Analytical and Bioanalytical Chemistry

, Volume 408, Issue 27, pp 7607–7615 | Cite as

Microscopic visualization of testosterone in mouse testis by use of imaging mass spectrometry

  • Shuichi Shimma
  • Henri-Obadja Kumada
  • Hisanori Taniguchi
  • Alu Konno
  • Ikuko Yao
  • Kyoji Furuta
  • Tadashi Matsuda
  • Seiji ItoEmail author
Research Paper
Part of the following topical collections:
  1. New Applications of Mass Spectrometry in Biomedicine


Testosterone is one of the androgens synthesized from cholesterol as a precursor in the Leydig cells of testes. Since the ionization efficiency of testosterone in matrix-assisted laser desorption ionization (MALDI) is quite low, visualization of testosterone by using MALDI-imaging mass spectrometry (MALDI-IMS) has been considered difficult. To overcome this problem, we used two types of on-tissue derivatization techniques, which were achieved by pyridine sulfur trioxide and Girard’s T (GT) reagent, to introduce a polar group into testosterone molecule with the aim to increase the sensitivity. Derivatization by use of GT reagent provided excellent results, superior to those obtained with pyridine sulfur trioxide, in terms of ionization efficiency, molecular specificity, and tissue damage. In GT derivatized testis tissues of mice treated with human chorionic gonadotropin (hCG), testosterone was broadly observed both inside and outside the seminiferous tubules by using an iMScope. To evaluate our imaging results, we performed quantification experiments of underivatized testosterone extracted from hCG-treated testes and control testes using LC-MS/MS. We confirmed the 256-fold concentration change between hCG-treated tissues and control tissues. We also confirmed the 228-fold change in detected peak intensities between hCG-treated tissue sections and control tissue sections in imaging results. We consider our tissue preparation methods for IMS provide high sensitivity with high precision. In addition, high-spatial definition IMS was also available, and we confirmed testosterone had mainly accumulated on the surface of the Leydig cells.

Graphical abstract

Girard’s T-testosterone (GT-Ts) provides the fragment ion at m/z 343.24. Clear GT-Ts signal was detected in hCG treated mouse testis not only as spectra but also as a mass image


Imaging mass spectrometry iMScope Testosterone Testis Derivatization Girard’s T reagent 



Girard’s T reagent


Girard’s T derivatized testosterone


Imaging mass spectrometry


Matrix-assisted laser desorption/ionization




Testosterone sulfate ester


α-Cyano-4-hydroxycinnamic acid



This work was supported in part by Grants-in-Aid for Scientific Research (B) (25293137 to SI) and for Challenging Exploratory Research (26670291 to SI) from the Japan Society for the Promotion of Science and by a MEXT-supported program for the Strategic Research Foundation at Private Universities (SI). We wish to thank Prof. E. Fukusaki (Osaka University) and visiting Prof. J. Iida (Shimadzu) for the use of facilities in Osaka University Shimadzu Analytical Innovation Research Laboratory. We also thank Ms. M. Suzuki for the assistance with the LC-MS/MS analyses.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2016_9594_MOESM1_ESM.pdf (1.4 mb)
ESM 1 (PDF 1430 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Shuichi Shimma
    • 1
  • Henri-Obadja Kumada
    • 2
  • Hisanori Taniguchi
    • 2
    • 3
  • Alu Konno
    • 4
    • 5
  • Ikuko Yao
    • 2
    • 4
    • 5
  • Kyoji Furuta
    • 6
  • Tadashi Matsuda
    • 3
  • Seiji Ito
    • 2
    Email author
  1. 1.Division of Advanced Science and BiotechnologyGraduate School of Engineering of Osaka UniversitySuitaJapan
  2. 2.Department of Medical ChemistryKansai Medical UniversityHirakataJapan
  3. 3.Department of Urology and AndrologyKansai Medical UniversityHirakataJapan
  4. 4.Department of Optical Imaging, Institute for Medical Photonics Research, Preeminent Medical Photonics Education & Research CenterHamamatsu University School of MedicineHamamatsuJapan
  5. 5.JST, ERATO, Sato projectTokyoJapan
  6. 6.Regeneration and Advanced Medical ScienceGifu University Graduate School of MedicineGifuJapan

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