Author's address (for correspondence): M Apparicio, Via de Acesso Prof. Paulo Donato Castellane, s/n, 14884-900, Jaboticabal-SP, Brazil. E-mail: firstname.lastname@example.org
The aim of the present study was to investigate the level of information on the chemical structures and relative abundances of lipids present in cat and dog oocytes by matrix-assisted laser desorption mass spectrometry (MALDI-MS). The MALDI-MS approach requires a simple analysis workflow (no lipid extraction) and few samples (two or three oocytes per analysis in this work) providing concomitant profiles of both intact phospholipids such as sphingomyelins (SM) and phosphatidylcholines (PC) as well as triacylglycerols (TAG). The lipids were detected in oocytes by MALDI using dihydroxybenzoic acid (DHB) as the matrix. The most abundant lipid present in the MS profiles of bitch and queen oocytes was a PC containing 34 carbons and one unsaturation [PC (34:1)]. Oocytes of these two species are characterized by differences in PC and TAG profiles detected qualitatively as well as by means of principal component analysis (PCA). Cat oocytes were mainly discriminated by more intense C52 and C54 TAG species and a higher number of unsaturations, indicating predominantly linoleic and oleic fatty acyl residues. Comparison of the lipid profile of bitch and queen oocytes with that of bovine oocytes revealed some similarities and also some species specificity: TAG species present in bovine oocytes were also present in bitches and queens; however, a more pronounced contribution of palmitic, stearic and oleic fatty acid residues was noticed in the lipid profile of bovine oocytes. MALDI-MS provides novel information on chemical lipid composition in canine and feline oocytes, offering a suitable tool to concomitantly monitor, in a nearly direct and simple fashion the composition of phospholipids and TAG. This detailed information is highly needed to the development of improved protocols for in vitro culture and cryopreservation of cat and dog oocytes.
Cryopreservation of oocytes has been employed for many species. However, for felines and canines, this technique is still considered experimental because of the low rates of survival and embryo development after thawing as a result of gametes' elevated cryosensibility, mostly attributed to their high lipid content (Luvoni et al. 1997; Turathum et al.2010). It is believed that the lipid content may affect cryosurvival of oocytes because the freezing-thawing process is accompanied by changes in physicochemical properties of intracellular lipids (Isachenko et al. 1998), which are responsible for uneven intracellular ice formation (Luvoni 2006; Turathum et al. 2010). Studies on the lipid composition of oocytes of these species may therefore contribute to improving the cryopreservation process as well as the advance of in vitro culture conditions.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful analytical technique for lipid chemical analysis and was recently applied for detecting sphingomyelins (SM), phosphatidylcholines (PC) and triacylglycerols (TAG) in single oocytes and embryos (Ferreira et al. 2010). MALDI-MS also allows single oocytes to be analysed, avoiding the pooling of tens to hundreds gametes for one single measurement. The use of MALDI-MS and related mass spectrometric protocols of mammalian oocytes and embryos is being rapidly incorporated in embryological research (Gonçalves et al. 2011; Ferreira et al. 2012a,b).
The aim of this work was to obtain the lipid profiles of dog and cat oocytes by MALDI-MS to address, for the first time, the chemical structure of lipid species present. This information allowed comparison with the lipid profile reported for bovine oocytes, a species with higher success in embryo development in vitro and commonly used for cryopreservation studies of oocytes and embryos.
Material and methods
All chemicals in this study were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise stated.
Ovaries were collected from three diestrous bitches (one Rottweiler and two mongrel bitches, age 1–7 years) and one queen in diestrous (cross-bred, 2 years old) by routine ovariohysterectomy. Stage of oestrus cycle was defined based on vaginal cytology and progesterone concentration. Oocytes were transported to laboratory where they were sliced in phosphate buffered saline supplemented with polyvinyl alcohol (PBS-PVA) to release the cumulus-oocyte complexes (COCs). Obtained oocytes were found to be immature and were graded according to morphological characteristics and only grade I COCs (dog, n = 18; cat, n = 10) were selected and stored in 0.5 ml tubes containing PBS solution at −20°C until analysis.
In vitro matured bovine oocytes (n = 3) were used just for visual comparison with the data obtained for canine and feline oocytes, because the lipid profile of bovine oocytes has been already reported under the same experimental conditions as used in this study (Ferreira et al. 2010).
Lipid analysis of single oocytes by MALDI-MS
Even though analysis of a single oocyte is possible, samples have been pooled to obtain high-quality mass spectra. Cumulus cells were removed by pipetting, and oocytes were washed in H2O/MeOH 1 : 1 (v/v) and placed (2–3 oocytes/spot) in the MALDI target plate. Samples were then covered with 1 μl of 2.5-dihydroxybenzoic acid (DHB 0.5 m) dissolved in pure methanol as the MALDI matrix. Then the MALDI target plate was immediately placed in a Q-ToF Premier (Synapt HDMS) mass spectrometer (Waters, Manchester, UK) equipped with a 200 Hz solid-state laser in the m/z 700–1200 range, operated in the reflectron and QTOF modes. The following were the typical operating conditions: laser energy 250 a.u. (arbitrary units), sample plate 20 V. Therefore, unmodified (i.e. no extraction procedure) oocytes have been directly analysed. All mass spectra were manually collected for approximately 1 min in the positive ion mode. Mass spectra were processed using the software MassLynx 4.1 (Waters Corp. Milford, MA, USA). Figure 1 illustrates the sample preparation procedure and information on lipids present in the MALDI mass spectra. Lipid species have been tentatively attributed based on our previous study (Ferreira et al. 2010) and on reported data from related studies (Table 1).
Table 1. Phospholipids (PL) and triacylglycerols (TAG) detected via MALDI-MS lipid fingerprinting of single bitch, queen and cow oocytes
Lipid ion (number of carbon atoms: unsaturations)
Attributions are based on earlier studies and MS/MS data (Leßig et al. 2004; Fuchs and Schiller 2008; Hayasaka et al. 2008; Ferreira et al. 2010). (L) linoleic acid; (Ln) linolenic acid; (O) oleic acid; (P) palmitic acid; (PC) phosphatidylcholines; (S) stearic acid.
[PC (34:1) + Na]+ and loss of N(CH3)3
[PC (32:0) + Na]+
[PC (34:1) + H]+
[PC 36:5) + H]+ and/or [PC (34:2) + Na]+
[PC (34:1) + Na]+
[PC (36:3) + H]+
[PC (38:7) +H]+, [PC (36:4) +Na]+
[PC (38:6) + H]+, [PC (36:3) +Na]+
[PC (38:5) + H]+, [PC (36:2) + Na]+
[PC (38:4) + H]+, [PC (36:1)+Na]+
[PC (38:4) + Na]+
[PPL (50:2) + Na]+
[PPO (50:1) + Na]+
[PLL (52:4) + Na]+
[PLO (52:3) + Na]+
[POO (52:2) + Na]+
[LLL (54:6) + Na]+
[LLO (54:5) + Na]+ and/or [OOLn (54:5) + Na]+
[OOO (54:3) + Na]+ and/or [SOL (54:3) + Na]+
[LLO (54:5) + K]+ and/or [OOLn (54:5) + K]+
The MALDI mass spectra of each sample were accumulated using MarkerLynx 4.1 software (Waters, Manchester, UK) and exported for principal component analysis (PCA by MarkerLynxTM XS, Waters, Manchester, UK). The following were the methodical parameters: mass tolerance = 0.5 Da, baseline noise = 50 and intensity threshold (count) = 1000 with deisotope MS data.
Moreover, lipid profiles obtained from canine and feline oocytes were qualitatively compared with those of bovine oocytes.
Figure 2 illustrates the applicability of MALDI-MS by displaying representative MALDI-MS spectra from oocytes of bitch, queen and cow. Mass spectra of bitch and queen oocytes were characterized mainly by abundant ions of m/z values corresponding to phosphatidylcholines containing 34 carbons (m/z 780.7–786.7) and 36 carbons (m/z 808.7–812.7). The main PC species detected was the PC (34:1), as the sodiated adduct of m/z 782.8 PC (34:1), its fragment of m/z 723.7 and the protonated molecule of m/z 760.8; however, many differences can be noticed in MS profile of dog and cat oocytes. Specifically, cat oocytes have more intense ions for PC containing 34 and 36 carbons (m/z 806.7, 818.7 and 810.7) and more intense TAG species, ions of m/z corresponding to C52 and C54 species and a higher number of unsaturations than dog gametes.
3D PCA reveals that there are differences in lipid profiles between dog and cat, but no variations among conspecific samples. The three principal components depicted in the 3D PCA plot explain more than 73% of the variance observed in the data (Figure 3).
Particularly for the canine and feline species, several difficulties with in vitro production of embryos occur when protocols for bovine embryo production in vitro are applied. Oocyte and embryo biology in the dog is quite unknown and different from that in other mammals (Chastant-Maillard et al. 2010).
Most information regarding lipid composition in oocytes and embryos collected so far has been obtained by means of staining methods, such as Nile Red, which quantify cytoplasmic lipids in general (Barceló-Fimbres and Seidel 2011) and gas chromatography (GC) that quantify by fatty acid (FA) residues extracted from the total sample lipid content (Lapa et al. 2011).
Recently, the MALDI-MS technique has been introduced for the direct and concomitant assess the profiles and chemical structures of both intact phospholipids (PL) and triacylglycerols present in single oocytes and embryos. These profiles are translated into lipid classes (sphingomyelin, phosphatidylcholines and triacylglycerols), number of carbons (which identify the fatty acyl residues attached to the glycerol backbone) and unsaturations present in the lipid molecules. This detailed information at the molecular level can be obtained without the need of pooling tens to hundreds of structures for a single measurement (Ferreira et al. 2010). MALDI-MS provides high levels of information, which are required for the understanding of the role of lipids in oocyte metabolism and cryopreservation survival.
MALDI mass spectra of bitch and queen oocytes were characterized mainly by abundant ions of m/z values corresponding to C34 PC (m/z 780.7–786.7) and C36 PC (m/z 808.7–812.7). The base ion for the three species was owing to PC (34:1), a membrane structural phosphatidylcholine species. By MALDI-MS, it was possible to detect this lipid species as [PC (34:1) + Na]+ ions of m/z 782.7, a fragment of m/z 723.7 (loss of choline) and the protonated molecule of m/z 760.7. This result is expected, because PC (34:1) is the main phospholipid present in the cellular membrane and has been observed among the most abundant lipid ions for oocytes from other mammalian species, such as human, sheep and cattle (Ferreira et al. 2010).
Characteristic differences can be noticed in the profiles of canine and feline oocytes, not only in unique ions with specific m/z values corresponding to individual lipid species, but also in ion abundances, indicating relative proportion differences among samples. The use of chemometrics for mathematical and statistical comparisons of the data has been carried out by PCA, which is a supervised test (Rantalainen et al. 2008). This strategy is commonly used to compare chemical fingerprinting profiles obtained by mass spectrometry (Ferreira et al. 2009) as it allows the simultaneous analysis of multiple variables and reduces data complexity, providing better visualization of differences contained in the data (Rantalainen et al. 2008). Thus, both qualitative and 3D PCA revealed species specificity in the characteristics of lipid profiles of these two species.
We have also visually compared the lipid species in canine and feline oocytes with those in bovine oocytes, the latter being similar to the findings of Ferreira et al. (2010). Our findings revealed some similarities among the lipid profiles of these three species: TAG species present in bovine oocytes were also identified in those of domestic carnivores, with the difference that in queens the ion abundances and the unsaturation levels were broader. However, in the bovine oocytes, it was noticed a more pronounced contribution of palmitic, stearic and oleic fatty acid residues.
Lipid attributions described herein must be confirmed by MS/MS experiments or by high mass resolution measurements. Nonetheless, our data indicate that MALDI-MS is able to detect the presence and determine the chemical structure (number of carbons present in the fatty acyl residues and unsaturations) of PC and TAG species in canine and feline oocytes. Future studies using a large number of oocytes are needed to elucidate how these differences in lipid profiles from bovine, bitch and queen oocytes could impact their cryosensitivity.
In conclusion, direct MALDI-MS with minimal sample preparation has been demonstrated to offer a rapid and efficient protocol for screening of the lipid composition in oocytes of the bitch and queen with detailed information on chemical structures (Cn and unsaturation levels) and concomitant detection of PC and TAG species. MALDI-MS offers a powerful diagnostic tool in studies aiming at improving cultural and cryopreservation requirements for in vitro development and cryobanking of canine and feline oocytes.
We thank CNPQ (PDJ 150184/2011-5) and FAPESP for the funding of this project.
Conflicts of interest
None of the authors have any conflicts of interest to declare.
Apparicio, Ferreira, Luvoni and Vicente contributed to design the study and analyzed the data. Apparicio, Ferreira, Tata, Santos, Alves, Mostachio, Pires-Butler, Motheo, Padilha, Pilau, Gozzo, Eberlin and LoTurco contributed during the experimental phase. All authors have contributed to drafting paper.