Introduction
Over the past decades major advances have been introduced to bovine embryo in vitro production systems due to improvement in culture methods and increased knowledge on preimplantation embryo physiology, ultrastructure and morphology (Gardner, Reference Gardner2008). However, a major obstacle for commercialization of in vitro-produced (IVP) embryos is the low effectiveness of cryopreservation, as these embryos show an increased sensitivity to chilling and freezing when compared with in vivo-produced embryos (Camargo et al., Reference Camargo, Boite, Wohlres-Viana, Mota, Serapiao, As, Viana and Nogueira2011). Therefore, attention has been focused on IVP embryos to identify changes that could be related to their lower viability after cryopreservation and that would allow more extensive use of this technology in cattle breeding programmes (Räty et al., Reference Räty, Ketoja, Pitkänen, Ahola, Kananen and Peippo2011).
During the cryopreservation process, embryonic cells are subjected to considerable physical/osmotic stress caused by abrupt changes in cell volume, as a consequence of water movement outside the cell and penetration of intracellular cryoprotectants (Leibo, Reference Leibo, Brackett, Seidel and Seidel1981). In order to maintain its physiological function, the lipid portion of cell membranes should be in a relatively fluid state. When the reduction in temperature occurs, there is a transition from liquid phase to a gel phase, termed lipid phase transition. During this process, the integrity of some membrane regions can be irreversibly damaged, culminating in cell death (Horvath & Seidel Jr., Reference Seidel2006).
The reduced cryotolerance of IVP embryos has been mainly associated with their excessive cytoplasmic lipid content (Horvath & Seidel Jr., Reference Seidel2006; Seidel, Reference Horvath and Seidel2006; Pereira et al., Reference Pereira, Baptista, Vasques, Horta, Portugal, Bessa, Silva, Pereira and Marques2007; Lapa et al., Reference Lapa, Marques, Alves, Vasques, Baptista, Carvalhais, Pereira, Horta, Bessa and Pereira2011). It is however yet not clear why and how this lipid accumulation occurs. There is evidence that lipid accumulation can be influenced by the use of an undefined culture medium, which is frequently supplemented with fetal calf serum (FCS), or as a result of abnormalities in embryo energy metabolism, which also affect the properties and stability of cell membranes (Dinnyes & Nedambale, Reference Dinnyes and Nedambale2009). Although the intracytoplasmic lipid accumulation can be detrimental to cryotolerance, lipids have also an important physiological role, as they are a reservoir of potential energy for early preimplantation development, before the activation of the embryo's own genome (Kim et al., Reference Kim, Kinoshita, Ohnishi and Fukui2001; Zeron et al., Reference Zeron, Sklan and Arav2002; Sturmey et al., Reference Sturmey, Reis, Leese and McEvoy2009).
Intracellular lipid droplets purified from various types of cultured and tissue cells are rich, not only in triacylglycerol (TAG) and cholesteryl esters esterified with a variety of different fatty acids, but also in the ether neutral lipid monoalk(en)yl diacylglycerol (MADAG). Despite representing only 1–2% of the total lipid in the droplet, the phospholipid (PL) composition includes diverse molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), ether-linked phosphatidylcholine (ePC) and ether-linked phosphatidyl-ethanolamine (ePE), but only small amounts of phosphatidylserine (PS) or sphingomyelin (SM) (Bartz et al., Reference Bartz, Li, Venables, Zehmer, Roth, Welti, Anderson, Liu and Chapman2007). These authors identified and quantified more than 160 phospholipid molecular species and suggested that the neutral and PL composition of lipid droplets was consistent with their direct role in lipid metabolism and in the intracellular traffic of membrane lipids. Then, the PLs influence physical properties of eukaryotic cell membranes, such as fluidity, permeability and thermal phase behavior (Van Meer et al., Reference Van Meer, Voelker and Feigenson2008). Although their role in cryopreservation success is still poorly understood, PL species have been recently suggested to be biomarkers of bovine blastocyst cryotolerance (Sudano et al., Reference Sudano, Santos, Tata, Ferreira, Paschoal, Machado, Buratini, Eberlin and Landim-Alvarenga2012). PC accounts for more than 50% of the PL content in most eukaryotic membranes and organizes spontaneously as a planar bilayer, in which each molecule has a nearly cylindrical geometry. Most PC molecules have one cis-unsaturated fatty acyl chain, which renders them fluid at room temperature. Nonetheless, the SMs have saturated tails (or trans-unsaturated) forming taller and narrower cylinders than PC lipids of the same chain length, and they pack more tightly and adopt the solid gel phase (Van Meer et al., Reference Van Meer, Voelker and Feigenson2008).
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been proposed as a comprehensive and informative method to obtain lipid profiles (Fuchs et al., Reference Fuchs, Süss and Schiller2011) of organisms such as oocytes and preimplantation embryos (Ferreira et al., Reference Ferreira, Saraiva, Catharino, Garcia, Gozzo, Sanvido, Santos, Turco, Pontes, Basso, Bertolla, Sartori, Guardieiro, Perecin, Meirelles, Sangalli and Eberlin2010; Apparicio et al., Reference Apparicio, Ferreira, Tata, Santos, Alves, Mostachio, Pires-Butler, Motheo, Padilha, Pilau, Gozzo, Eberlin, Lo Turco, Luvoni and Vicente2012; Sudano et al., Reference Sudano, Santos, Tata, Ferreira, Paschoal, Machado, Buratini, Eberlin and Landim-Alvarenga2012). MALDI-MS is appropriate for such task as it enables the detection and identification of intact molecules of most TAGs and PLs. This approach is also highly sensitive, allowing data acquisition even from a single oocyte and blastocyst (Ferreira et al., Reference Ferreira, Saraiva, Catharino, Garcia, Gozzo, Sanvido, Santos, Turco, Pontes, Basso, Bertolla, Sartori, Guardieiro, Perecin, Meirelles, Sangalli and Eberlin2010).
Considering that IVP embryo survival post-cryopreservation rates can be also related to membrane lipid changes, this study was conducted with the goal of evaluating the membrane PL profile by MALDI-MS in IVP bovine embryos submitted to vitrification.
Materials and methods
Unless stated otherwise, all chemicals were purchased from Sigma (Sigma Chemical Co., St Louis, MO, USA) and were tested for cell or embryo culture. Methanol (ACS/HPLC grade) was purchased from Burdick and Jackson (Muskegon, MI, USA) and 2,5-dihydroxybenzoic acid (DHB) was purchased from ICN Biomedicals (Aurora, OH, USA). Ultrapure water purified by a Direct-Q water system (Millipore, Bedford, MA, USA) was used for the preparation of culture media and solvents.
Embryo in vitro production
Bovine ovaries from slaughtered cows obtained in a local abattoir were transported to the laboratory. Intact cumulus–oocyte complexes (COCs) were aspirated from antral follicles (2–8 mm in diameter). COCs (n = 639, in three replicates) with at least four layers of cumulus cells and homogenous cytoplasm were selected for the experiment. These cells were washed and cultured in in vitro maturation (IVM) medium, which was Tissue Culture Medium (TCM)-199 (Gibco, Invitrogen Co., Grand Island, NY, USA) supplemented with 10% (v/v) FCS (Gibco, Invitrogen Co.), 0.2 mM sodium pyruvate, 25 mM sodium bicarbonate, 50 μg/ml amikacin, 0.5 μg/ml FSH (Pluset; Hertape Calier, Juatuba, MG, Brazil), and 100 IU/ml hCG (Vetecor, Hertape Calier, Juatuba, MG, Brazil). The IVM culture was carried out in wells containing 500 μl of IVM medium (50 oocytes per well) in a 24-well cell culture cluster (Costar 3526, Corning Incorporated, NY, USA), for 22–24 h, at 38.5ºC and under an atmosphere of 5% CO2 in air, with maximum humidity.
Matured oocytes were subjected to in vitro fertilization (IVF) with frozen semen from a single batch of a Nelore bull (Bos taurus indicus). Motile spermatozoa were obtained by centrifugation of frozen–thawed semen on a Percoll (GE Healthcare, Uppsala, Uppsala County, Sweden) discontinuous density gradient (250 μl of 45% Percoll over 250 μl of 90% Percoll, in a 1.5 ml microtube) for 5 min at 2500 g, at room temperature. The supernatant was discarded, and the spermatozoa were counted on a haemocytometer and then resuspended in IVF medium (TALP supplemented with 0.2 mM Na-pyruvate, 6 mg/ml fatty acid-free bovine serum albumin (BSA), 25 mM sodium bicarbonate, 13 mM Na-lactate, 50 μg/ml amikacin, 40 μl/ml PHE solution and 10 μg/ml heparin) to obtain a final concentration of 2 × 106 cells/ml. Finally, 4 μl of the sperm suspension was added to each 90 μl droplet. COCs (25 per droplet) and sperm were co-incubated for 18 h, under the same temperature and atmospheric conditions used for IVM. The day of fertilization was defined as day 0.
Following fertilization, the presumptive zygotes were stripped from the cumulus cells by vortexing and transferred to in vitro culture (IVC) medium, which consisted of modified synthetic oviductal fluid (mSOF) (Vajta et al., Reference Vajta, Rindom, Peura, Holm, Greve and Callesen1999) supplemented with 50 μg/ml amikacin, 5 mg/ml fraction fatty acid-free BSA and 2.5% (v/v) FCS. The culture was carried out in 24-well cell culture cluster under the same atmosphere conditions used for IVM. Zygotes were incubated up to 72 h post-insemination (hpi) for the assessment of cleavage rate. Embryo development rates were recorded at 168 hpi, at which time expanded blastocysts were ready for cryopreservation by the vitrification method.
All materials used for vitrification/thawing procedures were supplied by Ingámed Ltda. (Perobal, PR, Brazil). These included the vitrification solutions VI-I and VI-II, the thawing solutions DV-I, DV-II and DV-III, the vitrification strips Vitri-Ingá and their plastic sheaths. The technique used for blastocysts vitrification and thawing was performed according to Almodin et al. (Reference Almodin, Minguetti-Camara, Paixao and Pereira2010) and Rocha-Frigoni et al. (Reference Rocha-Frigoni, Leão, Nogueira, Accorsi and Mingoti2014). After thawing, the embryos were subsequently cocultured in SOF supplemented with 2.5% of FCS and 5 mg/ml of BSA, under atmosphere of 5% CO2 in air, during 24 h. Thereafter, embryo survival rates were evaluated and only the viable embryos were collect for MALDI-MS analysis.
MALDI-MS analysis
Viable embryos were collected before (fresh embryos) and after vitrification, transferred to microtubes containing 200 μl of methanol HPLC 50% in aqueous solution, and stored at –20°C overnight. For the MALDI-MS analyses, embryos were deposited individually at the centre of each MALDI target spot in the sample plate. Before analysis, 1 μl of 1.0 M organic matrix 2,5 dihydroxybenzoic acid (DHB) in methanol was deposited over each spot and the samples were left at room temperature until complete crystallization. The mass spectra data were obtained in the 700–1200 mass-to-charge ratio (m/z) range, in the positive ion and reflectron modes (MALDI(+)-MS) using an Autoflex III mass spectrometer (Bruker Daltonics, USA) equipped with smart beam laser technology at a frequency of 200 until signals in the region of interest were obtained. Mass spectra were observed when the laser hit the sample and then disappeared due to the consumption of the sample. FlexAnalysis 3.0 software (Brucker Daltonics) was used to process the mass spectra. The most abundant ions that were clearly distinct from noise after the exclusion of isotopic peaks were examined from each spectrum and used as the starting point to search for m/z values corresponding to lipids, and were also taken to be the starting point for the statistical analysis.
Statistical analysis
In total, 56 embryos were used for this study (40 fresh and 16 vitrified blastocysts). For lipid MS profiles, multivariate and univariate statistical models were used. Only the m/z values that were clearly distinct from noise in the mass spectra were included in the statistical analysis. Data were processed using the software Pirouette v.3.11 (Infometrix Inc., Woodinville, WA, USA) for principal component analysis (PCA) and also for support-vector machine (SVM) analysis, a non-probabilistic binary linear classifier that predicts, for each given input, which of two possible classes forms the output (Cortes & Vapnik, Reference Cortes and Vapnik1995).
For the SVM analysis, samples were divided into training set (21 fresh and nine vitrified embryos) and test set (19 fresh and seven vitrified embryos). The training set was used to build and optimize the SVM model. The classifier was then applied to the test set in order to determine the model's ability to discriminate samples. As Table 1 shows, the accuracy for the training set was larger than for the test set, as was expected.
Table 1 Performance of the classification of support-vector machine (SVM) models
Sensitivity, specificity and accuracy of the model were evaluated from SVM model and calculated according to the formulae:
Sensitivity = TP/(TP + FN) (1)
Specificity = TN/(TN + FP) (2)
Accuracy = (TP + TN)/(TP + FN + TN + FP) (3)
In the formulae above, the acronym TP (true positive) refers to the number of fresh blastocysts correctly classified as fresh blastocysts, while FN (false negative) is the number of fresh blastocysts classified as vitrified blastocysts. TN (true negative) refers to the number of vitrified blastocysts correctly classified as vitrified blastocysts, while FP (false positive) is the number of vitrified blastocysts classified as fresh blastocysts. Sensitivity is considered to be the rate at which the SVM model correctly classifies fresh blastocysts as fresh blastocysts. Specificity is the fraction of vitrified blastocysts correctly classified. The accuracy is the measure of the quality of the classification model, and represents the ratio of correctly assigned objects.
Based on the multivariate statistical analysis discrimination results, the most important ions that explained the variance of the data were submitted to analysis of variance (ANOVA) using MATLAB software (Math Woks Inc., Natick, Massachusetts, USA) to evaluate the significance of each ion of fresh and vitrified groups. Data are presented as mean ± standard deviation (SD). The level of statistical significance was set at P-value < 0.05.
Results
In respect to the embryo production in this work, cleavage and blastocyst rates were 80.3 and 47.9%, respectively. Blastocyst rate was estimated based on the number of oocytes in each maturation well. The percentage of embryo survival at 24 hours post-thawing, based on the blastocyst integrity and re-expansion, was 65.9%.
Each single bovine blastocysts were subjected to MALDI-MS analysis in the positive ion mode using DHB, as previously described (Sudano et al., Reference Sudano, Santos, Tata, Ferreira, Paschoal, Machado, Buratini, Eberlin and Landim-Alvarenga2012). This study has been based on the detection of lipid profiles, which allow for relative quantification of the detected molecular ions based on their intensities changes. Internal standards were not used. In addition to our previous works on single oocyte/embryo analysis by MALDI using this strategy (Ferreira et al., Reference Ferreira, Saraiva, Catharino, Garcia, Gozzo, Sanvido, Santos, Turco, Pontes, Basso, Bertolla, Sartori, Guardieiro, Perecin, Meirelles, Sangalli and Eberlin2010; Apparicio et al., Reference Apparicio, Ferreira, Tata, Santos, Alves, Mostachio, Pires-Butler, Motheo, Padilha, Pilau, Gozzo, Eberlin, Lo Turco, Luvoni and Vicente2012; Sudano et al., Reference Sudano, Santos, Tata, Ferreira, Paschoal, Machado, Buratini, Eberlin and Landim-Alvarenga2012; Tata et al., Reference Tata, Sudano, Santos, Landim-Alvarenga, Ferreira and Eberlin2013), diverse MALDI-MS applications are based in this approach and have been found to have good correlation with liquid chromatography–tandem mass spectrometry (LC-MS/MS) data (Hankin & Murphy, Reference Hankin and Murphy2010; Berry et al., Reference Berry, Hankin, Barkley, Spraggins, Caprioli and Murphy2011).
The lipid profile of fresh and vitrified blastocysts has been compared by PCA, an unsupervised method (Wold et al., Reference Wold, Esbensen and Geladi1987), and then by SVM, a supervised model. This approach was used because unsupervised statistics was not sufficient to completely clarify the differences in the lipid profiles present between the experimental groups.
As shown in Fig. 1, the first three principal components explain 39.82% of total data variance. Fresh blastocysts samples formed one cluster, while vitrified blastocysts samples formed three small clusters. As the sample's individual variability complicated the interpretation, SVM was applied. The prediction accuracy on the training set was 100% and the test set was 73.08%. The SVM model could assign correctly 19 of the 26 test samples, as presented in Table 1. Therefore, by SVM, the differences between lipid profiles of fresh and vitrified blastocysts could be better characterized.
Figure 1 Principal component analysis (PCA) plot for the MALDI-MS data of fresh (▼) and vitrified (·) embryos. PC 1: principal component 1; PC 2: principal component 2; PC 3: principal component 3.
Comparison of lipid profiles by MALDI or small molecules by other ionization methods in biological samples does not need to include internal or external references as it is not an absolute, but rather a semi-quantitative, comparison. The chemical fingerprinting comparison approach has been applied not only in oocyte and embryo analysis, but also for prediction of embryo implantation potential (Cortezzi et al., Reference Cortezzi, Cabral, Trevisan, Ferreira, Setti, Braga, Figueira, Iaconelli, Eberlin and Borges2013), proof of origin of oils (Saraiva et al., Reference Saraiva, Cabral, Eberlin and Catharino2009; Cabral et al., Reference Cabral, Sevart, Spindola, Coelho, Sousa, Queiroz, Foglio, Eberlin and Riveros2013) and biodiesel (Eberlin et al., Reference Eberlin, Abdelnur, Passero, de Sa, Daroda, de Souza and Eberlin2009).
Lipid attribution of significant ions indicated by the SVM was performed using the LIPID MAPS database (Fahy et al., Reference Fahy, Sud, Cotter and Subramaniam2007) and previous results of MALDI-MS lipid analysis of embryos performed under similar conditions (Ferreira et al., Reference Ferreira, Saraiva, Catharino, Garcia, Gozzo, Sanvido, Santos, Turco, Pontes, Basso, Bertolla, Sartori, Guardieiro, Perecin, Meirelles, Sangalli and Eberlin2010; Sudano et al., Reference Sudano, Santos, Tata, Ferreira, Paschoal, Machado, Buratini, Eberlin and Landim-Alvarenga2012). The nomenclature used starts with the class abbreviation followed by the total number of carbons and double bonds in the acyl residues attached to the glycerol backbone in parenthesis.
Data obtained by MALDI-MS was submitted to multivariate statistical analysis and results revealed a significant difference of seven ions (m/z values of 703.5, 722.5, 744.5, 758.5, 762.5, 790.5 and 810.5) between fresh and vitrified embryos. The relative abundance of ions at m/z 703.5, 722.5, 758.5, 762.5, 790.5 and 810.5 was higher in vitrified embryos compared with the fresh group. In fresh embryos, only the ion with a m/z 744.5 was more abundant (Fig. 2).
Figure 2 Boxplot graphic showing the differences in the relative abundance of the seven significant ions (m/z values of 703.5, 722.5, 744.5, 758.5, 762.5, 790.5 and 810.5) of fresh and vitrified embryos. Bottom, middle and top lines of each box correspond to the 25th percentile, 50th percentile (median) and the 75th percentile, respectively. Data were obtained by MALDI-MS and analysed by multivariate statistical analysis.
Other ions detected by the model presented no significant differences in their abundances in both experimental groups. For example, the ions with a m/z 845.6 and 1049.7 displayed similar abundances for vitrified or fresh embryos, whereas the ions with m/z 785.7 and 741.5 showed slightly different abundances in both groups, that is, they increased in abundance only 1.05 times on vitrified group and 1.2 times on fresh embryos, respectively (data not shown). A representative MALDI-MS spectra for each experimental group is shown in Fig. 3.
Figure 3 Representative MALDI-MS for each experimental group. (A) fresh group; (B) vitrified group.
The seven significant ions detected by the multivariate statistical analysis were submitted to ANOVA to evaluate the significance of each ion of fresh and vitrified groups. Table 2 shows the m/z values and assignments given to these significant ions for the model, comparing the fresh and vitrified bovine embryos. Higher relative abundance of ions with m/z 722.5 (20 times), 762.5 (1.8 times), 790.5 (3 times) and 810.5 (2.7 times) was observed in vitrified embryos compared with the fresh ones. In fresh embryos, only the ion with a m/z 744.5 was more abundant (3.4 times). Although no difference was observed in the relative abundance of ions with m/z 703.5 and 758.5 (P > 0.05) after evaluation by the univariate analysis, all the seven ions detected in the multivariate statistical analysis (listed in Table 2) were considered to be important to explain the variance of the data for discrimination of fresh and vitrified groups.
Table 2 Phospholipids tentatively identified via MALDI(+)-MS of fresh and vitrified bovine embryos
Identification is based on earlier studies and MS/MS data from our group (Ferreira et al., Reference Ferreira, Saraiva, Catharino, Garcia, Gozzo, Sanvido, Santos, Turco, Pontes, Basso, Bertolla, Sartori, Guardieiro, Perecin, Meirelles, Sangalli and Eberlin2010; Sudano et al., Reference Sudano, Santos, Tata, Ferreira, Paschoal, Machado, Buratini, Eberlin and Landim-Alvarenga2012) and Lipidmaps (www.lipidmaps.org). PC, phosphatidylcholines; SD, standard deviation; SM, sphingomyelin.
Discussion
In the present study, the membrane PL profile of fresh bovine embryos produced in vitro was compared with the one from vitrified embryos by MALDI-MS. Due to conditions established for the analysis, mainly membrane lipids such as PC and SM were detected. To our knowledge, this is the first report on lipid membrane changes in bovine IVP blastocysts associated with the cryopreservation process.
The cytoplasm membrane is extremely sensitive to low temperature (Arav et al., Reference Arav, Zeron, Leslie, Behboodi, Anderson and Crowe1996) and its lipid composition influences its physical properties, particularly membrane fluidity (Kim et al., Reference Kim, Kinoshita, Ohnishi and Fukui2001). The study of the membrane lipid profile is therefore fundamental to fully comprehend the mechanisms by which cryopreservation affects embryo survival. Changes in the lipid structural composition of the membranes, which are not possible to detect using staining procedures, are readily detected by MS (Ferreira et al., Reference Ferreira, Eberlin, Hallett and Cooks2012; Sudano et al., Reference Sudano, Santos, Tata, Ferreira, Paschoal, Machado, Buratini, Eberlin and Landim-Alvarenga2012; Tata et al., Reference Tata, Sudano, Santos, Landim-Alvarenga, Ferreira and Eberlin2013). The direct analysis by MALDI-MS of single and intact preimplantation embryos was initially standardized by Ferreira et al. (Reference Ferreira, Saraiva, Catharino, Garcia, Gozzo, Sanvido, Santos, Turco, Pontes, Basso, Bertolla, Sartori, Guardieiro, Perecin, Meirelles, Sangalli and Eberlin2010). This strategy allows us to obtain reproducible and characteristic lipid fingerprints from single oocytes or preimplantation embryos in a procedure involving no extraction, no chemical manipulation and no pre-separation protocols. MALDI-MS is sensitive enough to enable analysis of individual intact embryos and to monitor the changes in membrane lipid composition in early stages of preimplantation development, thereby serving as screening method for studies of biology and biotechnologies of reproduction (Tata et al., Reference Tata, Sudano, Santos, Landim-Alvarenga, Ferreira and Eberlin2013).
Significant differences in PC and SM profiles in vitrified blastocysts compared with fresh blastocysts have been detected by MALDI-MS using SVM as a statistical approach, indicating that cryopreservation affects membrane lipids dynamics. Among the changes, that of the ions with m/z 722.5 assigned as [PC (40:3) + Na]+, m/z 758.5 [PC (34:2) + H]+, 762.5 [PC (34:0) + H]+, 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ or [PC (36:1) + Na]+, as well as the ion with a m/z 703.5, assigned as [SM (16:0) + H]+ were the most significant. Results suggest that a putative remodeling and/or turnover of membranes occurs readily after vitrification and during the subsequent incubation for an additional 24 h.
In fresh embryos, only the ion with a m/z 744.5, tentatively attributed as [PCp (34:1) + H]+ or PCe (34:2) + H]+, was the most abundant. Interestingly, ions with a m/z 744.5 indicate the presence of lipid species that contain fatty acid residues linked through ether-type bonds (alkyl ether or plasmalogens, indicated by the lowercase ‘e’ and ‘p’, respectively) to the glycerol structure, which are lipids that are more resistant to enzymatic degradation and that are commonly found in sperm.
In the membrane of mammalian cells, PC is the most abundant PL (Vance & Tasseva, Reference Vance and Tasseva2013). Kaplan & Simoni (Reference Kaplan and Simoni1985) determined the effect of low temperature on the rate of PC transport from its site of synthesis, the endoplasmic reticulum, to the plasmatic membrane. They observed a significant effect of temperature reduction, as little or no transport occurred at 0°C, but transport resumed when cells were warmed to 25°C. Thus, the increase in relative abundance of ions attributed to be PC after vitrification, when the temperature is reduced abruptly, can be a response of the embryonic cells by repairing damaged plasmatic membrane by synthesis of PC.
In addition to their structural role, the SM also participates in cell signaling. For example, products of SM metabolism, such as ceramide, sphingosine, sphingosine-1-phosphate and diacylglycerol are important cellular effectors, and give to SM a role in cellular functions, such as apoptosis (Merrill et al., Reference Merrill, Schmelz, Dillehay, Spiegel, Shayman, Schroeder, Riley, Voss and Wang1997; Huwiler et al., Reference Huwiler, Kolter, Pfeilschifter and Sandhoff2000). Cellular stress responses in general may be mediated by ceramide formation, presumably secondary to sphingomyelin hydrolysis (Merrill et al., Reference Merrill, Schmelz, Dillehay, Spiegel, Shayman, Schroeder, Riley, Voss and Wang1997). The binding of TNF-α or Fas ligand to their respective receptors activates sphingomyelinase, which catalyzes the sphingomyelin degradation to produce ceramide (Wiegmann et al., Reference Wiegmann, Schütze, Kampen, Himmler, Machleidt and Krönke1992). Ceramide is also a regulator of programmed cell death, so-called apoptosis, induced by TNF-α and Fas (Merrill et al., Reference Merrill, Schmelz, Dillehay, Spiegel, Shayman, Schroeder, Riley, Voss and Wang1997). Once ceramide is produced, it can be recycled back to sphingomyelin by transfer of the phosphocholine headgroup of PC within the Golgi apparatus and by releasing diacylglycerol as a byproduct (Huwiler et al., Reference Huwiler, Kolter, Pfeilschifter and Sandhoff2000).
Based on the outlined findings, it can be hypothesized that the increment in the relative abundance of [SM (16:0) + H]+ of the m/z 703.5 on vitrified embryos can be a response to the vitrification process. Sudano et al. (Reference Sudano, Paschoal, Rascado, Magalhães, Crocomo, de Lima-Neto and Landim-Alvarenga2011) observed that the stress generated by cryopreservation procedures corresponded to an increase of 20.3 to 40.8% in blastomere apoptosis rate, when comparing fresh with vitrified IVP blastocysts. It is known that apoptosis has important roles both in embryonic development and organism homeostasis. Preimplantation embryonic development is a naturally dynamic process that involves cell proliferation, differentiation, and apoptosis (Paula-Lopes & Hansen, Reference Paula-Lopes and Hansen2002). However, a disproportionate incidence of this apoptosis was associated with the reduction of embryo viability (Byrne et al., Reference Byrne, Southgate, Brison and Leese1999).
Although the conditions established for the present analysis allowed mainly the detection of membrane lipids and we have not quantified the lipid droplets, we should also consider that excessive cytoplasmic lipid content is correlated with a reduction in cryotolerance (Horvath & Seidel Jr., Reference Seidel2006; Seidel, Reference Horvath and Seidel2006; Pereira et al., Reference Pereira, Baptista, Vasques, Horta, Portugal, Bessa, Silva, Pereira and Marques2007; Lapa et al., Reference Lapa, Marques, Alves, Vasques, Baptista, Carvalhais, Pereira, Horta, Bessa and Pereira2011). Then, the composition, localization, amount and function of each one of the embryo's lipid is a remarkably complex issue. Typification and monitoring of some lipid changes during the developmental stages and/or culture conditions should contribute to improve cryopreservation and in vitro culture conditions of mammalian embryos.
In summary, as discussed above, the significant changes on lipid profile regarding PL, observed mostly in vitrified embryos, suggest that the cryopreservation process can alters the membrane due to some putative remodeling and/or turnover, as well as a response of the embryonic cells repairing damaged plasmatic membrane and/or increased apoptosis. On the other hand, it should be also noted that only the viable embryos were evaluated after thawing in the present study. Therefore, we cannot exclude a possibility of a selection of a subset of cryotolerant embryos and, if so, the observed changes could not be due to the vitrification process itself. However, regardless of the changes in membrane lipid profile are the cause or effect of embryo survival after thawing; an understanding of the lipid profile is fundamental to address the difficulties of post-cryopreservation embryo survival. Previous data from Sudano et al. (Reference Sudano, Santos, Tata, Ferreira, Paschoal, Machado, Buratini, Eberlin and Landim-Alvarenga2012) have suggested that PC abundance in embryos seems to function as a prospective biomarker for cryopreservation success. Then, in agreement with their hypothesis, present data clearly demonstrated a significant difference in the membrane lipid profile, mainly the PC profile, of fresh and vitrified/thawed embryos produced in vitro.
In conclusion, our results indicated that the vitrification process affects membrane lipid profile of bovine embryos, and that these alterations can be properly monitored by MALDI-MS. The monitoring of lipid profiles may be a useful tool to examine the effect of cryopreservation on membrane homeostasis and to improve cryopreservation success. It may also be a suitable tool to investigate embryonic metabolic response to injury or changes in environmental conditions.
Acknowledgements
We thank Ingámed Ltda. (Perobal, PR, Brazil) for providing all materials used for the vitrification/thawing procedures. BCSL was supported by an MS scholarship from CNPq, Brazil.
