Fourier-Transform Infrared micro-spectroscopy (µFT-IR) was used for an in vitro investigation on spermatozoa (SPZ) samples separately collected from caput and cauda of mouse epididymis. SPZ are characterized by deep biochemical changes during the transit along the epididymis and they can constitute ideal candidates for a µFT-IR investigation thanks to the ability of this technique in analyzing cells at a molecular level. For FT-IR spectral analysis SPZ pellets were resuspended in 0.9% NaCl solution and drops of few microliters were used for spectra acquisition. For this purpose, a Perkin Elmer Spectrum One FT-IR spectrometer equipped with a Perkin Elmer Multiscope system infrared microscope (Mercury Cadmium Telluride detector) was used. Spectral acquisitions were performed in specular-reflection mode with thin layers of the sample (2 ml) put on metallic IR-reflective surface. Infrared spectra were obtained for the two different kinds of samples in the wavenumber range 4000-500 nm. The use of a univariate analysis allowed us to use average spectra. Differences were evidenced in the infrared spectra from caput and cauda SPZ and biochemical changes in protein, nucleic acid, lipid, and carbohydrate content of cells were observed. In order to have quantitative information about the changes occurring in the infrared spectra related to different experimental conditions, the ratios between the area of selected peaks were evaluated. This ratiometric approach can be useful for assessing changes in protein, nucleic acid, lipid, and carbohydrate components.
The present investigation indicates that µFT-IR can constitute a valuable tool for monitoring in an easy and fast way the changes suffered by SPZ during the transit along the epididymis.