M. Romek

Lipid content of non-cultured and cultured pig embryo.

The objectives of the study were: (i) to work out a precise and efficient method for quantitative analysis of lipid content and (ii) to quantitatively determine the lipid content in non-cultured and cultured pig embryos. The experiment was carried out on pig embryos from zygote to late blastocyst stages produced in vivo and embryos collected at the zygote stage and then cultured in vitro up to blastocyst stage. Embryos were fixed, dehydrated, embedded in epoxy resin and cut into semi-thin sections to analyse the quantity of lipids in fat droplets. Stained sections were then analysed with Cavalieri and point counting methods to evaluate the following stereological parameters of the embryo: total embryo volume - V(e), volume density of cytoplasm per unit volume of embryo - Vv(c,e), volume density of lipid droplets per unit volume of embryo cytoplasm - Vv(fat,c) and total volume of lipid droplets per whole embryo - V(fat). Values of Vv(fat,c) and V(fat) remained unchanged up to the morula stage, but decreased significantly at blastocyst and late blastocyst stages both in cultured and non-cultured embryos. Volume density of lipid droplets per unit volume of embryo cytoplasm and total volume of lipid droplets for cultured embryos showed statistically significant differences between late blastocyst and almost all other stages. Comparisons of Vv(fat,c) in embryos at the same stages of development but differing in origin of embryos (non-cultured or cultured) show that statistically significant differences exist for all analysed stages. In conclusion, differences in lipid content observed in pig embryos were dependent on the developmental stage of the embryo as well as the culture conditions (i.e. cultured and non-cultured embryos at the same stage of development).

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.

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.

Mitochondrial activity and morphology in developing porcine oocytes and pre-implantation non-cultured and cultured embryos.

Mitochondria are important determinants of developmental competence for oocytes and embryos owing to their central role in cellular metabolism, yet mitochondrial activity and morphometry during early porcine development have not been quantified. In this study, we examined the membrane potential Δψ(m) and the surface density Sv(in,m) of the inner mitochondrial membrane in pig oocytes and pre-implantation embryos using fluorescent probes and confocal microscopy. Mitochondria and their cristae were also examined by transmission electron microscope. Δψ(m) was consistently low from immature oocytes up to morulae and increased significantly in the early blastocyst before decreasing at the expanded blastocyst stage. This stage-dependent pattern of Δψ(m) changes differs from that reported for other mammals. We also determined that Δψ(m) is lower in cultured when compared to non-cultured porcine early blastocysts. Sv(in,m) was higher in immature oocytes than mature oocytes and remained constant up to the 4- to 8-cell embryo stage. It increased significantly at morula and early blastocyst stages. No differences in Sv(in,m) were found between developmentally matched non-cultured and cultured embryos. These results indicate that the inner mitochondrial membrane potential and surface density change significantly during pre-implantation porcine development in relation to metabolic alterations of the embryo. It is possible that modification of Δψ(m) by manipulating culture conditions may improve the performance of embryos that develop in vitro.

Silicone-stabilized liposomes as a possible novel nanostructural drug carrier.

Development of silicone stabilized liposomes which can serve as novel drug nanocarriers is presented. Silicone precursor 1,3,5,7-tetramethylcyclotetrasiloxane (D4(H)) was introduced into the bilayer of the cationic liposomes prepared from egg yolk phosphatidylocholine (PC) and double-tailed dimethyldioctadecylammonium bromide (DODAB). The silicone material was created inside of the liposomal bilayer in the base-catalyzed polycondensation process of the D4(H) what was confirmed employing (29)Si solid-state MAS NMR and FTIR measurements. Surfactant lysis experiments revealed that resulted systems can be effectively stabilized. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements demonstrated that the silicone-stabilized liposomes have typical lipid vesicles morphology and mean hydrodynamic diameters in the range of about 110nm. They have considerably lower tendency for aggregation than the pristine liposomes. The permeability of vesicles can be tuned by introducing various amounts of silicone precursor into the liposome bilayer, as confirmed in calcein-release studies. The effect of fetal bovine serum (FBS) on the stability of liposomes was also tested in in vitro studies. Biological studies revealed that resulted liposomes can be considered as possible drug nanocarriers because they are not toxic to human skin fibroblasts (HSFs) and mouse embryonic fibroblasts (MEFs).

Quantification of Connexin43 Gap Junctions in Porcine Myometrium by Confocal Microscopy and Stereology

The aim of this study was to quantify the number and size of connexin43 (Cx43) gap junctions in the circular and longitudinal layers of myometrium of the non-pregnant pig. We developed a novel approach to measure the mean surface area (s), numerical density (N(v)) and surface density (S(v)) of gap junctions using confocal microscopy and stereological analysis. Immunolabelled Cx43 gap junctions were measured in the subendometrial and deep regions of the circular layer and in the longitudinal layer of the myometrium of pre-pubertal pig and mature pig at pre-ovulatory and secretory stages of the oestrous cycle. In the circular subendometrial region, all investigated stereological parameters of Cx43 gap junctions (s, N(v) and S(v)) were significantly higher (p<0.05) than those of the circular deep region and the longitudinal layer in all three groups of animals. These results indicate the large-scale heterogeneity of the number and size of Cx43 gap junctions across the myometrium in non-pregnant pig and emphasize the existence of functional diversity among myometrial cells.

Nucleobases functionalized quantum dots and gold nanoparticles bioconjugates as a Fluorescence Resonance Energy Transfer (FRET) system – synthesis, characterization and potential applications

Fluorescence resonance energy transfer (FRET) system based on functionalized CdTe-guanine and AuNPs-cytosine bioconjugates for the model nucleobase - guanine detection was developed. Thioglycolic acid coated cadmium telluride quantum dots (QDs) conjugated with guanine and sodium 3-mercapto-1-propanesulfonate stabilized gold nanoparticles (AuNPs) capped by cytosine were obtained and fully characterized. Successful formation of the materials was confirmed by UV-Vis, fluorescence and FTIR spectroscopies. Composition of the conjugates was also characterized with elemental analysis and XPS. By employing a guanine-cytosine interaction the bonding between these complementary nucleobases attached to the nanoparticles leads to the formation of QDs-guanine-AuNPs-cytosine assembly, with the size about 7 nm as demonstrated using atomic force microscopy. That enables an effective FRET from functionalized QDs to AuNPs since both, the required distance and the spectral characteristics of donor-acceptor pair were secured. However, it was shown that in the presence of guanine-model molecule which inhibits the interaction between conjugated QDs and AuNPs the FRET is efficiently hampered. Thus monitoring the changes in the restoring fluorescence signal allows to assay the free guanine concentration. Importantly, we have demonstrated the sensitivity and selectivity of the obtained FRET-based system towards guanine. Moreover, in order to confirm the feasibility of the proposed material for nucleobase detection in the real biological samples the developed nanoparticles were also evaluated under simulated urine conditions. The presented strategy of FRET-based conjugated system preparation might be easily used for the development of another nucleobases selective detection and thus opens many possibilities for the determination of biomolecules in the real samples.