Abstract
Sperm cells progressive motility is the most important parameter involved in the fertilization process. Sperm middle piece contains mitochondria, which play a critical role in energy production and whose proper operation ensures the reproductive success. Notably, sperm progressive motility is strictly related to mitochondrial membrane potential (MMP) and consequently to mitochondrial functionality. Although previous studies presented an evaluation of mitochondrial function through MMP assessment in entire sperm cells samples, a quantitative approach at single-cell level could provide more insights in the analysis of semen quality. Here we combine laser scanning confocal microscopy and functional fluorescent staining of mitochondrial membrane to assess MMP distribution among isolated spermatozoa. We found that the sperm fluorescence value increases as a function of growing progressive motility and that such fluorescence is influenced by MMP disruptors, potentially allowing for the discrimination of different quality classes of sperm cells in heterogeneous populations.
Highlights
Human spermatozoa rely mainly on two metabolic pathways to obtain energy: anaerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS)[1,2]
The obtained pellet was incubated with 200 nM solution of MitoTracker in order to obtain a selective fluorescence from the inner mitochondria membrane in the middle piece[21]
A signal to noise ratio comparable with the one obtained for MT-G was observed at two-time higher excitation power. 200 nM was selected as the best concentration of MT-G to stain only active mitochondria from live sperm cells with the lowest progressive motility, and to distinguish them from the noise
Summary
Human spermatozoa rely mainly on two metabolic pathways to obtain energy: anaerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS)[1,2]. Sperm mitochondria are unique organelles that participate in many crucial processes, the major being the synthesis of adenosine triphosphate (ATP) through the sophisticated mechanism of the oxidative phosphorylation (OXPHOS)[1,2] This process requires the coordinated operation of two main components: the respiratory chain and the ATP-synthase, both located in the inner mitochondrial membrane. Several studies reported that mitochondrial functionality is associated with sperm quality, especially with sperm motility, an essential factor in the diagnosis of male infertility. With respect to MitoTracker, JC-1 has the peculiarity of creating agglomeration that red-shifts the emission wavelength With this approach, Paoli et al were able to demonstrate that both human sperm motility and viability are associated with MMP. The link between high MMP and high viability and motility is clear from the literature, to the best of our knowledge currently available approaches only distinguish between the existence or not of a detectable fluorescence signal from the analyzed cells
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