industrial processing, might have potential use as food supplements for their therapeutic effects. In this study the hypolipidaemic and hepatoprotective effects of ethanolic (EEA) and aqueous (AEA) extracts from asparagus by-products were evaluated in mice fed a high-fat diet (HFD). Continuous HFD feeding caused obvious hyperlipidaemia and liver damage in mice. However, both EEA and AEA significantly decreased the levels of body weight gain, serum total cholesterol and serum low-density lipoprotein cholesterol in hyperlipidaemic mice when administered at a daily dose of 200 mg kg(-1) for 8 weeks. Also, serum high-density lipoprotein cholesterol levels were evidently increased in the AEA-treated group. Moreover, both EEA and AEA dramatically decreased the activities of alanine and aspartate transaminases in serum. Finally, superoxide dismutase activity and total antioxidant capacity were increased and malondialdehyde level and the distribution of lipid droplets decreased in liver cells of both EEA- and AEA-treated mice. The findings of this study suggest that both EEA and AEA have strong hypolipidaemic and hepatoprotective properties and could be used as supplements in healthcare foods and drugs or in combination with other hypolipidaemic drugs.

Cloning and characterization of a new gene from the PAT protein family, in a marsupial, the stripe‐faced dunnart (Sminthopsis macroura)

Recent studies of PAT proteins in Drosophila and Xenopus have revealed significant roles for this family of proteins in the polarized transport of lipid droplets and maternal determinants during early embryogenesis. In mammals, PAT proteins are known to function mainly in lipid metabolism, yet research has yet to establish a role for PAT proteins in mammalian embryogenesis. Oocytes and early cleavage stages in Sminthopsis macroura show obvious polarized cytoplasmic distribution of organelles, somewhat similar to Drosophila and Xenopus, suggesting that a PAT protein may also be involved in S. macroura embryonic development. In the present study, we identified a new marsupial gene for PAT family proteins, DPAT, from S. macroura. Expression analyses by RT-PCR and whole mount fluorescent in situ hybridization revealed that DPAT expression was specific to oocytes and cleavage stage conceptuses. Analysis of the localization of lipid droplets during S. macroura early embryonic development found a polarized distribution of lipid droplets at the two- and four-cell stage, and an asymmetric enrichment in blastomeres on one side of conceptuses from two- to eight-cell stage. Lipid droplets largely segregate to pluriblast cells at the 16-cell stage, suggesting a role in pluriblast lineage allocation.

Ultrastructural Characteristics of Non‐matured and In Vitro Matured Oocytes Collected from Pre‐pubertal and Adult Domestic Cat Ovaries

The present study describes the ultrastructural characteristics of cat oocytes before maturation and after 12- and 24-h in vitro maturation (IVM). Oocytes were recovered from pre-pubertal and adult queen ovaries after ovariohysterectomy and a proportion were stored in glutaraldehyde at 4 degrees C until examination by transmission electronic microscopy (TEM). Those selected for maturation were cultured before TEM in DMEM for 12 and 24 h at 38 degrees C in a humidified environment of 5% O(2), 5% CO(2) and 90% N(2). Specimens were divided into six groups: non-matured oocytes from pre-pubertal queens (PP0), non-matured oocytes from adult queens (A0), 12-h in vitro matured oocytes from pre-pubertal queens (PP12), 12-h in vitro matured oocytes from adult queens (A12), 24-h in vitro matured oocytes from pre-pubertal queens (PP24) and 24-h in vitro matured oocytes from adult queens (A24). Across the treatment groups, it was possible to observe differences in the thickness of the perivitelline space, the penetration of cumulus cell projections forming a junctional complex, distribution and density of small vesicles, lipid droplets, microvilli, mitochondria and cortical granules and variable degrees of development of Golgi complexes. These findings demonstrated that ultrastructural analysis of oocytes matured in vitro is a valuable tool to evaluate oocyte cytoplasmic maturation and that this IVM protocol was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre-pubertal and adult cat oocytes.

The role of LMNA in adipose: a novel mouse model of lipodystrophy based on the Dunnigan-type familial partial lipodystrophy mutation

We investigated the role of LMNA in adipose tissue by developing a novel mouse model of lipodystrophy. Transgenic mice were generated that express the LMNA mutation that causes familial partial lipodystrophy of the Dunnigan type (FPLD2). The phenotype observed in FPLD-transgenic mice resembles many of the features of human FPLD2, including lack of fat accumulation, insulin resistance, and enlarged, fatty liver. Similar to the human disease, FPLD-transgenic mice appear to develop normally, but after several weeks they are unable to accumulate fat to the same extent as their wild-type littermates. One poorly understood aspect of lipodystrophies is the mechanism of fat loss. To this end, we have examined the effects of the FPLD2 mutation on fat cell function. Contrary to the current literature, which suggests FPLD2 results in a loss of fat, we found that the key mechanism contributing to the lack of fat accumulation involves not a loss, but an apparent inability of the adipose tissue to renew itself. Specifically, preadipocytes are unable to differentiate into mature and fully functional adipocytes. These findings provide insights not only for the treatment of lipodystrophies, but also for the study of adipogenesis, obesity, and insulin resistance.