Autologous whole ram seminal plasma and its vesicle-free fraction improve motility characteristics and membrane status but not in vivo fertility of frozen-thawed ram spermatozoa

Motility characteristics (assessed subjectively and with computer-assisted semen analysis) and membrane status (after staining with chlortetracycline) of washed and non-washed frozen-thawed ram spermatozoa were evaluated after incubation in buffer and buffer containing autologous whole seminal plasma or one of its two fractions: the pellet of membrane vesicles obtained by ultracentrifugation (and used at three times normal protein concentration) or the vesicle-free supernatant fraction. Whole seminal plasma and supernatant, but not membrane vesicles, improved the motility characteristics of spermatozoa after 3 and 6 h of post-thaw incubation compared with the control buffer. Resuspension and incubation with whole seminal plasma, supernatant or membrane vesicles lowered the proportion of acrosome-reacted frozen-thawed spermatozoa compared with the control buffer. Unwashed frozen-thawed semen from three rams, incubated with autologous whole seminal plasma or its fractions and inseminated using cervical or intrauterine artificial insemination, had no effect on pregnancy rates of ewes in synchronized oestrus. However, fertility was higher after laparoscopic than cervical insemination (44.9 vs 12.3%, p < 0.001). In conclusion, resuspension and incubation of frozen-thawed ram spermatozoa in autologous whole seminal plasma or its vesicle-free supernatant fraction improved their motility characteristics and, with membrane vesicles, membrane status, but these benefits were not reflected in improved fertility after cervical or intrauterine insemination. © 2007 The Authors

Supplementary Material for:A Paediatric Acute Promyelocytic Leukaemia Patient Harbouring a Cryptic PML-RARA Insertion due to a Complex Structural Chromosome 17 Rearrangement

Acute promyelocytic leukaemia with <i>PML-RARA</i> fusion is usually associated with the t(15;17)(q24.1;q21.1) translocation but may also arise from complex or cryptic rearrangements. The fusion usually resides on chromosome 15 but occasionally on others. We describe a cryptic <i>PML-RARA </i>fusion within a novel chromosome 17 rearrangement. We performed interphase fluorescence in situ hybridisation (FISH) using a dual-fusion <i>PML-RARA </i>probe, followed by reverse transcriptase-polymerase chain reaction (RT-PCR) for <i>PML-RARA</i>, karyotyping, and metaphase FISH using <i>RARA</i>break-apart, locus-specific, and subtelomere probes for chromosome 17. An 850K SNP microarray was also employed. Interphase and metaphase FISH showed atypical results involving a single <i>PML</i>-<i>RARA</i> fusion, no second fusion, but instead separate diminished <i>PML</i> and <i>RARA</i> signals. RT-PCR confirmed <i>PML-RARA </i>fusion; however, karyotyping detected only an altered chromosome 17. Metaphase FISH showed the single fusion and diminished 5′<i> RARA</i> signals located unexpectedly in the subtelomeric short-arm and long-arm regions of the rearranged chromosome 17, respectively. SNP microarray revealed no copy number abnormality. This paediatric patient with <i>PML-RARA</i> fusion reflects a cryptic insertion that resides within a complex and novel chromosome 17 rearrangement. This rearrangement likely arose via 7 chromosome breaks with the insertion occurring first followed by sequential paracentric and then pericentric inversions