Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation

Abstract BACKGROUND: Reports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation. METHODS: Protein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis. 2-D gels were stained with Sypro Ruby. Map images of DC and SU spermatozoa were compared using dedicated software. Intensities of a given spot were considered different between DC and SU when their group mean differed by >1.5-fold (p<0.05, Anova). RESULTS: No differences were observed for 853 spots, indicating a 98.7% similarity between DC and SU. Five spots were DC>SU and 1 was SU>DC. Proteins present in 3 of the differential spots could be identified. One DC>SU spot contained lactate dehydrogenase C and gamma-glutamylhydrolase, a second DC>SU spot contained fumarate hydratase and glyceraldehyde-3-phosphate dehydrogenase-2, and a SU>DC spot contained pyruvate kinase M1/M2. CONCLUSIONS: The differences in protein levels found on comparison of DC with SU spermatozoa indicate possible dissimilarities in their glycolytic metabolism and DNA methylation and suggest that DC cells may have a better capacitation potential

Effects of the Insemination of Hydrogen Peroxide-Treated Epididymal Mouse Spermatozoa on γH2AX Repair and Embryo Development

BACKGROUND: Cryopreservation of human semen for assisted reproduction is complicated by cryodamage to spermatozoa caused by excessive reactive oxygen species (ROS) generation. METHODS AND FINDINGS: We used exogenous ROS (H(2)O(2)) to simulate cryopreservation and examined DNA damage repair in embryos fertilized with sperm with H(2)O(2)-induced DNA damage. Sperm samples were collected from epididymis of adult male KM mice and treated with capacitation medium (containing 0, 0.1, 0.5 and 1 mM H(2)O(2)) or cryopreservation. The model of DNA-damaged sperm was based on sperm motility, viability and the expression of γH2AX, the DNA damage-repair marker. We examined fertility rate, development, cell cleavage, and γH2AX level in embryos fertilized with DNA-damaged sperm. Cryopreservation and 1-mM H(2)O(2) treatment produced similar DNA damage. Most of the one- and two-cell embryos fertilized with DNA-damaged sperm showed a delay in cleavage before the blastocyst stage. Immunocytochemistry revealed γH2AX in the one- and four-cell embryos. CONCLUSIONS: γH2AX may be involved in repair of preimplantation embryos fertilized with oxygen-stressed spermatozoa

MRI of the kidney—state of the art

Ultrasound and computed tomography (CT) are modalities of first choice in renal imaging. Until now, magnetic resonance imaging (MRI) has mainly been used as a problem-solving technique. MRI has the advantage of superior soft-tissue contrast, which provides a powerful tool in the detection and characterization of renal lesions. The MRI features of common and less common renal lesions are discussed as well as the evaluation of the spread of malignant lesions and preoperative assessment. MR urography technique and applications are discussed as well as the role of MRI in the evaluation of potential kidney donors. Furthermore the advances in functional MRI of the kidney are highlighted

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation