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.
Accumulating evidence demonstrates that cilia play important roles in a variety of processes in embryogenesis. For functional survey of larval cilia at the cellular level, we exploited the simple cell organization of tadpole larvae in the ascidian Ciona intestinalis. Immunofluorescent microscopy showed distribution of cilia not only in previously described tissues but also in a subpopulation of ependymal cells in the sensory vesicle, gut primordium, papillae, apical trunk epidermal neurons, and the endodermal strand. Transmission electron microscopy revealed a variety of axonemal structures, including a 9+0 structure similar to vertebrate primary cilia, a 9+0 structure with electron-dense materials in the center, a 9+2 structure with no dynein arms, and an axoneme with a disorganized structure at the distal end. Extensive description of cilia in the present study gives important insights into the evolution of the ciliary structure and provides a basis for analysis of ciliary functions in establishment of chordate body plan.
In ascidian Ciona intestinalis, a subset of trunk epidermal neurons were shown to possess external network of neural projections. To characterize a more complete network in naturally hatched (chorionated) larvae, we visualized the structure with a confocal laser scanning microscope. High resolution images revealed the huge network consisting of several subnetworks in whole-larval tunic. We named this network the ASNET (ascidian dendritic network in tunic). The ASNET was dynamically generated and collapsed during larval stages. Interestingly, one of the subnetworks found around apical trunk epidermal neurons was bilaterally asymmetric. In caudal epidermal neurons, transmission electron microscopy revealed that 912 axonemes were accompanied by a vesicle-containing mass in the ASNET arbor, but the distal end of the arbor contained only the vesicle-containing fibrous mass and no 912 axonemes. The characteristics of the ASNET suggest that it forms a unique outer body network in the ascidian larval tunic.
Vessel painting is one of the most accessible and cost-effective techniques for visualising vasculature by fluorescence microscopy. In this method, the hydrophobic carbocyanine dye DiIC18 labels the plasma membrane via insertion of its alkyl chains into the lipid bilayer. A major disadvantage of this procedure is that it does not stain veins and some microvessels in mouse brain. Furthermore, DiIC18 molecules can aggregate during perfusion, thereby occluding arteries and reducing the success rate and reproducibility of the experiment. To overcome these problems, we developed an improved vessel painting procedure that employs neutral liposomes (NLs) and DiIC12. NLs prevented DiI aggregation under physiological conditions whereas DiIC12 showed enhanced dye incorporation into liposomes and consequently increased staining intensity. Using this method, we successfully labelled all major blood vessel types in the mouse brain, including both veins and microvessels. Thus, liposome-mediated vessel painting is a simple and efficient method for visualising vasculature.
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