DNA fragmentation dynamics in fresh versus frozen thawed plus gradient-isolated human spermatozoa.

The aim of this study was to compare the rate of sperm DNA fragmentation (rSDF; increase of SDF over time) in fresh and gradient isolated frozen-thawed semen samples from male sperm donors of proven fertility. SDF was assessed in the two samples obtained from the same fifteen male donors after 0.5, 1.5, 4.5, 6, 24, 48, and 72 h of incubation in a humidified atmosphere of 5% CO(2) in air at 37 degrees C. Analysis was performed based on chromatin dispersion patterns evaluated using the Halosperm kit. No significant differences in SDF were obtained when fresh and gradient-isolated frozen-thawed spermatozoa were compared at baseline. However, the rSDF shown by the two samples differed and gradient-isolation selected for sperm subpopulations showing a lower variance for SDF. At the individual level, sperm selection by density gradient purification in frozen-thawed samples did not improve the levels of SDF when compared with the values obtained in fresh samples at baseline.

Simultaneous observation of DNA fragmentation and protein loss in the boar spermatozoon following application of the sperm chromatin dispersion (SCD) test.

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% H(2)O(2) for 48 hours at 37 degrees 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 degrees 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.

Low levels of chromosomal differentiation between the grasshoppers Chorthippus brunneus and Chorthippusjacobsi (Orthoptera; Acrididae) in northern Spain.

The grasshopper species Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae) form a hybrid zone in northern Spain. These species probably diverged while isolated in southern refugia during one of the recent ice ages, and are clearly distinguished by morphology and male calling song. However, in contrast to other Chorthippus taxa that form hybrid zones in Europe, these two species cannot be reliably distinguished on the basis of characteristics of the karyotype such as heterochromatin banding patterns and composition, as revealed by C-banding and fluorochrome staining. Silver staining also reveals the presence of two autosomal nucleolar organiser regions (NORs) in both species. However, differentiation between C. brunneus and C. jacobsi was revealed on the X chromosome using fluorescent in situ hybridisation (FISH). C. brunneus individuals showed additional rDNA sequences on the X chromosome that were not observed in any C. jacobsi individuals. These sequences are not transcribed, indicating either mutational silencing of an ancestral NOR on the X chromosome, or the transposition of non-functional sequences from the autosomes. The implications of these results for the evolution of NOR number in Chorthippus are discussed.