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.
The mammalian ovary regulates several different processes, most significantly the development, maturation, and release of oocytes for fertilization. This organ is also responsible for the synthesis and secretion of hormones that are essential for follicular growth and reproductive maintenance. In this latter context, complex chemical events occur in a coordinated temporal order to maintain the rhythm of ovarian function . For example, inflammation pathways will occur in response to hormone stimulation, by the production lipid molecules such as prostaglandins and eicosanoids that will degrade follicle tissue and promote ovulation . While there are differences in the timing and sequence of developmental events among mammals, across species the ovaries develop in an embryologically similar manner .
Current analysis of ovarian function relies on the use of gene expression to evaluate the follicle steroidogenesis and to demonstrate their relationship with such processes as apoptosis, cell regulation, and DNA fragmentation [4,5,6,7]. Recent approaches based on mass spectrometry have assisted the knowledge of ovary physiology, indicating metabolic differences with respect to follicle size and response to hormone stimulation [8, 9]. In addition, mass spectrometry analysis of follicular components, such as cumulus cells, has improved comprehension on how these cells can modulate oocyte development .
Imaging mass spectrometry can provide additional understanding of ovarian function due to the possibility of mapping the locations of small molecules in tissue sections, which has been applied in reproductive and developmental studies [11, 12]. In this regard, ovarian lipids are an untapped source of information not only for their role as membrane compounds and their energy storage properties, but also as signaling and steroidogenic molecules [13,14,15]. The physiological dynamics and distribution of lipids in the ovaries are crucial for follicle and oocyte development, and as such they represent a limiting factor for fertility.
Desorption electrospray ionization mass spectrometry (DESI-MS) imaging is well suited to the direct analysis of small molecules such as lipids and metabolites in biological samples. It maps the spatial distributions of small molecules in unmodified tissue sections (no fixatives, no matrix deposition) [16,17,18]. DESI-MS has been previously explored in reproductive biology to profile single oocytes and embryos, through the detection of free fatty acids (FFA), phospholipids (PL), cholesterol-related molecules, and triacylglycerol (TAG) in murine, bovine, and porcine models [19, 20]. DESI-MS lipid profiles of oocytes and early stage embryos allowed following changes related to the developmental stage, in vitro culture and cryopreservation [19, 20].
Increased knowledge of ovarian molecular mechanisms is key to the further development of assisted reproduction technologies for domestic animals and humans, such as in vitro generated gametes, ovary superovulation protocols, and fertility restoration, naturally or via artificial ovaries . In this research, we combine the information on the distribution of numerous lipids from DESI-MS imaging with optical microscopy to study ovarian lipid content and distribution. This approach demonstrates that ovarian lipid distribution represents a dynamic complexity that associates structure and physiology in different species, and that specific lipids may have key roles in ovarian function.
Frozen ovaries from cows (N = 8) and sows (N = 3) were purchased from Animal Technologies (Tyler, TX, USA). BCB and CD-1 strain mouse ovary samples (N = 12) at 0.5 and 3.5-days post-ovulation were obtained from super-ovulated animals housed at the Purdue University Center for Cancer Research’s Transgenic Mouse Core Facility (under Purdue Animal Care and Use Committee protocol 11-060).
Samples were frozen and stored at − 80 °C without using fixative reagents. Cow and sow ovaries were sliced into 20-μm thick sections using a Shandon SME Cryotome cryostat (GMI, Inc., Ramsey, MN, USA) and thaw mounted onto a microscope glass slide (Erie Scientific, Portsmouth, NH). Due to their small size, mouse ovaries were embedded in optimal cutting temperature compound (OCT compound) in an array. Ovarian sections were stored at − 80 °C until analysis, when they were kept to room temperature and dried in a desiccator for ∼ 15 min prior to analysis.
DESI-MS imaging settings and chemical imaging visualization
A Thermo Finnigan LTQ (San Jose, CA) linear ion trap mass spectrometer equipped with a home-built DESI ion source coupled to an imaging stage platform was operated in the negative ion mode in the m/z range of 200 to 1000, scan time 1.14 s, injection time 500 ms, and 2 microscans per spectrum, with 5 kV applied to the solvent syringe needle. The DESI solvent dimethylformamide, acetonitrile (DMF-ACN) 1:1 (v/v), flow rate 0.7 μL min−1, is a morphologically friendly solvent that allows H&E staining and morphological analysis after MS . Figure 1 illustrates the workflow and sample processing for the DESI-MS imaging experiments.
A 300 μm lateral resolution was used on the moving stage. Ion images were constructed using Image Creator (in-house built software) and BioMAP 184.108.40.206 provided by Novartis Institutes for BioMedical Research (freeware). Each pixel of the image corresponds to one mass spectrum. By selecting ions of one individual m/z value as characteristic of particular compounds (typically the deprotonated ion in negative ionization mode), it is possible to represent the localization and intensity of each compound using a false color scale. The false color scale used is show at Fig. 1c, where black/purple/blue color are related to lower ion intensities, while green color is intermediate and yellow/red indicates the highest ion intensities for a given m/z value.
Since the spray solvent composition used for DESI-MS imaging was morphologically friendly, it was possible to stain the tissue sections after MS analysis by hematoxylin and eosin (H&E). Optical images from the DESI-MS imaged tissue sections were acquired in a Microscope SM-LUX Binocular Bright field (Leitz, Wetzlar, Germany). The optical images guided attention in the accumulated mass spectra to regions of interest (ROI), namely, ovarian stroma, follicular epithelium, and corpora lutea (CL) for further analysis.
DESI-MS data acquired in .raw format were converted using an in-house program into a format compatible with Biomap (Novartis Institutes for BioMedical Research, http://www.maldi-msi.org). Different anatomical regions were selected manually via the ROI function in Biomap. The mass list was then exported from Biomap into .txt files which were compiled in a spreadsheet (Excel, Microsoft). The .txt and .xlsx (Excel) files were then imported into Matlab (The MathWorks, Inc., USA) and multivariate data processing was performed using in-house Matlab routines. Spectra were normalized using the standard normal variate (SNV) transformation, correcting for both baselines shift and global intensity variation . Tentative lipid attributions were performed based on MS/MS analysis (Table 1) and full scan supported by data in previously published DESI-MS images of biological tissues [24,25,26,27,28,29].
Results and discussion
Representative spectra from the major anatomical substructures of the bovine ovary (Fig. 2) display a range of free fatty acids (FFAs) and phospholipids. The most abundant FFAs detected (as negative ions) in the ovarian tissue were palmitic acid (m/z 255), oleic acid (m/z 281), arachidonic acid (m/z 303), and docosahexaenoic acid (m/z 327). The abundance of FFA was such that proton-bound dimers of two fatty acid anions were prevalent in the mass spectra (gas-phase dimer formation is positively associated with concentration). The most abundant lipid ions are seen in CL where FA dimers and lysophospholipids (LPA) are observed in the m/z range of 500–700.
Representative mass spectra showed higher abundances of free fatty acids (FFA) and phospholipids (PL) in corpora lutea and follicular walls for all three species (Fig. 2 and in Electronic Supplementary Material (ESM), Figs. S1–S4). These results corroborate literature data in which matrix-assisted laser desorption/ionization mass spectrometry imaging demonstrates differences in lipid spatial distribution, mainly between the follicles and interstitial tissue in swine ovaries. In addition, the presence of FFA in these follicles was associated with the metabolic activity required for oocyte competence during oogenesis, which is associated with intense follicular cells proliferation, steroidogenesis, and development to the blastocyst stage [30, 31].
Mass spectra for selected regions of interest (ROI) with estimated 200 μm location precision were guided by the overlay of H&E and DESI-MS as indicated in Fig. 3 where examples of overlays for bovine, mouse, and swine ovaries are shown. By looking at individual ion images (Fig. 3, data not shown for all three species) it is possible to observe that specific lipids, such as m/z 885 tentatively identified as phosphatidylinositol, PI(38:4), m/z 788 corresponding to the phosphatidylserine, PS(36: 1), and free oleic acid (C18:1, of m/z 281) are concentrated in follicles and in the CL. These lipids were also observed to be prominent in earlier DESI-MS analysis of swine oocytes  and zebrafish embryos . In bovine, PI(38:4) and PS(36:1) have been previously related to the proliferation and cell migration in implantation embryos imaged by MALDI-MS . The presence of these lipids in CL have been associated with prostaglandin stimulation during the ovarian mid-late luteal phase, and macrophages activity during CL apoptosis [34, 35].
With respect to lipid location and abundance, the three species showed strong similarities as well as some differences (Fig. 4). The ion of m/z 885.7, corresponding to PI(38:4), was abundant in the CL of all species, especially in mice. However, the lipid PS(40:5), m/z 836, was widely distributed in both stroma and CL of bovine ovaries but was detected only in the stroma of swine ovaries, and was mainly detected in the CL of mice. Stearic acid, m/z 283, was abundant in the follicular walls and the CL the bovine ovary, but only in the stroma and CL of the swine ovary and in the stroma of mice ovaries.
The variability of lipids localization between particular structures in the three species may be related to the functional variability of ovaries of different animal species [36,37,38,39]. Also, such variability may be associated with lipids role in specific structures (CL, follicular walls and stroma), which are responsible for processes as cell proliferation and apoptosis, and ovarian steroidogenesis . Cows characteristically have one dominant follicle that matures for fertilization, and subordinate follicles that will suffer apoptosis . Presumably, the presence of PS(40:5) and stearic acid in the bovine follicle could be an indication of such mechanisms, considering PS role in apoptosis and stearic acid inhibition of cell proliferation in bovine large follicles . On the other hand, polyovulatory species (swine and mice) have small antral follicles that will all develop into large follicles . In these species, PS(40:5) and stearic acid were absent in follicular cells.
The visualization of specific ions, as described above, was complemented by the statistical analysis of regions of interest (ROI) selected from cattle (n = 8), swine (n = 3), and mouse (n = 5) ovaries (including pre-pubertal and cycling adults) (Fig. 5a, b). ROI for stroma (n = 54), follicles (n = 89), and corpora lutea (n = 61) from the three species were selected based on morphological structures and mass spectra were analyzed using principal component analysis. Morphological structures could be differentiated (> 90% prediction rate, Fig. 5c) independent of the species, indicating that lipid constitution is conserved between the species evaluated.
Similar to Fig. 2 (mass spectra only bovine species), Fig. 6 displays the average mass spectra by morphological features for all species and Table 1 summarizes the main lipids observed in stroma, in CL and in follicles. Ions from the ROI of the three species indicated as discriminant for CL, follicle wall, and ovarian stroma by PCA were also analyzed using univariate analysis (ANOVA), to examine the significance of particular lipids between morphological structures. For example, adrenic acid (p value = 1.7 × 10−8) and the PS(40:5) of m/z 836 (p value = 8.9 × 10−9) were higher in CL and follicles than in stroma, adding more evidence of the conserved ovarian lipid composition of these mammalian species.
The FA C22:4 (decosatetraenoic or adrenic acid, m/z 331) is a naturally occurring polyunsaturated FA formed through a 2-carbon chain elongation of arachidonic acid in swine and cattle. A box-and-whisker plot (ESM Fig. S5) shows differences in the abundance of m/z 303 (arachidonic acid) and m/z 331 (adrenic acid) in swine and bovine ovarian structures, indicating higher abundance of these lipids in CL. For all three types of ovaries, arachidonic acid seems to play an essential role in ovulation and fertility, in addition to assist the development of the CL [42,43,44,45]. Moreover, adrenic acid may play a role in bovine luteal phase .
For the mouse oocytes only, we were able to obtain data at different stages after ovulation. Ion images superimposed on the H&E stained mouse ovary array (1 to 14 days after ovulation), are shown in Fig. S4 (see ESM). Several lipid species and the fatty acids palmitic (C16:0), linoleic (C18:2), oleic (C18:1), stearic (C18:0), EPA (C20:5), arachidonic (C20:4), DHA (C22:6), DPA (C22:5), and decosatetraenoic acid (C22:4) and a number of their dimer combinations were observed in high intensity. Signaling lipids such as phosphatidylserines PS(36:1), PS(38:4), and PS(40:4), as well as PI(36:4) and PI(38:4) were found to be concentrated in the CL, although irregularly. The presence of these lipids may be related to physiological processes of oocyte growing, cell proliferation, and follicular development, followed by CL formation after ovulation [42, 47].
By comparing the ion images of the mouse ovaries collected 0.5 and 3.5 days after ovulation, it is apparent that most of the detected lipids are concentrated in the CL, which presents high metabolic activity. The distribution of some specific lipid species varies among different CL in the same ovary and between the two developmental stages of CL activity. In CL at 0.5 day post-ovulation, FA species and FA dimers occur in higher relative intensities compared to the same species in CL at day 3.5 post-ovulation. Among the complex lipids, PI(38:4) was the dominant lipid species in the mass spectra of mouse CL and it was also present in the bovine and porcine ovarian stroma. Interestingly, C22:4 has slightly higher relative intensities in 3.5-day-old ovaries, which may play a role in luteal phase similarly to bovine ovaries [46, 48]. For complex lipids, tandem MS experiments for PS(38:4) were performed and indicate that there is a mixture of isomers of these fatty acyl combinations, while PI (38:4) likely consists either of one arachidonic (20:4) and one stearic (18:0) acid esterified to a glycerol backbone, or one adrenic (22:4) and one palmitic acid (16:0).
We acknowledge limitations in that the research presented here does not address some key physiological aspects of the ovarian physiology. We aimed at a high-level species comparison. Also, the samples used were from animals without precise record of the day of the cycle as well as breed for the bovine and swine, and we also have evaluated a limited time window for the mouse samples. Nonetheless, the results introduce new insights into the ovarian lipid content and roles, as well as point to the informative value DESI-MS imaging if applied to more controlled conditions and to specific physiological questions.
In this study, we were able to explore the location of lipids in swine, cattle, and mouse ovarian lipid composition at different physiological stages with organ anatomical information by relying on the non-destructive nature of DESI-MS imaging method of analysis. This approach allowed access in an untargeted way to specific lipid compositions and their distributions, as well as guiding the selection of the regions of interest for statistical analysis. Even though differences in the location of some lipids were observed, the multivariate statistical approach showed that corpora lutea, follicular wall (epithelium), and ovarian stroma have similar lipid signatures independent of the species, indicating conserved lipid-based physiological mechanisms of follicle recruitment. Major differences in lipid profiles were correlated with steroidogenic structures, demonstrating that specific lipids are related to corpora lutea development. Regarding lipid pathways, the abundance of adrenic acid suggests the importance of homo-prostaglandins in ovarian physiology. This finding was further supported by univariate and multivariate statistics.
All .raw files and Biomap-compatible DESI-MS imaging files are freely available via MassIVE (MSV00008462), a mass spectrometry data repository.
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We thank Dr. Francesca E. Duncan for her valuable comments on an initial draft of this manuscript.
This study received funding from Grant No. UL1TR002529 (A. Shekhar, PI) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical, and Translational Sciences Award; from the National Institute of Biomedical Imaging and Bioengineering, NIH Grant R21EB015722; from the National Institute of Allergy and Infectious Diseases, NIH Grant R01AI122298; and from the Purdue University Center for Cancer Research Small Grants Program.
The present study has used mice samples from the Purdue University Center for Cancer Research’s Transgenic Mouse Core Facility. The study received approval from the Purdue Animal Care and Use Committee (protocol 11-060).
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Cordeiro, F.B., Jarmusch, A.K., León, M. et al. Mammalian ovarian lipid distributions by desorption electrospray ionization–mass spectrometry (DESI-MS) imaging. Anal Bioanal Chem 412, 1251–1262 (2020). https://doi.org/10.1007/s00216-019-02352-6
- Mass spectrometry
- DESI-MS imaging