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Merging optical images of tissue sections with the spatial distributions of molecules seen by imaging mass spectrometry is a powerful approach to better understand the metabolic roles of the mapped molecules. Here, we use histologically friendly desorption electrospray ionization–mass spectrometry (DESI-MS) to map the lipid distribution in tissue sections of ovaries from cows (N = 8), sows (N = 3), and mice (N = 12). Morphologically friendly DESI-MS imaging allows the same sections to be examined for morphological information. Independent of the species, ovarian follicles, corpora lutea, and stroma could be differentiated by principal component analysis, showing that lipid profiles are well conserved among species. As examples of specific findings, arachidonic acid and the phosphatidylinositol PI(38:4), were both found concentrated in the follicles and corpora lutea, structures that promoted ovulation and implantation, respectively. Adrenic acid was spatially located in the corpora lutea, suggesting the importance of this fatty acid in the ovary luteal phase. In summary, lipid information captured by DESI-MS imaging could be related to ovarian structures and data were all conserved among cows, sows, and mice. Further application of DESI-MS imaging to either physiological or pathophysiological models of reproductive conditions will likely expand knowledge of the roles of specific lipids and pathways in ovarian activity and mammalian fertility.

Stable suspensions of gold nanorods in 4-cyano-4′-pentylbiphenyl (5CB) have been prepared by capping the nanoparticles with polyethylene glycol (PEG). Small angle X-ray scattering has been used to characterize the orientational order of the gold nanorods and the properties of any aggregates. It was found that the nanorods had a very high degree of orientational order with respect to the director of the 5CB matrix and this could be redirected by an applied electric field at 2kHz. Two different PEG molecular weights were investigated and it was found that the lower (1.9kDa) gave exceptionally high order parameters, above 0.9.

Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacylglycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural information of great interest. This paper describes the concept and presents the results of lipid profiling by mass spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids, cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism, particularly in early embryo development and cell differentiation research.

Knowledge of the lipids present in individual preimplantation embryos is of interest in fundamental studies of embryology, in attempts to understand cellular pluripotency and in optimization of in vitro culture conditions necessary for the application and development of biotechnologies such as in vitro fertilization and transgenesis. In this work, the profiles of fatty acids and phospholipids (PL) in individual mouse preimplantation embryos and oocytes were acquired using an analytical strategy based on desorption electrospray ionization mass spectrometry (DESI-MS). The methodology avoids sample preparation and provides information on the lipids present in these microscopic structures. Differences in the lipid profiles observed for unfertilized oocytes, two- and four-cell embryos, and blastocysts were characterized. For a representative set of embryos (N = 114) using multivariate analysis (specifically principal component analysis) unfertilized oocytes showed a narrower range of PL species than did blastocysts. Two- and four-cell embryos showed a wide range of PLs compared with unfertilized oocytes and high abundances of fatty acids, indicating pronounced synthetic activity. The data suggest that the lipid changes observed in mouse preimplantation development reflect acquisition of a degree of cellular membrane functional and structural specialization by the blastocyst stage. It is also noteworthy that embryos cultured in vitro from the two-cell through the blastocyst stage have a more homogeneous lipid profile as compared with their in vivo-derived counterparts, which is ascribed to the restricted diversity of nutrients present in synthetic culture media. The DESI-MS data are interpreted from lipid biochemistry and previous reports on gene expression of diverse lipids known to be vital to early embryonic development.

Background & Aims Early embryogenesis involves cell fate decisions that define the body axes and establish pools of progenitor cells. Development does not stop once lineages are specified; cells continue to undergo specific maturation events, and changes in gene expression patterns lead to their unique physiological functions. Secretory pancreatic acinar cells mature postnatally to synthesize large amounts of protein, polarize, and communicate with other cells. The transcription factor MIST1 is expressed by only secretory cells and regulates maturation events. MIST1-deficient acinar cells in mice do not establish apical-basal polarity, properly position zymogen granules, or communicate with adjacent cells, disrupting pancreatic function. We investigated whether MIST1 directly induces and maintains the mature phenotype of acinar cells. Methods We analyzed the effects of Cre-mediated expression of Mist1 in adult Mist1 –deficient ( Mist1 KO ) mice. Pancreatic tissues were collected and analyzed by light and electron microscopy, immunohistochemistry, real-time polymerase chain reaction analysis, and chromatin immunoprecipitation. Primary acini were isolated from mice and analyzed in amylase secretion assays. Results Induced expression of Mist1 in adult Mist1 KO mice restored wild-type gene expression patterns in acinar cells. The acinar cells changed phenotypes, establishing apical-basal polarity, increasing the size of zymogen granules, reorganizing the cytoskeletal network, communicating intercellularly (by synthesizing gap junctions), and undergoing exocytosis. Conclusions The exocrine pancreas of adult mice can be remodeled by re-expression of the transcription factor MIST1. MIST1 regulates acinar cell maturation and might be used to repair damaged pancreata in patients with pancreatic disorders.

Lipid profiling by mass spectrometry (MS) is increasingly used for the analysis of oocytes, embryos, and other reproductive cells. This analytical approach has several advantages, such as simple preparation (no need to perform extraction or separation), low detection limits (no need of sample pooling), and detection of structurally intact and diverse lipids. Many degrees of freedom are ensured by MS techniques (e.g. with the adoption of diverse ionization sources, mass analyzers, data acquisition systems), and this broadens the classes of lipids that can be detected and identified. Tandem or high-resolution MS experiments are normally performed for chemical characterisation. However, the use of novel approaches is a constant need to obtain deeper structural insights into lipids of biological interest, resulting in an information-rich dataset. Here we propose the use of multiple stages of MS for lipid profiling, specifically MS/MS data domain (i.e. ion mapping) experiments, so that comprehensive structural and relationship information (i.e. classes) can be extracted from a dataset. Indeed, the data generated have 2 dimensions of mass (i.e. precursor and product ions) and one of ion intensity, resulting in a datacube structure. Cutting through the datacube in different ways allows the extrapolation of (i) chemical composition of specific compounds (i.e. product scans) and (ii) pattern recognition for compounds that share identical neutral or charged fragments loss (i.e. neutral loss and precursor scans, respectively). The global chemical information enclosed into the datacube can be also processed by means of multiway statistical analyses to chemically characterise cells and cellular compartments. Preliminary data have been acquired, and the development of statistical tools for data processing is ongoing. Bovine and rat embryos were used for the experiments and analysed by extraction spray ambient MS. Experiments were performed with a Thermo Finnigan LTQ linear ion trap. Dimethylformamide-acetonitrile (1 : 1 v/v) was used as spray solvent. The ion mapping experiment was configured to scan ions of mass-to-charge (m/z) ratio 700 to 900 and perform MS/MS every m/z 1.5 window with a collision energy of 25 (arbitrary units). Fragments were detected in the m/z range of 150 to 900. Chemical differences are present between bovine and rat embryos, of note are palmitic, oleic, and stearic acids. The application of ion mapping to characterise species-specific and developmental dynamics regarding lipid composition is currently under investigation.

Mass spectrometry (MS) allows the detection and structural characterisation of intact molecules such as fatty acids and complex lipids. Desorption electrospray ionization (DESI) is an ambient ionization technique used for MS analysis and profiling and imaging of drugs, metabolites and lipids directly from biological samples with no sample preparation. With the recent introduction of morphologically friendly DESI-MS solvent systems, it is also possible to acquire DESI-MS data non-destructively. Due to the extractive nature of these solvent combinations, enough ion intensity can be generated to chemically profile samples of microscopic dimensions. The objective of this work was to perform chemical profiling on intact bovine blastocysts by DESI-MS, focusing on lipid distributions. Blastocysts produced in vitro were washed 3 times in PBS + 0.1% polyvinyl alcohol to remove lipids present in the culture medium, were placed in PBS/methanol 50% and stored under –20°C for 1 week. For DESI-MS analysis, the embryos were simply placed in glass slides and allowed to dry at room temperature. Mass spectra were acquired in the negative ion mode at the mass/charge range from m/z 150 to 1000, using as solvents a combination of 1:1 (vol/vol) ethanol:dimethylformamide (DMF) or acetonitrile:DMF. The mass spectrometer used was a LTQ linear ion trap mass spectrometer controlled by Xcalibur 2.0 software (Thermo Fisher Scientific, San Jose, CA, USA). The lipid species detected included deprotonated free fatty acids such as palmitic acid (m/z 255.2), stearic acid (m/z 283.2), arachidonic acid (m/z 311.2) and docosanoic acid (m/z 339.3). Free fatty acid dimers appear in the region from m/z 500 to 650 and complex lipids represented mainly by glycerophospholipid classes appear in the region from m/z 700 to 1000 and include phosphatidylinositols (PI 38:1; m/z 788.7), phosphatidylserines (PS 36:1, m/z 885.7) and also the chlorinated phosphatidylcholines (PC 36:1; m/z 794.7). After recording the mass spectra, embryos could still be observed in the glass slide with evident dehydration due to the action of the organic solvent. Since lipid composition of bovine embryos is closely related to cryosensitivity and due to the limited amount of analytes (each embryo is estimated to have a mass of 15 pg of total lipids) lipid analysis usually involves the pooling of individuals to have a large enough amount of analytes. Traditionally, gas chromatography is used for fatty acid residue analysis in oocytes and embryos pooled are submitted to lipid extraction and derivatization. Mass spectrometry by DESI, however, allows direct analysis of intact and single embryos and the profiling of not only free fatty acids but also complex lipids, represented mainly by 3 glycerophospholipid classes (PC, PI and PS). We envisage that DESI-MS will likely become a routine tool for the analysis of lipid composition in mammalian embryos and will contribute significantly to the development of culture systems that produce embryos with higher cryoresistance. Support from the Purdue University Center for Cancer Research Small Grants Program is gratefully acknowledged.

Lipids and metabolites can be directly detected from biological samples by desorption electrospray ionization mass spectrometry (DESI-MS). The introduction of morphologically friendly solvent systems allows nondestructive analysis of samples and because of its extractive nature, ion intensities obtained are adequate for profiling microscopic samples. This work reports the individual chemical profiling of mouse oocytes and diverse pre-implantation developmental stages of embryos by DESI-MS. Mice were superovulated with 5 IU pregnant mare serum gonadotropin (PMSG) IP followed 46 h later by 5 IU hCG IP and mated or not for embryo and oocyte recovery, respectively. Mice were killed and their oviducts removed and flushed at different intervals post-hCG to collect oocytes and embryos at different stages of development. Two-cell embryos were also cultured in vitro to obtain additional samples. For MS analysis, samples (n = 63) were taken from the flushing or culture medium, washed briefly in distilled water and placed on a glass slide for same-day analysis. Mass spectra were acquired in the negative ion mode at the mass/charge range of m/z 150 to 1000 using 1:1 (v/v) acetonitrile/DMF as a solvent system in an LTQ linear ion trap mass spectrometer (ThermoFisher Scientific, Pittsburgh, PA). Mass spectra from oocytes (n = 11) as well as zygotes (n = 9), 2-cell (n = 11), 4-cell (n = 1), 8- to 16-cell (n = 19) embryos and blastocysts (n = 12) showed the presence of deprotonated free fatty acids such as palmitic (m/z 255.2), linoleic (m/z 281.2), arachidonic (m/z 303.3) and docosahexanoenic (m/z 327.3), as well as prominent fatty acid dimers in the region of m/z 500 to 650, such as m/z 563.5 (oleic acid dimer) and glycerophospholipids in the m/z 700 to 1000 range. These complex lipids were represented by chlorinated phosphocholines such as PC 34:1, m/z 794.7; phosphatidylinositols such as PI 38:4, m/z 885.8; phosphatidylethanolamines such as PE 38:3, m/z 768.7; and phosphatidylserines such as PS 38:4, m/z 810.7. After analysis, samples observed on the glass slide displayed evident dehydration caused by the action of the organic solvent. Chemical profiling was more difficult for oocytes and blastocysts compared with other developmental stages, probably because of the presence of the intact zona pellucida in oocytes, whereas blastocysts were more prone to lysis during the rapid water wash, leading to a decrease in signal intensity. Optimization of conditions, comparison of MS spectra from a greater number of samples of different developmental stages by multivariate statistics and the influence of in vitro vs in vivo conditions are currently being investigated. Although small molecules have been shown to play a role in the direction of cell fate, the mechanisms involved are poorly understood. Perspectives for the application of this novel DESI-MS technique include the ability to study chemical signatures of hierarchical pluripotency observed during early embryonic development including embryonic stem cell differentiation. Support from the Purdue University Center for Cancer Research Small Grants Program is gratefully acknowledged.