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

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.