Abstract
Raman microspectroscopy (RM) and polarization sensitive digital holographic imaging (PSDHI) are valuable analytical tools in biological and medical research, allowing the detection of both biochemical and morphological variations of the sample without labels or long sample preparation. Here, using this multi-modal approach we analyze in vitro human sperm capacitation and the acrosome reaction induced by heparin. The multimodal microscopy provides morphofunctional information that can assess the sperms ability to respond to capacitation stimuli (sperm function). More precisely, the birefringence analysis in sperm cells can be used as an indicator of its structural normality. Indeed, digital holography applied for polarization imaging allows for revelation of the polarization state of the sample, showing a total birefringence of the sperm head in non-reacted spermatozoa, and a birefringence localized in the post-acrosomal region in reacted spermatozoa. Additionally, RM allows the detection and spectroscopic characterization of protein/lipid delocalization in the plasma and acrosomal membranes that can be used as valuable Raman biomarkers of sperm function. Interestingly, these spectral variations can be correlated with different time phases of the cell capacitation response. Although further experimentation is required, the proposed multimodal approach could represent a potential label-free diagnostic tool for use in reproductive medicine and the diagnosis of infertility.
Highlights
Sperm cell capacitation is an essential step in the fertilization process; it represents one of the most important aspects of semen assessment[1,2]
Owing to longitudinally oriented protein filaments, human sperm cells are naturally birefringent when observed under polarized light, while after the acrosome reaction, the local protein organization disaggregates leading to loss of the birefringence in the acrosomal region[18]; reacted spermatozoa show a partial head birefringence[19], typically in the post-acrosomal region
CTC-staining patterns were classified into three types according to the method described by Fraser et al.[43,44]: uncapacitated-spermatozoa, bright fluorescence detected over the whole region of the sperm head; capacitated-spermatozoa, fluorescence detected on the sperm head but not over the post-acrosomal region; and acrosome-reacted-spermatozoa, weak fluorescence observed over the sperm head with a bright band sometimes present in the equatorial region (Fig. 1a)
Summary
Sperm cell capacitation is an essential step in the fertilization process; it represents one of the most important aspects of semen assessment[1,2]. (i) The trigger event is the removal of the decapacitation factors (i.e. glycoproteins)[4] These proteins are present in the epydidimal fluid and in the seminal plasma, stabilizing the sperm membrane and keeping spermatozoa in a non-capacitated state along the lower female tract. Spermatozoa incubated with heparin are subjected to a cascade of events leading to specific modifications of the lipid/protein content and localization in the acrosomal region, mainly involving the plasma and outer acrosomal membranes, as described above. Moody and colleagues validated a Cap-Score Sperm Function Test[15] based on the analysis of the glycolipid (monosialotetrahexosylganglioside GM1) localization patterns, reflecting the capacitated state in human sperm These assays may classify an infertile man according to the type and degree of his spermatogenetic defect, these protocols provide limited information about how well a sperm will function in in vitro or in vivo settings. RM has been used to detect protein denaturation, DNA fragmentation and lipid peroxidation in human sperm cells[30,39,41,42]
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