Computational protocol: CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization

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Protocol publication

[…] The assays were performed in a modified Zigmond chamber consisting of two wells separated by a 2-mm partition wall (). One of the wells (W1) was filled with capacitated mouse sperm and the other one (W2) with medium either alone (control) or with attractants. Then, a 1D attractant concentration gradient was formed across the bridge in the partition wall. 15 min after sealing the chamber, sperm movement was recorded at 30 frames/s in the bridge with a Coolpix L20 camera (Nikon) in an Eclipse TS100 microscope (Nikon) using LWD 20×/0.40 Ph1 ADL ∞/1.2 WD 3.0 objective lenses. Sperm tracks were then analyzed with the ImageJ software (version 1.38; National Institutes of Health) and the MtrackJ plugin. The percentage of “oriented sperm” was calculated for 150 analyzed tracks per treatment with the SpermTrack software (version 4.0, Universidad Nacional de Córdoba). For each sperm track, orientation toward W2 was calculated as a ratio between the distances traveled over the X and Y axes (ΔX/|ΔY|). When the value of the ratio was >1, the spermatozoon was considered oriented to W2. A CRISP1 dose–response curve (ranging from 1 pM to 10 µM) was performed. Medium alone was used as a negative control and 100 pM progesterone or COC-conditioned medium (COC from five oviducts in 100 µl, overnight at 37°C) was used as a positive control. The pattern of movement was evaluated by means of the fractal dimension (FD; ). As FD values were analyzed at 30 Hz, the motility pattern of spermatozoa was classified as linear (FD < 1.3), transitional (1.3 < FD < 1.8), or hyperactivated (FD > 1.8; ). […]

Pipeline specifications

Software tools ImageJ, MTrackJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens