New cytogenetic data on bladder carcinoma cell line (CHA89) revealed by M-FISH analysis.
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Chromosome Aberrations↗
Biomedical subjects
D Boucher
Publications and source records attributed to D Boucher.
Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa.
Semen cryopreservation is an important procedure in the treatment of human infertility. However, the ability of spermatozoa to survive freeze/thaw processes varies between patients. Cryopreservation-induced stress may result in membrane injury with consequent loss of sperm motility and viability. We investigated the relationship between the physico-chemical state of the human sperm membranes and their tolerance to cryopreservation. Conventional characteristics of 20 semen samples were analysed before and after cryopreservation as well as their membrane fluidity assessed by measuring the fluorescence polarization anisotropy, which is inversely proportional to the fluidity. Correlation between fluidity and post-thaw recoveries of motile and viable spermatozoa were examined. Results showed that membrane anisotropy markedly varies between patients. In cryopreserved spermatozoa, anisotropy values were significantly higher than in fresh spermatozoa. Furthermore, recovery of motile and viable spermatozoa after freeze/thaw was strongly correlated with anisotropy of fresh spermatozoa (P < 0.05). The higher the membrane fluidity was before freezing, the better was the response of spermatozoa to cryopreservation. The results indicate that the freeze/thaw process results in a rigidifying effect on the sperm membrane and suggest that sperm adaptability to freeze/thaw-induced stress could be dependent on their initial membrane fluidity. The latter finding has practical implications for predicting the response of spermatozoa following freezing and thawing and for improving the recovery of viable spermatozoa.
Cell Membrane↗
Sex chromosome mosaicism in males carrying Y chromosome long arm deletions.
Microdeletions of the long arm of the Y chromosome (Yq) are a common cause of male infertility. Since large structural rearrangements of the Y chromosome are commonly associated with a 45,XO/46,XY chromosomal mosaicism, we studied whether submicroscopic Yq deletions could also be associated with the development of 45,XO cell lines. We studied blood samples from 14 infertile men carrying a Yq microdeletion as revealed by polymerase chain reaction (PCR). Patients were divided into two groups: group 1 (n = 6), in which karyotype analysis demonstrated a 45,X/46,XY mosaicism, and group 2 (n = 8) with apparently a normal 46,XY karyotype. 45,XO cells were identified by fluorescence in-situ hybridization (FISH) using X and Y centromeric probes. Lymphocytes from 11 fertile men were studied as controls. In addition, sperm cells were studied in three oligozoospermic patients in group 2. Our results showed that large and submicroscopic Yq deletions were associated with significantly increased percentages of 45,XO cells in lymphocytes and of sperm cells nullisomic for gonosomes, especially for the Y chromosome. Moreover, two isodicentric Y chromosomes, classified as normal by cytogenetic methods, were detected. Therefore, Yq microdeletions may be associated with Y chromosomal instability leading to the formation of 45,XO cell lines.
Adult↗
Prostasomes inhibit the NADPH oxidase activity of human neutrophils.
Prostasomes are particular lipid vesicles secreted by the prostate in human semen and involved in several physiological functions such as the improvement of sperm motility or immunomodulation. We have previously shown that they reduced the overall reactive oxygen species (ROS) production of seminal polymorphonuclear neutrophils (PMN). The present study was conducted to define the mechanism by which prostasomes inhibit the ROS production of blood and seminal PMN. The luminol chemiluminescence measuring total ROS production of blood PMN stimulated by either a phorbol ester (PMA) or a chemoattractant peptide, formyl-Met-Leu-Phe (fMLP) was significantly inhibited by prostasomes. The NADPH oxidase activity of the PMN was measured by 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1, 2-a]pyrazin-3-one (MCLA) chemiluminescence. Prostasomes inhibited the NADPH oxidase activity of blood or seminal PMN and increased the lag-phase of the enzyme after PMA stimulation. Prostasomes also inhibited significantly the NADPH oxidase activity of fMLP stimulated blood PMN, but the inhibition was not significant for seminal PMN. The lipid composition of blood PMN was analysed and compared to the lipid composition of prostasomes. This showed that prostasomes had a high cholesterol:phospholipid molar ratio and a high proportion of sphingomyelin. Together with the fact that prostasomes can rigidify the plasma membrane of blood PMN, these results led us to postulate that prostasomes inhibit the NADPH oxidase activity of PMN by lipid transfer from the prostasomes to the plasma membrane of the PMN.
Cholesterol↗
Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.
Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.
Animals↗
Screening for microdeletions on the long arm of chromosome Y in 53 infertile men.
About 30% of couple infertilities are of male origin. They appear in some cases de novo and are considered idiopathic. The aim of our work was to evaluate, in these cases, the prevalence of microdeletions of the long arm of chromosome Y, within the AZF a, b and c regions using molecular biology techniques. Men with azoospermia or oligozoospermia resulting from hereditary, endocrine or obstructive causes, or with a constitutional cytogenetic abnormality were excluded. Fifty-three infertile men with azoospermia or oligozoospermia, as determined by a spermiogram, were studied. Of these, 34 were idiopathic and 7 exhibited a past history of genital infection or biological abnormalities, suggesting partial obstruction of the genito-urinary tract. A further 8 men had a varicocele and 11 cases with a history of cryptorchidism were also studied. Peripheral blood DNA was extracted from each patient, then amplified by multiplex PCR with STS genomic markers from the three Y chromosome AZF zones. PCR products were then analysed on agarose gels. In view of the difficulty of confirming the absence of a signal in molecular biology, each case suspected of having a deletion was checked by multiplex PCR through coamplification with the SRY marker. Five men with microdeletions of the long arm of the Y chromosome were diagnosed among the 53 patients. All of them included the AZFc zone and the intragenic DAZ gene markers. Furthermore, a larger Y chromosome deletion encompassing the 3 AZF zones was diagnosed, and confirmed by cytogenetic analysis. All Y chromosome microdeletions were observed in the 34 truly idiopathic azoospermia/oligozoospermia cases, corresponding to a proportion of 14.7% (or 9.4% considering the whole population of 53 infertile men). The relatively high proportion of microdeletions found in our series suggests the need for strict patient selection to avoid unnecessary screening for long arm Y chromosome microdeletions.
Adult↗
Influence of seminal plasma on cryopreservation of human spermatozoa in a biological material-free medium: study of normal and low-quality semen.
The objective was to evaluate the efficiency of a biological material-free medium and the role of seminal plasma (SP) in the cryopreservation of human spermatozoa. Normal semen samples and low-quality semen samples were used for this study. After centrifugation of 300 microL fractions of whole semen, pellets were resuspended either in autologous SP or in a chemically defined medium (BM) supplemented or not with 3% bovine serum albumin (BSA); after 15 min at 37 degrees C, the samples were diluted (V/V) with cryoprotective medium (30 mM NaCl; 22 mM sodium citrate, 19.4 mM fructose; 80 mM glutamine; 14%, V/V, glycerol) and maintained for 15 min at room temperature before freezing. Assessment of viability and motility was performed using fresh semen (T0), after centrifugation and resuspension prior to adding the cryoprotectant (T15), after adding the cryoprotectant (T30) and after freezing and thawing (Tpost). In all three resuspending media used, sperm viability and motility (forward and total) decreased (p < 0.05) during both the equilibration period especially before addition of the cryoprotective medium (between T0 and T15) and during the freeze-thaw process comparison between T30 and Tpost. The recovery of viable and motile spermatozoa (post-thaw values/values of fresh samples) was higher (p < 0.05) in normal semen than in low-quality semen. In both groups, the recovery was slightly, but significantly, higher with SP than with BM and the presence of BSA has no beneficial effect. To conclude, these data suggest that SP may reduce the deleterious effects of cryopreservation. Nevertheless cryopreservation of spermatozoa in a medium containing neither SP nor biological substances could offer an acceptable cryoprotection of spermatozoa to be used in assisted fertilization procedures, especially for intracytoplasmic sperm injection.
Animals↗
Screening for cystic fibrosis transmembrane conductance regulator gene mutations in men included in an intracytoplasmic sperm injection programme.
The present study was undertaken to evaluate the frequency and nature of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in infertile patients undergoing intracytoplasmic sperm injection. A total of 90 patients were screened for a panel of 10 mutations in the CFTR gene frequently involved in congenital absence of the vas deferens (CAVD); the patients included 14 with azoospermia and CAVD, 39 patients with azoospermia without CAVD (n = 39) and 37 patients with severe oligozoospermia. The length of the polymorphic polypyrimidine tract (allele 5T, 7T and 9T) in the intron 8/exon 9 splice-acceptor site was also determined. In 10 out of 14 patients with CAVD, CFTR mutations were found; nine patients had one DeltaISOdiaDeltaF508 mutation and one patient had two CFTR mutations (N1303K/R117H). Allele 5T was present in eight of these patients. In six patients, 5T was the non-DeltaISOdiaDeltaF508 allele and in two patients there was no known CFTR mutation. None of the CFTR mutations were observed in patients with azoospermia without CAVD or with severe oligozoospermia and the frequency of allele 5T was 3.6% (three out of 78 alleles) and 1.35% (one out of 74 alleles) respectively. Our observation suggests that the CFTR gene is not involved in either spermatogenesis or in the pathology of the genital tract, except for CAVD.
Adult↗
Chronic interstitial nephritis due to 5-aminosalicylic acid.
Nephrotoxicity has recently been reported with the use of 5-aminosalicylic acid (5-ASA) which has structural similarities to phenacetin and aspirin. The present paper describes 2 cases of interstitial nephritis and 1 case of end-stage failure associated with 5-ASA treatment. The first patient presented with severe renal failure which was partially reversed with 5-ASA discontinuation and steroid therapy. The second had severe renal failure (serum creatinine 469 mmol/l) but renal function stabilized with 5-ASA withdrawal. The third patient had end-stage renal failure and underwent hemodialysis and a successful kidney transplant.
Adult↗
Clinical evaluation of the CDI-100 in-line hematocrit/saturation monitor.
This study was undertaken to evaluate the accuracy, reliability, consistency and biases of the CDI-100 saturation monitor when compared with a blood gas analyzer. The advantage of continuous in-line monitoring is that the perfusionist has continuous updates as to the patient's changing physiologic state. During this study, if the sample readout of the CDI-100 was off by greater than 10% from that of the Gem-Premier, the CDI-100 parameter was recalibrated. The accuracy of the CDI-100 was fair (greater than 10% of the samples needed recalibration) with regards to the initial sample comparisons. Recalibration was needed 67% of the time for the hematocrit and 35% for the saturation. The reliability of the CDI-100 was good (no equipment failure). The CDI-100 was consistent. It consistently overestimated both the hematocrit and saturation. This overestimation is the bias of the monitor. We recommend recalibration of the CDI-100 during clinical use to insure greater accuracy.
Aged↗
Effects of losartan and captopril on endothelin-1 production in blood vessels and glomeruli of rats with reduced renal mass.
Recently, we have reported that endothelin-1 (ET-1) production is increased in blood vessels and glomeruli of rats with chronic renal failure. This study was design to investigate the role of angiotensin II (Ang II) in endogenous ET-1 production in rats with reduced renal mass. One week after subtotal (5/6) nephrectomy, uremic rats were divided into three groups, and received either no treatment, the Ang II subtype 1 receptor (AT1) antagonist losartan (10 mg/kg/day), or the angiotensin-converting enzyme inhibitor (ACE-I) captopril (30 mg/kg/day) for 6 weeks. Sham-operated rats were used as controls and received no treatment. The levels of immunoreactive ET-1 (ir-ET-1) in plasma and urine, as well as in vascular and renal tissues, were determined by radioimmunoassay (RIA) after extraction. In uremic rats, losartan and captopril completely prevented the increase in systolic blood pressure. At week 6, plasma ir-ET-1 was similar in the different groups of uremic rats and in the controls. However, ir-ET-1 concentration in the mesenteric arterial bed, the thoracic aorta, preglomerular arteries, and glomeruli, as well as urinary ir-ET-1 excretion were significantly greater in uremic-untreated rats compared to controls (P < .01). Treatment of uremic rats with losartan or captopril reduced irET-1 concentration in the thoracic aorta and preglomerular arteries (P < .05), but ir-ET-1 concentration in the mesenteric arterial bed was unchanged. Although both drugs completely prevented the increase in proteinuria, losartan but not captopril significantly reduced ir-ET-1 concentration in glomeruli (P < .05) and normalized urinary ir-ET-1 excretion. This indicates that increased ET-1 production in blood vessels and glomeruli of uremic rats is modulated, at least in part, by Ang II through the AT1 receptor. The beneficial effects of the AT1 antagonist losartan could be attributable to the attenuation of Ang II-induced ET-1 production in this rat remnant kidney model of chronic renal failure, whereas those of the ACE-I captopril are not related to changes in ET-1 production in glomeruli.
Angiotensin II↗
Influence of oxygen tension on function of isolated spermatozoa from ejaculates of oligozoospermic patients and normozoospermic fertile donors.
Oxygen radical generation is known to be detrimental to sperm function. An example of a reactive oxygen species-associated male pathology is oligozoospermia in which fertilization and pregnancy rates are low in in-vitro fertilization (IVF) programmes. As the extent of the modifications induced by reactive oxygen species (ROS) depends on several factors, notably from oxygen tension in the incubation medium, the aim of this study was to examine the influence of a low (5%) rather than atmospheric (20%) oxygen tension in the incubator gas phase on the function of Percoll-selected spermatozoa from ejaculates of oligozoospermic patients and normozoospermic fertile donors. After incubation for several hours in a gas phase of either 5% CO2/90% N2/5% O2 or 5% CO2/95% air (20% O2), none of the parameters investigated, e.g. movement characteristics, potential of spermatozoa to acquire hyperactivated motility, to undergo the acrosome reaction when challenged with a calcium ionophore and to fuse with zona-free hamster oocytes, was significantly different between the two oxygen tensions in fertile donors. In contrast, among oligozoospermic patients, the motility parameters, the percentage of hyperactivated motility and of induced-acrosome reaction were significantly improved under a gas phase of 5% O2 compared with those observed under an atmosphere of 20% O2 (P < 0.05). Exposure to 5% rather than 20% oxygen tension also induced a significant increase in the percentage of penetration of zona-free hamster eggs after capacitation for 17 h, but no difference was found in the mean number of bound spermatozoa per oocyte. After incubation for 24 h, a significantly higher survival rate was observed under 5% compared with 20% oxygen tension. These results show that the use of a low oxygen tension rather than air might improve spermatozoan competence of oligozoospermic patients during IVF programmes.
Acrosome Reaction↗
Antioxidant capacity of prostasomes in human semen.
Prostasomes are human-specific lipid vesicles originating from the prostate and present in seminal plasma. They are involved in a number of biological functions such as sperm motility and immunomodulation by seminal plasma. The aim of our study was to investigate whether prostasomes play a role in the antioxidant status of seminal plasma. In cell suspensions obtained after elimination of seminal plasma by centrifugation, reactive oxygen species (ROS) as measured by luminol-enhanced chemiluminescence were mainly produced by polymorphonuclear neutrophils (PMN). The addition of prostasomes to these cell suspensions lowered the overall ROS production in the basal state and after stimulation with phorbol ester. This action could not be explained by a ROS-scavenging capacity of the prostasomes, as demonstrated by their inability to scavenge ROS produced by 2,2'-azobis-2-amidinopropane dihydrochloride. Using electron spin resonance, we could assess the influence of prostasomes on the plasma membranes of blood PMN and show that it was characterized by an increase in the correlation-relaxation time of the probe 16-doxyl-stearic acid inserted in the membranes. Thus, prostasomes caused a rigidification of blood PMN membranes. These results strongly suggest that the effect of prostasomes in semen could result from their interaction with PMN.
Antioxidants↗
[Search for microdeletions in the long arm of chromosome Y in 48 infertile men].
About 30% of infertilities are from male origin. They appear in some cases de novo and considered idiopathic. The aim of our work is to evaluate, in these cases, the Y chromosome long arm microdeletion prevalence within the AZF a, b and c regions by molecular biology technics. Were excluded from our study, azoo-oligospermia from hereditary, endocrine, obstructive origins or with a constitutional cytogenetic abnormality. 48 infertile men with a spermogram-proven azoo-oligospermia were studied. Among them, 30 were idiopathics, 8 out of them exhibited a genital infection past history or biological abnormalities suggesting partial obtruction of the genito-urinary tractus. 8 cases of varicocela and 10 of cryptorchidia were also studied. Peripheral blood DNA was extracted from each patient, then amplified by multiplex PCR with STS genomic markers from the 3 Y chromosome AZF zones. PCR products were then analysed on agarose gels. Considering the difficulty to affirm the absence of a signal in molecular biology, each suspicion of deletion was checked by multiplex PCR complication with the SRY marker. 5 Y chromosome long arm microdeletions were diagnosed among our 48 patients. All of them included the AZFc zone and the intragenic DAZ gene markers. Moreover a larger Y chromosome deletion encompassing the 3 AZF zones was diagnosed, and confirmed by the cytogenetic analysis. All the Y chromosome microdeletions were observed in the 22 truly idiopathic azoo/oligospermia, corresponding to a proportion of 22.7% which falls to 10.4% considering the whole population of 48 studied people (closer to the published data). The relatively high proportion of microdeletions found in our series, underlines the need of a strict patient selection to avoid unnecessary search for long arm Y chromosome microdeletions.
Chromosome Deletion↗
Birth after combination of cryopreservation of sperm recovered from urine and intracytoplasmic sperm injection in a case of complete retrograde ejaculation.
OBJECTIVE: To assess whether sperm recovered from postejaculatory urine and cryopreserved can be used successfully for intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: Laboratory of Developmental and Reproductive Biology, University Hospital. PATIENT(S): A couple with male infertility resulting from complete retrograde ejaculation. INTERVENTION(S): Freezing of sperm recovered from urine and subsequent use for ICSI. MAIN OUTCOME MEASURE(S): Survival, maintenance of fertilization, and pregnancy potential of sperm recovered from urine and cryopreserved. RESULT(S): On thawing, the sperm were still alive (32% viability) and very slightly motile (2% sluggish motility). Of 16 oocytes injected, 10 (62.5%) produced normal, cleaving embryos. A twin pregnancy with the birth of two healthy infant girls was achieved after the transfer of three embryos. CONCLUSION(S): The birth of normal, healthy female infants after ICSI with sperm recovered from postejaculatory urine and then cryopreserved demonstrates the usefulness of freezing such sperm even if motility is very low, to avoid surgery in the patient.
Adult↗
Interaction of kinesin motor domains with alpha- and beta-tubulin subunits at a tau-independent binding site. Regulation by polyglutamylation.
Interaction of rat kinesin and Drosophila nonclaret disjunctional motor domains with tubulin was studied by a blot overlay assay. Either plus-end or minus-end-directed motor domain binds at the same extent to both alpha- and beta-tubulin subunits, suggesting that kinesin binding is an intrinsic property of each tubulin subunit and that motor directionality cannot be related to a preferential interaction with a given tubulin subunit. Binding features of dimeric versus monomeric rat kinesin heads suggest that dimerization could drive conformational changes to enhance binding to tubulin. Competition experiments have indicated that kinesin interacts with tubulin at a Tau-independent binding site. Complementary experiments have shown that kinesin does not interact with the same efficiency with the different tubulin isoforms. Masking the polyglutamyl chains with a specific monoclonal antibody leads to a complete inhibition of kinesin binding. These results are consistent with a model in which polyglutamylation of tubulin regulates kinesin binding through progressive conformational changes of the whole carboxyl-terminal domain of tubulin as a function of the polyglutamyl chain length, thus modulating the affinity of tubulin for kinesin and Tau as well. These results indicate that microtubules, through tubulin polymorphism, do have the ability to control microtubule-associated protein binding.
Animals↗