Intracytoplasmic Sperm Injection by Gianpiero D. Palermo & E. Scott Sills

Author:Gianpiero D. Palermo & E. Scott Sills
Language: eng
Format: epub
Publisher: Springer International Publishing, Cham

7.2.1.3 Enzymatic Treatment

The original protocol, as well as most currently used protocols, include an exposure to the broad-spectrum serine protease Proteinase K to remove protamines, thereby allowing sperm DNA to decondense and migrate [13]. However, there are discrepancies in this technique: incubation with Proteinase K can be done directly in the lysis solution or as an additional step after lysis, performed at different concentrations, either at room temperature or 37 °C, and for durations varying from 3 to 18 h [8]. Prolonged incubations with high concentrations of Proteinase K have been reported to increase the baseline DNA damage observed with sperm [17]. Whether this is attributable to de novo DNA damage or further supercoil relaxation is still unclear. Other protocols include two enzymatic digestion steps with RNAse and Proteinase K [18]. Such variations in the procedure can greatly affect the sensitivity of the assay and aggravate the inability to compare data between laboratories. More recently, protocol modifications introduced incubation with the chaotropic agent LIS instead of Proteinase K [14, 15, 19]. This permitted reduction of both experimental time and background DNA damage [15]. Hence, LIS appears to be the better option as it shortens and simplifies the process and prevents agarose softening as, unlike Proteinase K, it is active at room temperature.

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