Methodology for Analysis of Caspase in Human Sperm Detection of mRNA Caspases

The presence of transcripts in human spermatozoa has been initially established using reverse transcription (RT)-PCR and in situ hybridization (ISH). Sperm pellets are suspended in medium and incubated with P-labeled uridine triphosphate (UTP) that can easily permeate the plasma membrane. The purity and quantity of RNA could be determined via the measurement of the absorbance at 280 and 260 nm, respectively. RNA could be visualized with UV irradiation after ethidium bromide staining in loading buffer and the total RNA from each sample should be used for RT-PCR protocol. Primary human dermal fibroblast could be used as controls. Using this approach, Grunewald et al. have demonstrated caspase-3 mRNA in human spermatozoa of fertile donors [54, 55]. Fluorometric Assays

Caspases can be detected in living spermatozoa by fluorescence-labeled inhibitors of caspases (FLICA). This method could be used to show spermatozoa with caspase activation in a semen sample. FLICA includes a green fluorescent label (FAM) and an amino acid peptide inhibitor that attaches to the active caspase. These inhibitors are cell permeable and nontoxic [56]. High specificity ofFLICA for detection of caspase activation has been shown [57]. Fluorescence microscopy or flow cytometry in spermatozoa has been used to detect FLICA. The application is easy to perform and varying fluorescence inhibitors of caspases are available; it has been proven that the evaluation of active caspase-3 is possible up to 10 days after staining of human sperm [58], Colorimetric Assays

Activated caspases can be detected on the same principles of fluorescence-labeled inhibitors. A chromophore is attached to the caspase inhibitor, and after cleavage by the active protease, it becomes fluorescent. This color could be evaluated by spec-trophotometer at 405 nm. This method is not suitable for single cell, but it has been used for the seminal ejaculate [14]. Western Blotting

This method is suitable for detailed analysis of caspase proenzyme, inactivated and activated caspase. Caspases-1, -3, -7, -8, -9, and -12 could be detected using western blotting. This approach requires an appropriate number of sperm for protein extraction. This may be the reason for the lack of detection of caspase-3 in human sperm in some reports [59] . Immunohistochemistry

Detection of caspases in testes was accomplished by immunohistochemistry methods [22, 26, 60-63]. Using this technique, caspases activation was detected during physiologic conditions such as germ cell proliferation and maturation [64] as well as environmental factors such as heat stress, irradiation, toxicants, or supraphysio-logical levels of prolactin [12].

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