卵母细胞脂肪酸及其对卵母细胞成熟和发育的影响

脂肪酸是一类具有重要生物学功能的营养物质,它们影响机体脂类的代谢、基因表达及细胞膜功能。而且,许多研究揭示,日粮脂肪酸浓度、种类及比例影响动物卵母细胞的质量。目前,利用代谢组学预测卵母细胞质量成为繁殖生物学研究的热点,但主要集中在葡萄糖、氨基酸代谢研究。动物卵母细胞含有大量的内源脂肪酸,但目前关于卵母细胞脂肪酸的代谢作用研究较少,而且脂肪酸还作为信号分子调控基因表达,同时也是生物膜的重要组成成分。基于此,该文综述了卵母细胞脂肪酸主要种类及来源、脂肪酸的生理作用及对卵母细胞成熟和发育的影响,为未来科学合理地调控卵母细胞脂肪酸组成和利用、促进卵母细胞成熟和发育,进而提高动物繁殖能力提供理论基础。     

猪卵母细胞玻璃化冷冻的研究进展

配子冷冻保存技术在动物繁殖育种中具有重要的意义,但猪卵母细胞的冷冻保存目前还很困难,主要表现为冻后继续发育能力低。这与影响卵母细胞玻璃化冷冻效果因素众多有关,如脂滴的存在使猪卵母细胞对冷冻非常敏感。冷冻保护剂的使用同时也产生了毒性作用。针对猪卵母细胞冷冻保存的特点,研究人员已研究出了一些新的方法来提高冷冻效果,如细胞骨架稳定剂的使用减少了冷冻对猪卵母细胞造成的损伤,通过改进冷冻载体提高了冷冻速率,从而提高了冷冻效果。     

低氧对脂肪细胞发育及脂质代谢调控研究进展

脂肪细胞的生长、分化与增殖贯穿整个生命过程,脂肪细胞中脂质代谢紊乱影响脂肪组织免疫和全身能量代谢。脂质代谢参与调控机体多种疾病的发生与发展,如高脂血症、非酒精性脂肪肝病、糖尿病和癌症等,对人和动物健康具有重大威胁。低氧诱导因子（hypoxia inducible factor,HIF）是介导机体组织器官中氧感受器的主要转录因子,HIF可调控脂质合成、脂肪酸代谢和脂滴形成并诱导疾病发生。但由于低氧程度、时间和作用方式的不同,对机体脂肪细胞发育和脂质代谢产生有害或有益的影响还无从定论。本文总结了低氧介导转录因子的调控作用以及对脂肪细胞发育和脂质代谢调控的研究进展,旨在揭示低氧诱导脂肪细胞代谢途径变化的潜在机制。     

脂滴膜转运蛋白Rab18与脂质代谢的关系研究

脂滴是动物细胞内储存脂质的一种亚细胞器。脂滴表面存在多种脂滴周围相关蛋白,参与脂质动态平衡的调节,防止脂质代谢异常的发生。其中,Rab18作为脂滴周围相关蛋白中的一种,在脂质代谢的调节、信号的转导、膜运输等多种生理功能中发挥重要作用。对于Rab18与脂质代谢之间关系的研究,为动脉粥样硬化、糖尿病、非酒精性脂肪肝及肥胖等多种代谢性疾病的防治发挥重要作用,本文就Rab18与细胞脂质代谢之间的关系研究作一综述。     

脂滴包被蛋白2在动脉粥样硬化中的作用

动脉粥样硬化性心血管疾病严重威胁着人类生命健康,其中脂质代谢异常和炎症反应是其重要的发病机制。脂滴是细胞内储存脂质的一种亚细胞器,其表面存在多种脂滴包被蛋白,参与调控脂质动态平衡。脂滴包被蛋白2(Plin2)作为脂滴包被蛋白的一种,在脂质代谢的调节、脂肪酸的氧化及炎症反应等多种生理功能中发挥重要作用。近年来,越来越多的研究发现Plin2在动脉粥样硬化的发生发展中扮演着重要的角色。因此,本文主要综述Plin2在胆固醇代谢、脂质合成、自噬和炎症反应等过程中发挥的作用,进一步阐述其与动脉粥样硬化之间的关系。     

脂滴与心力衰竭的研究进展

脂滴(LDs)是脂肪组织的基本单位,广泛存在于真核生物胞浆中,其表面覆盖以Perilipin 1(Plin1)为主的脂滴包被蛋白,在脂滴表面发挥屏障作用,保护脂滴内部甘油三酯及胆固醇酯免受胞浆中脂肪酶分解。近年来研究表明脂滴与心力衰竭关联密切,脂滴包被蛋白缺失可引起机体脂质代谢异常,进而引起心肌细胞结构改变,最终导致心力衰竭发生。这些研究为认识脂滴与心力衰竭间关系提供了依据,本文将脂滴与心力衰竭的研究进展进行归纳。     

骨骼肌的脂质代谢及其与胰岛素抵抗的研究进展

一系列的研究表明骨骼肌细胞内甘油三酯(intramyocellular triacyglycerol,IMTG)和胰岛素抵抗之间有密切联系。许多因素可调控IMTG的动态变化,如甘油三酯水解酶、激素敏感性脂肪酶、甘油一酯脂肪酶和脂滴结合蛋白等。而IMTG的代谢中间产物甘油二酯、神经酰胺和脂肪酸在骨骼肌中的聚集也与胰岛素抵抗密切相关。脂滴和线粒体对于细胞内甘油三酯及其代谢产物的周转速度起关键作用,对胰岛素抵抗也起着重要的作用。     

PAT家族蛋白在细胞内脂滴代谢过程中的作用

哺乳动物细胞内的甘油三酯是以脂滴的形式贮存的,现在有很多证据表明,脂滴参与多种代谢过程,因而被看作胞内有功能的细胞器。脂滴含有甘油三酯构成的脂质核心,脂核表面覆盖有单层磷脂,在单层磷脂内镶嵌着在结构上具有相关性的PAT家族蛋白,包括perilipin、ADRP、TIP47和S3—12。本文就这些蛋白在甘油三酯水解和脂滴合成中的调节作用加以综述。     

脂肪甘油三酯水解酶的研究进展

胰岛素抵抗等代谢疾病的发生与脂代谢紊乱密切相关。细胞中的脂肪主要储存在一个以中性脂为核的细胞器——脂滴(lipid droplet,LD)中。脂肪甘油三酯水解酶(adiposetriglyceride lipase,ATGL)是在脂滴上发现的水解甘油三酯的脂肪酶。除脂肪组织外,ATGL也广泛存在于骨骼肌等多种非脂肪组织中,并发挥着重要的生理功能。越来越多的研究表明,ATGL与中性脂质贮存异常、胰岛素抵抗等代谢疾病密切相关。运动可以通过改变ATGL的表达起到调控脂代谢的作用,进而在防治胰岛素抵抗等代谢疾病中发挥作用。     

Leptin过表达对猪前脂肪细胞脂滴形成的研究

研究Leptin过表达对猪前脂肪细胞脂滴形成的影响,旨为进一步研究Leptin与脂质代谢相关的分子机制奠定理论基础。选取Leptin过表达与野生型猪皮下脂肪组织,在无菌条件下分离前脂肪细胞进行传代培养并诱导分化形成脂滴,通过油红O和Bodipy染色后观察脂滴面积并分析脂质的含量,利用Q-PCR检测脂滴形成相关基因mRNA的表达水平。诱导4 d后可分化形成脂滴,油红O和Bodipy染色的结果显示,Leptin过表达猪前脂肪细胞脂滴数量和甘油三酯含量显著低于野生型(P0.05);且脂质合成相关基因PPARγ、SCAP、SREPB1和PLIN2的表达水平显著低于野生型(P0.01)。结果表明,过表达Leptin可促使猪前脂肪细胞中PPARγ、SREPB1、SCAP、PLIN2基因的表达下调,进而抑制脂滴形成。     

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.     

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 microg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.     

Oleic acid prevents detrimental effects of saturated fatty acids on bovine oocyte developmental competence

Mobilization of fatty acids from adipose tissue during metabolic stress will increase the amount of free fatty acids in blood and follicular fluid and, thus, may affect oocyte quality. In this in vitro study, the three predominant fatty acids in follicular fluid (saturated palmitic and stearic acid and unsaturated oleic acid) were presented to maturing oocytes to test whether fatty acids can affect lipid storage of the oocyte and developmental competence postfertilization. Palmitic and stearic acid had a dose-dependent inhibitory effect on the amount of fat stored in lipid droplets and a concomitant detrimental effect on oocyte developmental competence. Oleic acid, in contrast, had the opposite effect, causing an increase of lipid storage in lipid droplets and an improvement of oocyte developmental competence. Remarkably, the adverse effects of palmitic and stearic acid could be counteracted by oleic acid. These results suggest that the ratio and amount of saturated and unsaturated fatty acid is relevant for lipid storage in the maturing oocyte and that this relates to the developmental competence of maturing oocytes.     

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.     

Lipoprotein lipase (LPL) is highly expressed and active in the ovary of European sea bass (Dicentrarchus labrax L.), during gonadal development

The oocytes of many fish species accumulate high amounts of neutral lipids as a caloric reserve for embryonic and larval development. We propose that lipoprotein lipase (LPL, EC 3.1.1.34) plays an important role in supplying the oocytes with fatty acids and we have cloned its cDNA from the ovary of sea bass, and determined the patterns of LPL activity and LPL mRNA expression in the ovary. The cDNA obtained was 3051 bp long with an open reading frame encoding 518 amino acids. The amino acid sequence has a high similarity and shows similar structural features to LPL of other species. Northern blot analysis revealed LPL expression in adipose tissue and gonads only. LPL activity and LPL mRNA expression in the ovary was very high in fish with a gonadosomatic index (GSI) above 5, coinciding with the appearance of a high number of lipid droplets in the ooplasm. The LPL mRNA expression was localised to the follicle cells surrounding the oocyte. Our results suggest that LPL is likely to play an important role in the incorporation of neutral lipids into the oocytes, and that follicle cells, in addition to participating in steroidogenesis, also may be important in building up oocyte lipid reserves.     

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.     

Lipid analysis of immature pig oocytes

The detailed analysis of the lipid composition of immature pig oocytes represents the first such study carried out on mammalian eggs. In order to undertake a large scale lipid analysis using conventional extraction and chromatographic techniques a procedure for mass harvesting relatively large numbers of pig oocytes (200-300 oocytes/ovary) was developed. The study revealed that triacylglycerol was the major lipid component (100.71 nmol/mg protein) followed by cholesterol (32.71 nmol/mg protein). Phosphatidylcholine constituted the major phospholipid component (27.83 nmol/mg protein). Pig oocytes contained relatively low proportions of phosphatidylethanolamine (16.41% total phospholipid) and relatively high proportions of lysophosphatidylcholine (4.68% total phospholipid). The free fatty acid pattern was strikingly similar to the fatty acid composition of phosphatidylcholine. This observation, in conjunction with the observed high levels of lysophosphatidylcholine and the low ratio of phosphatidylethanolamine to phosphatidylcholine, suggests a fast rate of phospholipid turnover in the immature pig oocyte. Analysis of fatty acids esterified to the individual phospholipids and neutral lipids has shown that in all the classes examined, particularly in the neutral lipid fractions, there are high levels of the saturated fatty acid palmitic acid (16:0) and the monounsaturated fatty acid oleic acid (18:1). Triacylglycerol, free fatty acids and most of the phospholipids, particularly phosphatidylethanolamine, are considerably enriched in n-6 polyunsaturated fatty acids, specifically linoleic (18:2), arachidonic (20:4) and adrenic (22:4) acids. This may indicate an ability of oocytes to synthesize prostaglandins and leukotrienes. The results show that the lipid environment of the immature pig oocyte may be adapted to the highly specialized requirements of the cell, promoting growth and development with a potential role in the regulation of maturation.     

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.     

Raman spectroscopy evidence of lipid separation in domestic cat oocytes during freezing

Although lipid droplets are believed to play an important role in cryopreservation of mammalian embryos and oocytes, the effect of low temperatures on lipid droplets and related mechanisms of cryodamage are still obscure. Here, we provide Raman spectroscopy evidence of lipid separation inside the lipid droplets in domestic cat oocytes during slow freezing. It was shown that at −25 °C lipids coexist in two separated phase states inside lipid droplets. The scale of detected domains was a few micrometers size. We also found that under certain conditions these areas have a specific spatial distribution. Lipids with high melting temperatures are distributed near the surface of lipid droplets while fusible lipids are located deep inside. Raman spectroscopy was found to be a prospective approach to study inhomogeneity of lipid phase transition in cells and to reveal effects of this inhomogeneity on cryopreservation of biological cells.     

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.