MASS SPECTROMETRY IMAGING STUDY OF LIPID METABOLITES IN THE ADULT MOUSE TESTIS

Significance of Topic

The testis is a highly specialized organ where lipid homeostasis supports both endocrine and exocrine functions essential for male fertility. Seminolipids, though comprising a small fraction of total lipids, play a pivotal role in germ cell maturation. Phosphatidylcholines and other glycerophospholipids contribute to membrane structure and signaling. Mapping the spatial distribution of these lipids at high resolution enables a deeper understanding of testicular physiology and potential pathological alterations.

Study Objectives and Overview

This work aimed to employ a multimodal mass spectrometry imaging (MSI) strategy—combining matrix‐assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI)—to chart the localization and relative abundance of lipid metabolites within adult mouse testis. The study compared positive and negative ion modes, evaluated spatial resolution effects, and leveraged statistical analyses to distinguish lipid signatures associated with different cell types and maturation stages of seminiferous tubules.

Methodology and Instrumentation

Sample Preparation and Ionization

• Mouse testis cryosections (10–12 µm) mounted on glass slides and stored at –80 °C.
• MALDI matrices: α-Cyano-4-hydroxycinnamic acid (5 mg/mL, ACN/H₂O 70/30) for positive mode; 9-aminoacridine (5 mg/mL, MeOH/H₂O 70/30) for negative mode.
• DESI solvent: 98% MeOH, 2% H₂O; no additional sample coating required.

Mass Spectrometry Imaging

• MALDI‐MSI: Waters SYNAPT G2-Si HDMS with Nd:YAG laser (355 nm), pixel size ~30 µm, mass range m/z 100–1200.
• DESI‐MSI: Waters Xevo G2-XS Q-ToF, pixel sizes 25 µm and 50 µm, flow rate 1.5 µL/min, capillary voltage 4.5 kV, nebulizing gas 5 bar.

Data Processing and Statistical Analysis

• Ion image reconstruction and region‐of‐interest (ROI) definition using HDI v1.5.
• Data mining in MassLynx, normalization and export to MetaboAnalyst.
• Unsupervised PCA and supervised OPLS-DA performed in EZ Info; comparative loadings and heatmaps generated.

Results and Discussion

Lipid Distribution by Ion Mode

• Negative MALDI: Seminolipids (e.g., m/z 795.53, 809.52, 767.50, 821.50, 823.50) localized predominantly within seminiferous tubule lumens; PI(38:4) enriched in Leydig and blood vessels.
• Positive MALDI and DESI: Phosphatidylcholines (PC 34:1, 34:2, 36:4, 38:5, 38:6) exhibited cell‐type specificity: early germ/Sertoli cells, mature germ cells, or Leydig/blood vessel regions.
• DESI positive mode uniquely detected triglycerides (m/z >900), absent in MALDI spectra.

Spatial Resolution Impact

• Pixel size of 25 µm in DESI yielded sharper ion images and finer structural details compared to 50 µm.

Multivariate Analysis

• PCA score plots revealed clear separation of ROI intensity profiles corresponding to three histological regions.
• Loadings plots identified key m/z values driving discrimination among cell types.

Benefits and Practical Applications

• In situ lipid mapping provides molecular insight into testis maturation stages and cellular heterogeneity.
• Complementary use of MALDI and DESI extends detectable lipid classes, including seminolipids, phospholipids, and triglycerides.
• Potential utility in reproductive biology research, biomarker discovery, and toxicology assessments targeting testicular function.

Future Trends and Applications

• Integration of higher spatial resolution techniques (e.g., sub‐micron MSI) for single‐cell lipidomics.
• Development of multimodal platforms combining MSI with immunohistochemistry or fluorescence imaging.
• Application of machine learning to automate ROI classification and lipid signature identification.
• Extension to human tissue studies for clinical and diagnostic purposes.
• Three‐dimensional reconstruction of lipid distributions across organ volumes.

Conclusion

This study demonstrated that MALDI and DESI MSI offer complementary views of testicular lipid architecture. Negative mode MALDI effectively visualized seminolipids and PIs, while positive mode DESI extended coverage to phosphatidylcholines and triglycerides. Spatially resolved lipid profiling enables discrimination of distinct cell populations and maturation stages within the mouse testis. Advances in spatial resolution and multimodal integration will further enhance our understanding of reproductive lipid biology.

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