Biomedical subjects

G Grizard

Publications and source records attributed to G Grizard.

At least 19 recordsLinked to original sources

PubMed2000

Separation and quantification of cholesterol and major phospholipid classes in human semen by high-performance liquid chromatography and light-scattering detection.

A high-performance liquid chromatographic method coupled with light-scattering detection for the separate and accurate quantification of cholesterol and main phospholipid classes was applied to human spermatozoa and seminal plasma (SP). This method is based on normal-phase chromatography with silica gel as stationary phase and a ternary gradient with hexane, mixtures of chloroform-methanol and water as mobile phase. Lipids are separated with a good resolution and a high reproducibility. About 5 x 10(6) spermatozoa or 25 microl of seminal plasma are sufficient to accurate quantitative analysis of phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidycholine (PC), sphingomyelin (SM) and cholesterol. PC is the predominant phospholipid class in spermatozoa (102+/-8 nmol/10(8) spermatozoa) whereas SM is the major in the SP (163+/-6 nmol/ml). Both in spermatozoa and SP, PI is the minor class of the phospholipids (12+/-1 nmol/10(8) spermatozoa and 24+/-2 nmol/ml). In conclusion, this method offers interesting perspectives for analysis of sperm lipid composition in semen samples with low quantities of spermatozoa.

Cholesterol↗

PubMed2000

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↗

PubMed2000

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↗

PubMed1999

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↗

PubMed1999

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↗

PubMed1998

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↗

PubMed1998

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↗

PubMed1997

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↗

PubMed1997

[The examination of sperm in the study of male fertility].

The laboratory investigation of male fertility is based on semen examinations and allows assessment of all events occurring between the onset of spermatogenesis and ejaculation. The sperm count and semen cytology remain the first-line examinations, but can be completed by more specific examinations allowing assessment of spermatozoan functions involved in passage along the female genital tract and penetration of the oocyte. However, apart from azoospermia, there is no absolute criterion beyond which semen can be considered to be infertile. The results must be interpreted by taking into account all these examinations which can provide element concerning the aetiology of infertility, helping to guide subsequent treatment. When the use of various in vivo treatments has failed, patients may require medically-assisted procreation (MAP) techniques. The type of MAP chosen will depend on the characteristics of the semen and the functional capacities of the spermatozoa.

Ejaculation↗

PubMed1997

Decrease in insulin and insulin-like growth factor I (IGF-I) binding to erythrocytes from patients with cystic fibrosis.

Cystic fibrosis, an autosomal recessive disease affecting exocrine glands, is associated in many cases with a severe undernutritional state, growth retardation and glucose intolerance. To obtain a better definition of the possible defects of insulin and insulin-like growth factor I (IGF-I) receptors, we investigated 125I-insulin and 125I-IGF-I binding to erythrocytes from patients with cystic fibrosis (n = 23) and controls (n = 13). Erythrocytes were isolated by Ficoll-Hypaque gradient centrifugation, and hormone binding was performed in cell suspensions of 3 x 10(9) cells/ml. Cystic fibrosis patients displayed a statistically significant 33% and 40% (p < 0.05) decrease of insulin and IGF-I binding, respectively, compared to controls. These alterations were due to an almost 50% reduction in the binding capacity of the high-affinity receptor compartment. Affinity constants were modified to a lesser extent, except for a two-fold decrease in K1 of the high-affinity compartment of insulin receptors. Interestingly, the decrease in insulin binding was proportional to the degree of growth failure. The statistical significance of hormone binding alterations was assessed in terms of the graphic distribution of individual affinity constants and binding capacity values. Although variable, 50 to 60% of cystic fibrosis patients displayed alterations in stoichiometric binding parameters located outside the area described by the 95% tolerance interval of controls. A major reduction in insulin and IGF-I binding in conditions of low and normal insulin and IGF-I plasma levels, respectively, as well as the correlation with the degree of growth failure in patients with cystic fibrosis, may contribute to an understanding of the pathogenesis of insulin resistance and glucose abnormalities in undernutritional states.

Adolescent↗

PubMed1996

Improvement of motility and fertilization potential of postthaw human sperm using glutamine.

The effectiveness of three amino acids, glutamine, proline, and histidine, and one amino acid-related compound, betaine, in preserving human sperm diluted v/v in a basal medium (BM) containing 14% glycerol during the freeze-thaw (FT) process was studied. At 80 mM in BM, only glutamine improved the 5- to 60-min postthaw total and progressive motilities and velocity. The presence of glutamine at 80 mM is not sufficient to achieve lower concentrations of the toxic agent glycerol in FT medium. Glutamine may therefore have a mechanism of protection on human spermatozoa that is independent from that of glycerol. The zona-free hamster egg penetration test showed that the percentage of eggs penetrated was significantly greater when spermatozoa were frozen-thawed with 80 mM glutamine in BM. Consequently, the presence of glutamine at 80 mM in a glycerol-FT medium maintains human sperm motility and fertilizing ability during the FT process.

Animals↗

PubMed1995

Cholesterol, phospholipids and markers of the function of the accessory sex glands in the semen of men with hypercholesterolaemia.

The effect of hypercholesterolaemia on the cholesterol and phospholipid content of spermatozoa and seminal plasma was studied. Testosterone and specific markers of the accessory sex glands were also measured. Semen samples from 11 hypercholesterolaemic patients (plasma cholesterol > 6.42 mmol/l, plasma triglycerides < 2 mmol/l) were compared with those of 11 normocholesterolaemic controls (plasma cholesterol < 5.14 mmol/l, plasma triglycerides < 2 mmol/l). Cholesterol, phospholipids and the molar ratio of cholesterol: phospholipids were not significantly different between the two groups of patients either in spermatozoa or in seminal plasma. In hypercholesterolaemic patients the total amount of carnitine in the ejaculate was significantly higher, but there were no significant differences in the levels of acid phosphatase or fructose. There were no significant differences in seminal plasma levels of testosterone in the two groups of subjects. These results suggest that hypercholesterolaemia has no effect on cholesterol and phospholipid levels in spermatozoa and does not cause gross modification of the secretory function of the accessory sex glands.

Acid Phosphatase↗

PubMed1994

Characterization of a monoclonal antibody to a human intra-acrosomal antigen that inhibits fertilization.

Among monoclonal antibodies (mAb) selected after the immunization of mice with human ejaculated spermatozoa, mAb I9G9 (IgG1 kappa) was found by immunoperoxidase staining to label most of the acrosome of human spermatozoa permeabilized with methanol-acetone. The antigen was poorly expressed on the surface of fresh ejaculated sperm, but was detectable on most viable sperm after 5-h incubation in medium containing human serum albumin (HSA) followed by 30-min incubation with the calcium ionophore A23187. This treatment resulted in acrosomal loss. Immunoelectron microscopy labeling with I9G9 mAb localized the antigen within the acrosome. Immunocytochemistry on testis sections showed that antigen was located in the round spermatids within the adluminal compartment of the seminiferous epithelium. Western blotting of sperm extract proteins showed that sperm intra-acrosomal (SIAA) recognized by I9G9 mAb had a polymorphism of immunogenic peptides from 16 to 35 kDa. Most of the antigenic peptides possessed an isoelectric point of approximately 5. When spermatozoa were treated with a series of protease inhibitors, the polymorphism of immunogenic peptides was reduced, suggesting that the multiple form of the antigen was due, at least in part, to proteolytic processing. In the testis, only a single peptide band of 35 kDa was detected with mAb I9G9. Studies of human tissue specificity by Western blotting showed that the epitope recognized by I9G9 mAb was present solely in ejaculated spermatozoa and the testis. I9G9 mAb did not agglutinate or immobilize sperm but inhibited the penetration of zona-free hamster ova by human sperm.

Acrosome↗

PubMed1994

[IGF(s) and testicular functions. Secretion and action of IGF-1 on Leydig cells].

Many in vitro and in vivo studies have demonstrated a presence of IGF-I in the testis. Testicular production of IGF-I and its mRNA is stimulated largely by gonadotropins; FSH acts on Sertoli cells while LH acts on Leydig cells. A regulation by local factors is also demonstrated; the role of GH remains unclear. In the testis, IGF-I has various effects but we only focussed this review on Leydig cells. Specific receptors have been characterized on these cells. They are up-regulated by LH/hCG (at the transcriptional level) and down regulated by IGF-I. The effect of GH is depend on the experimental models used. In addition in this paper, we have shown in adult rats that the IGF-I level in the testicular interstitial fluid and the IGF-I receptors on Leydig cells are modified by fasting. Both in vivo and in vitro, IGF-I increased LH/hCG receptors and the steroïdogenic response to hCG by a mechanism which is poorly understood yet.

Animals↗

PubMed1992

Changes in carnitine and acetylcarnitine in human semen during cryopreservation.

L-Carnitine and acetylcarnitine concentrations were determined in spermatozoa and seminal plasma from 15 men, in both fresh ejaculate and frozen-thawed semen with cryoprotective medium. Sperm motility was also evaluated. In fresh samples, the levels of carnitine and acetylcarnitine in seminal plasma were comparable whereas in spermatozoa, acetylcarnitine predominated. Cryopreservation did not change the carnitine and acetylcarnitine levels in seminal plasma nor the carnitine concentration in spermatozoa; by contrast, the acetylcarnitine level in spermatozoa was decreased in 14 cases (110 +/- 8 versus 210 +/- 20 nmol/10(8) cells). This decrease in acetylcarnitine content was greater during semen dilution in cryoprotectant than after the freezing/thawing process. Motility was also decreased in all cases after the freezing/thawing process. These results suggest that acetylcarnitine recovery in spermatozoa is further evidence of the deleterious effect of the cryoprotective medium in the cryopreservation of semen.

Acetylcarnitine↗

PubMed1992

Influence of rat testicular macrophages on Leydig cell function in vitro.

The influence of co-cultures of rat testicular macrophages and Leydig cells (LC) on LC morphology and steroidogenesis was investigated with and without macrophage stimulation by a bacterial lipopolysaccharide (LPS). LC showed an elongated form in the presence of stimulated testicular macrophages. In the presence of non-stimulated testicular macrophages a significant inhibition of testosterone production was observed (decrease of 33%) from 48 h in co-culture while an increase of 16% was obtained at the same culture time, after stimulation of macrophages by LPS. When LC were treated with testicular macrophage-conditioned media (MCM) obtained from LPS-treated macrophages, they became fusiform and there was stimulation (78%) of steroid production. After human FSH stimulation (1-1000 mIU ml-1), MCM from testicular macrophages was no more effective in enhancing testosterone production by LC than was media from untreated LC. Similar experiments with LPS were conducted with macrophages of peritoneal origin. Peritoneal macrophages stimulated or not by LPS in co-cultures with LC or peritoneal MCM did not significantly modify testosterone production. However, these cells were able to modify LC morphology when LPS-MCM was added to LC-culture medium. The present results suggest strongly that testicular macrophage-LC interactions could be important in the control of LC steroidogenesis.

Animals↗

PubMed1992

Comparative study of the binding of prolactin and growth hormone by rabbit and human lung cell membrane fractions.

The specific binding capacities for human prolactin (hPRL) and human growth hormone (hGH) were examined in human lung membrane preparations at different developmental stages. A parallel study was carried out on rabbit lung preparations to compare binding parameters. Lung tissues were obtained from 15 fetuses of 16-38 weeks after spontaneous or therapeutic abortion and from 7 adults (lobar resection surgery). A histological study was systematically performed with a radial alveolar count in the fetuses with suspected hypoplasia. Binding analysis was performed on both intact membrane preparations and MgCl2-treated membranes, using [125I]hGH and [125I]hPRL as tracers. In the rabbit lung, specific [125I]hGH binding was found. Scatchard analysis revealed a single class of binding sites (affinity constant: 2.6 +/- 0.8 x 10(9) l/nmol and number of binding sites: 9.5 +/- 4.3 fmol/mg protein for adult rabbit; 1.85 +/- 0.5 x 10(9) l/nmol and 27.6 +/- 3.0 fmol/mg protein for 25-day-old rabbit fetuses, respectively). In contrast, [125I]hPRL did not specifically bind to lung membrane preparations. In the human lung, no consistent specific binding sites for [125I]hPRL or [125I]hGH (less than 0.5%/mg protein) were detected in adults and in 11 of the 15 fetuses. In 4 fetuses, little specific binding was observed (0.59-1.9%/mg protein) for [125I]hGH and (1.5%/mg protein) for [125I]hPRL. There was no correlation with histological lung structure. Our findings confirm the presence of specific binding sites for GH in the adult rabbit lung and demonstrate such binding in the fetal rabbit lung. In contrast, our results showed no significant binding for PRL and GH in the human lung, suggesting that these hormones do not play a direct physiological role in human lung growth and maturation.

Animals↗

PubMed1991

Insulin-like growth factor I (IGF-I) and insulin binding to erythrocytes of normal prepubertal children and adults.

Erythrocyte insulin-like growth factor I (IGF-I) and insulin receptors were characterized in 10 normal prepubertal children (5 girls and 5 boys) aged 4-11 yrs and 10 normal adults (4 women and 6 men) aged 32-47 yrs. erythrocytes were purified from 5 ml of blood by Ficoll-Paque gradient centrifugation. Reticulocytes count in the erythrocyte suspensions were lower than 1%. Insulin and IGF-I binding assays were performed simultaneously. Maximal percent binding of [125I] labelled IGF-I was significantly higher in prepubertal children than in adults (8.7 +/- 0.7% versus 6.2 +/- 0.5% at a concentration of 5 x 10(9) erythrocytes/ml). Scatchard analysis revealed the high affinity constant was better in prepubertal children (Ka = 4.6 +/- 1.3 nM-1 versus 1.8 +/- 0.2 nM-1), whereas the binding capacity was similar (5.8 +/- 1.1 versus 7.7 +/- 0.8 high affinity binding sites/cell). In both groups, unlabelled IGF-I inhibited tracer-binding half maximally at about 1 nM. Insulin was 100-fold less potent. In adults, specific binding of [125I] labelled IGF-I was higher in women (7.6 +/- 0.7%) than in men (5.3 +/- 0.4%). No significant difference was observed in maximal specific binding of [125I] labelled insulin between prepubertal children (8.2 +/- 0.5%) and adults (7.2 +/- 0.7%). In both groups, competition by unlabelled insulin for [125I] labelled insulin binding gave 50% displacement for approximately 0.25 nM and IGF-I was about 80-fold less potent. Both IGF-I and insulin binding parameters were not significantly correlated with plasma hormone levels. In prepubertal children, the high-affinity IGF-I receptors number decreased with increasing high-affinity insulin receptors number.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗