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Simultaneous Observation of DNA Fragmentation and Protein Loss in the Boar Spermatozoon Following Application of the Sperm Chromatin Dispersion (SCD) Test

JOSE LUIS FERNÁNDEZ

STEVE D. JOHNSTON

From the ^* Departamento de Biología, Universidad Autónoma de Madrid, Madrid, Spain; the Sección de Genética y Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo, Coruña, Spain; and the School of Animal Studies, The University of Queensland, Gatton, Australia.

Correspondence to: Dr Jaime Gosálvez, Departamento de Biología, Unidad de Genética, Edificio de Biología, Universidad Autónoma de Madrid, C/Darwin no 2, 28049 Madrid, Spain (e-mail: jaime.gosalvez{at}uam.es).

DNA fragmentation and the nuclear protein matrix in boar spermatozoa were simultaneously assessed using a specific variant of the sperm chromatin dispersion (SCD) test that allows direct visualization of DNA and nuclear proteins under standard conditions of chemical lysis. Nuclear proteins remaining after lysis were stained with the fluorochrome 2,7-dibrom-4-hydroxy-mercury-fluorescein for specific protein staining. DNA and nuclear protein were stained in control-untreated (no lysis) and treated sperm cells (lysis), resulting in the identification of 3 cell types: type 1: nonlysed (control-untreated) cells; type 2: lysed cells showing nonfragmented DNA; and type 3: lysed cells showing fragmented DNA. DNA damage was also purposely induced by incubating the sperm in 0.015% H₂O₂ for 48 hours at 37°C; the cells were correspondingly stained for DNA fragmentation and protein. Nonlysed control sperm (type 1) nuclei showed no halos and stained strongly for protein in the postacrosomal region. Lysed spermatozoa with nonfragmented DNA (type 2) showed evidence of restricted DNA loop dispersions at the caudal extremity of the sperm head and a more homogenous but similar distribution of protein matrix in comparison with untreated spermatozoa. Lysed spermatozoa with fragmented DNA (type 3) exhibited large halos of DNA loops and a loss of the nuclear protein matrix component. Sperm cells exposed to 48 hours' incubation at 37°C and then treated with the lysing agent showed a concurrent and progressive loss of nuclear protein in association with correspondingly increased levels of DNA fragmentation. Discriminant analysis of quantitative fluorescence using digital image analysis and conducted after SCD processing revealed that DNA fragmentation and protein could be evaluated in an automated system. Ninety-seven percent of the total analyzed cells were accurately classified according to previously defined cell types (1, 2, and 3). The results of the current study demonstrated a synergistic relationship between that of nuclear protein alteration and DNA damage in the boar sperm cell. The importance of abnormal nuclear protein alteration to DNA fragmentation and any related effect on fertility remains to be investigated.

     Key words: Sperm chromatin structure, spermatogenesis, DNA damage, sperm nuclear proteins, reproduction