Ariane I. de Agostini

Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential

BackgroundExternalization of phosphatidylserine (EPS) occurs in apoptotic-like spermatozoa and could be used to remove them from sperm preparations to enhance sperm quality for assisted medical procreation. We first characterized EPS in sperms from infertile patients in terms of frequency of EPS spermatozoa as well as localization of phosphatidylserine (PS) on spermatozoa. Subsequently, we determined the impact of depleting EPS spermatozoa on sperm quality.MethodsEPS were visualized by fluorescently-labeled annexin V binding assay. Double staining with annexin V and Hoechst differentiates apoptotic from necrotic spermatozoa. We used magnetic-activated cell sorting using annexin V-conjugated microbeads (MACS-ANMB) technique to remove EPS spermatozoa from sperm prepared by density gradient centrifugation (DGC). The impact of this technique on sperm quality was evaluated by measuring progressive motility, viability, and the integrity of the mitochondrial membrane potential (MMP) by Rhodamine 123.ResultsMean percentages of EPS spermatozoa were 14% in DGC sperm. Four subpopulations of spermatozoa were identified: 70% alive, 3% early apoptotic, 16% necrotic and 11% late apoptotic or necrotic. PS were localized on head and/or midpiece or on the whole spermatozoa. MACS efficiently eliminates EPS spermatozoa. MACS combined with DGC allows a mean reduction of 70% in EPS and of 60% in MMP-disrupted spermatozoa with a mean increase of 50% in sperm survival at 24 h.ConclusionHuman ejaculates contain EPS spermatozoa which can mostly be eliminated by DGC plus MACS resulting in improved sperm long term viability, motility and MMP integrity. EPS may be used as an indicator of sperm quality and removal of EPS spermatozoa may enhance fertility potential in assisted medical procreation.

Normal levels of anticoagulant heparan sulfate are not essential for normal hemostasis

Endothelial cell production of anticoagulant heparan sulfate (HS(act)) is controlled by the Hs3st1 gene, which encodes the rate-limiting enzyme heparan sulfate 3-O-sulfotransferase-1 (3-OST-1). In vitro, HS(act) dramatically enhances the neutralization of coagulation proteases by antithrombin. The in vivo role of HS(act) was evaluated by generating Hs3st1(-/-) knockout mice. Hs3st1(-/-) animals were devoid of 3-OST-1 enzyme activity in plasma and tissue extracts. Nulls showed dramatic reductions in tissue levels of HS(act) but maintained wild-type levels of tissue fibrin accumulation under both normoxic and hypoxic conditions. Given that vascular HS(act) predominantly occurs in the subendothelial matrix, mice were subjected to a carotid artery injury assay in which ferric chloride administration induces de-endothelialization and occlusive thrombosis. Hs3st1(-/-) and Hs3st1(+/+) mice yielded indistinguishable occlusion times and comparable levels of thrombin.antithrombin complexes. Thus, Hs3st1(-/-) mice did not show an obvious procoagulant phenotype. Instead, Hs3st1(-/-) mice exhibited genetic background-specific lethality and intrauterine growth retardation, without evidence of a gross coagulopathy. Our results demonstrate that the 3-OST-1 enzyme produces the majority of tissue HS(act). Surprisingly, this bulk of HS(act) is not essential for normal hemostasis in mice. Instead, 3-OST-1-deficient mice exhibited unanticipated phenotypes suggesting that HS(act) or additional 3-OST-1-derived structures may serve alternate biologic roles.

Human Follicular Fluid Heparan Sulfate Contains Abundant 3-O-Sulfated Chains with Anticoagulant Activity

Anticoagulant heparan sulfate proteoglycans bind and activate antithrombin by virtue of a specific 3-O-sulfated pentasaccharide. They not only occur in the vascular wall but also in extravascular tissues, such as the ovary, where their functions remain unknown. The rupture of the ovarian follicle at ovulation is one of the most striking examples of tissue remodeling in adult mammals. It involves tightly controlled inflammation, proteolysis, and fibrin deposition. We hypothesized that ovarian heparan sulfates may modulate these processes through interactions with effector proteins. Our previous work has shown that anticoagulant heparan sulfates are synthesized by rodent ovarian granulosa cells, and we now have set out to characterize heparan sulfates from human follicular fluid. Here we report the first anticoagulant heparan sulfate purified from a natural human extravascular source. Heparan sulfate chains were fractionated according to their affinity for antithrombin, and their structure was analyzed by 1H NMR and MS/MS. We find that human follicular fluid is a rich source of anticoagulant heparan sulfate, comprising 50.4% of total heparan sulfate. These antithrombin-binding chains contain more than 6% 3-O-sulfated glucosamine residues, convey an anticoagulant activity of 2.5 IU/ml to human follicular fluid, and have an anti-Factor Xa specific activity of 167 IU/mg. The heparan sulfate chains that do not bind antithrombin surprisingly exhibit an extremely high content in 3-O-sulfated glucosamine residues, which suggest that they may exhibit biological activities through interactions with other proteins.

Coordinate Expression of Anticoagulant Heparan Sulfate Proteoglycans and Serine Protease Inhibitors in the Rat Ovary: A Potent System of Proteolysis Control

Abstract During the reproductive cycle, ovarian follicles undergo major tissue-remodeling involving vascular changes and proteolysis. Anticoagulant heparan sulfate proteoglycans (aHSPGs) are expressed by granulosa cells during the development of the ovarian follicle. The function of aHSPGs in the ovary is unknown, but they might be involved in proteolysis control through binding and activation of serine protease inhibitors. To identify functional interactions between aHSPGs and heparin-binding protease inhibitors in the follicle, we have coordinately localized aHSPGs, antithrombin III, protease nexin-1, and plasminogen activator inhibitor-1 in the rat ovary during natural and gonadotropin-stimulated cycles. Anticoagulant HSPGs were visualized by autoradiography of cryosections incubated with 125I-antithrombin III, and protease inhibitors were assessed by immunohistochemistry and Northern blot hybridization. Anticoagulant HSPGs were expressed in follicles before ovulation, were transiently decreased in postovulatory follicles, and were abundant in the corpus luteum, mainly on capillaries. Anticoagulant HSPGs were colocalized with protease nexin-1 in follicles from the early antral stage until ovulation, with antithrombin III in the preovulatory stage and after ovulation, and with plasminogen activator inhibitor-1 in the corpus luteum. These data demonstrate that aHSPGs are critically expressed in the ovary to interact sequentially with protease nexin-1, antithrombin III, and plasminogen activator inhibitor-1 during the cycle. The specificity of these inhibitors is shifted toward thrombin inhibition in the presence of heparin, suggesting that aHSPGs direct their action to control fibrin deposition in the follicle. The occupation of aHSPGs antithrombin-binding sites by mutant R393C antithrombin III, injected in the ovarian bursa, decreased ovulation efficiency, further supporting the involvement of aHSPGs in the ovulation process.

Synthesis of Anticoagulantly Active Heparan Sulfate Proteoglycans by Glomerular Epithelial Cells Involves Multiple 3-O-Sulfotransferase Isoforms and a Limiting Precursor Pool

Endothelial and other select cell types synthesize a subpopulation of heparan sulfate (HS) proteoglycans (HSPGs), anticoagulant HSPGs (aHSPGs) that bear aHS-HS chains with the cognate 3-O-sulfated pentasaccharide motif that can bind and activate anti-thrombin (AT). Endothelial cells regulate aHSPG production by limiting levels of HS 3-O-sulfotransferase-1 (3-OST-1), which modifies a non-limiting pool of aHS-precursors. By probing kidney cryosections with 125I-AT and fluorescently tagged AT we found that the glomerular basement membrane contains aHSPGs, with the staining pattern implicating synthesis by glomerular epithelial cells (GECs). Indeed, cultured GECs synthesized aHS with high AT affinity that was comparable with the endothelial product. Disaccharide analyses of human GEC (hGEC) HS in conjunction with transcript analyses revealed that hGECs express predominantly 3-OST-1 and 3-OST-3A. aHS production has not been previously examined in cells expressing multiple 3-OST isoforms. This unusual situation appears to involve novel mechanisms to regulate aHS production, as HS structural analyses suggest hGECs exhibit excess levels of 3-OST-1 and an extremely limiting pool of aHS-precursor. A limiting aHS-precursor pool may serve to minimize aHS synthesis by non-3-OST-1 isoforms. Indeed, we show that high in vitro levels of 3-OST-3A can efficiently generate aHS. Non-3-OST-1 isoforms can generate aHS in vivo, as the probing of kidney sections from 3-OST-1-deficient mice revealed GEC synthesis of aHSPGs. Surprisingly, Hs3st1-/- kidney only expresses 3-OST isoforms having a low specificity for aHS synthesis. Thus, our analyses reveal a cell type that expresses multiple 3-OST isoforms and produces minimal amounts of aHS-precursor. In part, this mechanism should prevent aHS overproduction by non-3-OST-1 isoforms. Such a role may be essential, as 3-OST isoforms that have a low specificity for aHS synthesis can generate substantial levels of aHSPGs in vivo.

Effect of plasma from patients with idiopathic nephrotic syndrome on proteoglycan synthesis by human and rat glomerular cells

In vivo and in vitro findings have shown that plasma of patients with idiopathic nephrotic syndrome (INS) contain factors that increase glomerular permeability to proteins. The effects of these factors on proteoglycan synthesis by glomerular cells are unknown. To investigate the effect of plasma from patients with INS (n = 23) and other glomerulopathies (n = 12) on the amount of proteoglycans synthesized by cultured rat mesangial cells and human glomerular epithelial cells, glomerular cells were cultured for 24 h with plasma from patients or control subjects, and incorporation of Na235SO4 in chondroitin dermatan sulfate and heparan sulfate was assessed using a cationic nylon membrane. The mean ratio of glycosaminoglycan produced by rat mesangial cells when in contact with plasma (5%) from INS patients to the amount produced when in contact with control plasma was 0.70 ± 0.06. The mean ratio of heparan sulfate was 0.58 ± 0.08. The decrease of heparan sulfate production was present in the cellular and in the extracellular fraction. It was observed when the cells were in contact with plasma from patients in relapse but not when in remission. No decrease of heparan sulfate production was observed with four of the five patients with membranous glomerulonephritis(ratio of 1.27 ± 0.03), IgA nephropathy (n = 5, ratio of 1.27± 0.03), and membranoproliferative glomerulonephritis (n = 2, ratio of 1.39 ± 0.34). When human glomerular epithelial cells were exposed to 5% plasma from INS patients in relapse (n = 9), the mean ratio of heparan sulfate was 0.62 ± 0.06 in the cellular fraction and 0.72 ± 0.08 in the medium. When in contact with plasma from patients in remission, no difference of glycosaminoglycan production was observed. A factor present in plasma from patients with INS during initial episodes or relapses is able to decrease the proteoglycan production of glomerular cells.

Role of cyclic AMP in idiopathic nephrotic syndrome: a pathway involving a decrease in glomerular cell heparan sulfates?

The physiopathological mechanisms of idiopathic nephrotic syndrome involve a circulating plasma factor and a decrease in HS in the glomerular basement membrane. Previous studies have demonstrated that plasma from patients with INS decreases glomerular cell HS in vitro. We examined the involvement of cyclic adenosine monophosphate (cAMP) in this interaction. We studied the effect of plasma from patients with INS on mesangial cell cAMP. We also determined mesangial cell HS when cAMP levels were modified using a cationic membrane after metabolic labeling. Cellular cAMP levels increased significantly when mesangial cells were incubated with plasma from patients with INS in comparison with control plasma (+77%, P = 0.01). Forskolin and IBMX, which increased cellular cAMP, decreased HS levels (−21 ± 9% and −15 ± 6% respectively, P < 0.05 for both), whereas dideoxyadenosine, which decreased cellular cAMP, increased HS levels (+24 ± 7%, P < 0.05). Plasma from patients with INS decreased glomerular cell HS in comparison with control plasma (−34 ± 8%, P < 0,05). This effect was abolished when cells were preincubated with ddAdo to prevent an increase in cAMP levels. We conclude that in mesangial cells, plasma from patients with INS increases cAMP levels, and that cAMP mediates a decrease in HS levels. Moreover, the action of plasma from patients on HS was inhibited when an increase in cAMP was prevented. cAMP may therefore be instrumental in the negative effect of the plasma factor on mesangial cell HS. J. Cell. Biochem. 78:363–370, 2000.

Increased Glomerular Cell Heparan Sulfates in vitro by Ciclosporin A: A Possible Explanation of Its Beneficial Effect in Idiopathic Nephrotic Syndrome

Background/Aim: In idiopathic nephrotic syndrome (INS), ciclosporin A (CsA) was shown to decrease proteinuria, an effect explained by its immunologic and hemodynamic actions. In order to determine whether CsA could have a direct action on glomerular cells, we studied the effect of CsA on glomerular cells in vitro, particularly on glycosaminoglcycans (GAG) and heparan sulfates (HS) which are decreased in INS patients. Methods: Human glomerular epithelial cells and rat mesangial cells were cultured at various concentrations of CsA. HS were quantified using a cationic membrane after metabolic labeling. Results: Mesangial cell GAG and HS and epithelial cell HS increased significantly when cells were cultured with CsA. For both cell types this increase was prevailing on the secreted fraction of HS in comparison with the cellular fraction. CsA induced also an increase in cellular cAMP levels, but the effect of CsA was not transduced via a cAMP pathway. Conclusions: CsA is able to increase glomerular GAG and HS in vitro. As this effect of CsA was the opposite effect on glomerular cells to the effect of plasma from INS patients, we conclude that this direct action of CsA on glomerular cells could explain in part the effect of this drug in decreasing proteinuria in INS.

Aglycone Ebselen and β-d-Xyloside Primed Glycosaminoglycans Co-contribute to Ebselen β-d-Xyloside-Induced Cytotoxicity

Most β-d-xylosides with hydrophobic aglycones are nontoxic primers for glycosaminoglycan assembly in animal cells. However, when Ebselen was conjugated to d-xylose, d-glucose, d-galactose, and d-lactose (8A-D), only Ebselen β-d-xyloside (8A) showed significant cytotoxicity in human cancer cells. The following facts indicated that the aglycone Ebselen and β-d-xyloside primed glycosaminoglycans co-contributed to the observed cytotoxicity: 1. Ebselen induced S phase cell cycle arrest, whereas 8A induced G2/M cell cycle arrest; 2. 8A augmented early and late phase cancer cell apoptosis significantly compared to that of Ebselen and 8B-D; 3. Both 8A and phenyl-β-d-xyloside primed glycosaminoglycans with similar disaccharide compositions in CHO-pgsA745 cells; 4. Glycosaminoglycans could be detected inside of cells only when treated with 8A, indicating Ebselen contributed to the unique property of intracellular localization of the primed glycosaminoglycans. Thus, 8A represents a lead compound for the development of novel antitumor strategy by targeting glycosaminoglycans.