CFAS EXCHANGE LECTURE: COMPARISON OF SPERM NUCLEAR DECONDENSATION PROTOCOLS USING UNBIASED SAMPLING AND DIGITAL IMAGE MORPHOMETRY.

Introduction: Human spermatozoa have unique, well-organized nuclear architecture different from somatic cells. Improperly packaged sperm chromatin will have a high probability of disrupting the particularly structured sequence of fertilization. The evaluation of nuclear organization in spermatozoa is challenging due to the extreme compactness of chromatin and the absolute necessity for induced nuclear decondensation (ND), which itself alters native sperm architecture. The purpose of our study was to identify sperm ND protocol that results in minimal disturbance of sperm architecture. Materials and Methods: Three widely used ND protocols for fluorescence in situ hybridization (FISH) were evaluated in this study: protocol 1 was combined denaturation and decondensation with 0.5N NaOH; in protocol 2, decondensation was achieved with 10mM dithiothreitol (DTT) and lithium diiodosalicylate (LIS); in protocol 3, 2.5mM DTT and heparin was used for ND. Protocols 2 and 3 were followed by denaturation with formamide at 75°C. The effect of denaturation and hybridization on spontaneous ND was also evaluated. Samples were counterstained with DAPI. Images were acquired using unbiased sampling and digital image morphometry analysis by Visiopharm Integrator System. Results were compared to neat samples using SPSS 16.0 statistical computer software. Results: Comparison of ND protocols is provided in the table. Mild decondensation was defined as 1.5 3 fold increase in nuclear area. Spontaneous ND occurred in an unexpectedly large proportion of spermatozoa during denaturation (74%) and hybridization (39%). Conclusions: Combined denaturation and decondensation with 0.5N NaOH demonstrated superior results providing uniform mild ND with over 80% of nucleus suitable for FISH sperm architecture assessment. The influence of spontaneous ND should be considered in any FISH preparation protocols. [ABSTRACT FROM AUTHOR]