Analytical Techniques in Order to Measure Sperm FISH
Due to the nature of the spermatozoa, FISH protocol for sperm analysis requires the following steps, which are also summarized in Figure 6.1.
Previous to the hybridization, spermatozoa must be fixed maintaining their morphology and allowing permeability to the DNA probes. After centrifugation with sperm washing media, the supernatant containing the seminal plasma is discarded and the pellet with the spermatozoa is fixed using Carnoy solution (methanol/glacial acetic acid = 3:1). The fixed spermatozoa are spread on glass slides avoiding overlapping.
Sperm heads have a tightly compacted nucleus due to the presence of disulfide bridges between protamines; this condensation of nuclear chromatin makes it inaccessible to DNA probes. To solve this
FIGURE 6.1 The steps in (a) sperm fixation and (b) hybridization.
problem, a pretreatment is performed by incubation with reducing agents and dehydration with ethanol. Reducing agents (e.g., dithiothreitol [DTT]) break the disulfide bonds and produce nuclear chromatin decondensation allowing subsequent hybridization with DNA probes.