Cell-to-cell transfer of glycosylphosphatidylinositol-anchored membrane proteins during sperm maturation

The majority of gene therapy protocols have used or plan to use retroviral vectors based upon murine leukaemia virus. These vectors are able to infect many different cell types, and the retroviral promoter, which is often used to control the expression of a therapeutic gene, is active in a wide range of different cell types. Safe and efficient gene transfer systems, whether based upon retroviruses or other agents, should deliver beneficial genes only to cells that require their therapeutic action, and these genes ideally should be expressed exclusively in such cells. In this paper, strategies for redirecting the infection spectrum of retroviral vectors in order to obtain cell-targeted gene delivery are discussed. These strategies include the engineering of the retroviral envelope protein, which, together with the availability of its cognate receptor, determines infectivity, and the use of proteins from other enveloped viruses of both retroviral and nonretroviral origin in the cell lines used to produce retroviral vector virus particles. Expression targeting can be achieved by limiting the expression of therapeutic genes to the cell type(s) of interest using promoters from genes that are normally active in these cells. This approach to targeting is illustrated using promoters from genes expressed in either the liver, the pancreas or the mammary gland as a means to limit gene expression specifically to the cell types that make up these organs. The successful utilization of new generations of targeted retroviral vectors in the clinic may well pave the way for superior gene delivery systems of the future that seek out their target cell, delivering a therapeutic gene to and expressing it only in such cells.     

Development of sperm motility patterns in the murine epididymis

The maturation of sperm motility in the epididymis of the mouse was assessed using a computer-assisted sperm analysis system. Spermatozoa were immotile in the most proximal regions of the epididymis but developed motility rapidly in the proximal caput epididymis; the percentage motility remained high thereafter. In the caput, flagellar beat was erratic with little progression, but in the proximal corpus region circular movement patterns were reflected in reduced linearity (LIN) and straightness (STR) of the sperm tracks, although velocities were little changed and wobble (WOB) increased. In the mid-corpus region, however, all velocities, LIN, STR and WOB, increased markedly. In more distal regions there was little change in these parameters. Distribution curves of the kinematic parameters of spermatozoa obtained from each region indicated that the most heterogeneous population was that from the mid-corpus epididymis; the most homogeneous was that from the mid-cauda region. Individual sperm tracks revealed slowly progressing spermatozoa in the distal caput, transforming to motion in small circles, interrupted by more linear progression. More distally, linear progression was interrupted by looping movements and a generally progressive path was observed thereafter, with less deviation from the average path as the spermatozoa matured. Spermatozoa displaying motion compatible with passing the uterotubal junction were first found in the proximal corpus epididymis, in agreement with earlier in-vivo fertilization studies on where fertilizing capacity is achieved with epididymal spermatozoa.     

Extraction of sp18 family membrane proteins on posterior head of bull sperm and the effect of anti-sp18 IgG on sperm motility and murine in vitro fertilization

BACKGROUND AND PURPOSE: We evaluated sucralfate, well-known in the treatment of gastric ulcers, in relation to its possible reduction of radiation-induced acute complications in the treatment of head and neck cancers. MATERIALS AND METHODS: One hundred two patients were randomized in a double-blind placebo-controlled prospective setting. All patients were treated to a minimum dose of 55 Gy in 5 weeks. Oral intake of sucralfate was started at the beginning of radiotherapy and continued during the whole treatment at a dose of 1 g six times a day. All patients were scored according to a scoring system developed in our department. Weight was checked once a week. RESULTS: Comparing the time course of the mean scores for subjective intolerance, mucositis, dysphagia, dermatitis and nausea, no statistically significant differences between the two treatment arms (sucralfate, n = 38; placebo, n = 45) were observed. The mean weight loss in the sucralfate arm was 1.6 +/- 3.4 kg while it was 1.3 +/- 2.0 kg in the placebo arm. Apart from gastrointestinal upset, the administration of sucralfate did not cause any side-effects. CONCLUSION: This trial produced no clinical evidence indicating that the oral intake of sucralfate reduces the acute radiation-induced side-effects. Therefore, we do not recommend the prophylactic use of sucralfate in patients with head and neck cancer treated by radiotherapy.     

Lipid domain structure of the plasma membrane revealed by patching of membrane components

Lateral assemblies of glycolipids and cholesterol, "rafts," have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T-lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.     

Changes in structural organization of surface membrane during erythrocyte maturation

The effect of concanavalin A and its succinylated derivative on cell agglutination and potassium compartmentation of mature and immature erythrocytes was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of rabbit erythrocytes leads to a change in the properties of the surface membrane, which results in an induction of cell agglutination and concomitant release of potassium from the cells. Both of the phenomena induced by concanavalin A are temperature dependent, and observed at above 15 degrees C. Divalent succinylated concanavalin A, lacking the inducing activity of surface glycoprotein cross-linking into patches and caps, caused neither cell agglutination nor change in the potassium compartmentation of erythrocytes and reticulocytes. In the case of immature reticulocytes, however, remarkable agglutination of the cells was induced without a change in the potassium compartmentation after treatment with tetravalent concanavalin A. It is suggested that changes in the molecular organization of the surface membrane occur in which potassium compartmentation of the reticulocytes becomes more susceptible to surface glycoprotein cross-linking during cellular maturation.     

Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 +/- 12.2, 40.6 +/- 20.8, 144 [corrected] +/- 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 +/- 6.4 to 33.8 +/- 4.8 to 70 +/- 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 +/- 7.8% in the proximal caput epididymis to 57.2 +/- 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Region-specific localization of retinoic acid receptor-alpha expression in the rat epididymis

BACKGROUND: Nitric oxide (NO), a recognized cell messenger for activating soluble guanylate cyclase, is produced by the enzyme NO synthase in a wide variety of tissues, including vascular endothelium and the central nervous system. The authors previously reported the possible involvement of the NO pathway in the anesthetic state by showing that a specific NO synthase inhibitor, nitroG-L-arginine methyl ester (L-NAME), dose dependently and reversibly decreases the minimum alveolar concentration (MAC) for halothane anesthesia. The availability of a structurally distinct inhibitor selective for the neuronal isoform of NO synthase, 7-nitro indazole (7-NI), allowed for the possibility of dissociating the central nervous system effects of neuronal NO synthase inhibition from the cardiovascular effects of endothelial NO synthase inhibition. METHODS: The effect of two structurally distinct inhibitors of NO synthase, L-NAME and 7-NI, on the MAC of isoflurane was investigated in Sprague-Dawley rats while concurrently monitoring the animals' arterial blood pressure and heart rate. L-NAME (1 to 30 mg/kg given intravenously, dissolved in 0.9% saline) and 7-NI (20 to 1,000 mg/kg given intraperitoneally, dissolved in arachis oil) were administered after determining control MAC and 30 min before determining MAC in the presence of NO synthase inhibitor. RESULTS: L-NAME and 7-NI caused a dose-dependent decrease from isoflurane control MAC (maximal effect: 35.5 +/- 2.5% and 43.0 +/- 1.7%, respectively) with a ceiling effect observed for both NO synthase inhibitors (above 10 mg/kg and 120 mg/kg, respectively). L-NAME administration significantly increased systolic and diastolic blood pressures (maximal effect: 39.9 +/- 2.2% and 64.3 +/- 4.0%, respectively), which were not accompanied by any changes in heart rate. 7-NI administration resulted in no changes in blood pressure and a small but clinically insignificant decrease in heart rate. CONCLUSIONS: Inhibition of the NO synthase pathway decreased the MAC for isoflurane, which suggests that inhibition of the NO pathway decreases the level of consciousness and augments sedation, analgesia, and anesthesia. The MAC reduction by two structurally distinct NO synthase inhibitors supports that this is a specific effect on NO synthase. Furthermore, the action of the neuronal NO synthase inhibitor 7-NI supports an effect selective for neuronal NO synthase and also avoids the hypertensive response of generalized NO synthase inhibitors.     

Constitutive transport between the trans-Golgi network and the plasma membrane according to the maturation model. A hypothesis

Here we examine the application of the cisternal/carrier maturation model to describe transport of cargo proteins from the Golgi apparatus to the plasma membrane. Interpretation of the available evidence in the light of carrier maturation suggests that the transport intermediates between these stations are large pleiomorphic carriers formed by maturation of the trans-Golgi compartment, rather than vesicles, as would be postulated by the vesicular shuttle model. Mature carriers move along microtubules towards the plasma membrane via a microtubule/(kinesin)-based motor system. The maturation and vesicular transport models are compared in terms of consistency with the available literature.     

Duration of spermatogenesis and sperm transit time through the epididymis in the Piau boar

The Piau boar is a rustic breed of economical importance in Brazil. The duration of spermatogenesis and sperm transit through the epididymis in Piau boars was estimated using intratesticular injections of tritiated thymidine. Animals were sacrificed 1 h, 7 days, 14 days, 21 days, 34 days, and 36 days after injections. Each cycle of spermatogenesis in Piau boars lasts 9 +/- 0.2 days. At least 9 days are necessary for spermatozoa to traverse the entire epididymis. Considering that the total duration of spermatogenesis takes about 4.5 seminiferous epithelium cycles, spermatogenesis was estimated to take 40.6 days. The primary spermatocytes life span is 13.5 days, while spermiogenesis in Piau boars lasts 14.5 days. Staging in Piau boars was based on tubular morphology system. The relative stage frequencies in these boars, based on approximately 1200 seminiferous tubule cross-sections for each animal, were as follows: stage 1, 11.7 +/- 0.7%; stage 2, 14.3 +/- 0.3%; stage 3, 5.4 +/- 0.1%; stage 4, 12.1 +/- 0.6%; stage 5, 9.6 +/- 0.4%; stage 6, 17.2 +/- 0.4%; stage 7, 15.4 +/- 0.8%; and stage 8, 14.3 +/- 0.9%. The duration of spermatogenic events and the relative stage frequencies in Piau boars differ slightly from those observed in improved swine breeds.     

Effects of epididymal occlusion on sperm maturation in the hamster

Combination chemotherapy with adriamycin and DTIC was used in 102 evaluable patients under 15 years of age who had previously treatted metastatic solid tumors. Responses, defined as 50% or more reduction in all tumor masses, occured in 10 out of 27 patients with neuroblastoma, 3 out of 8 patients with Wilms tumor, 7 out 15 patients with Ewing sarcoma, 2 out of 6 patients with osteosarcoma, 5 out of 13 patients with rhabdomyosarcoma, and 15 out of 33 patients with miscellaneous tumors which included a patient who had a complete regression of an extensive juvenile angiofibroma. Response rate to combination chemotherapy with adriamycin and DTIC in patients with Ewing sarcoma was significantly superior to the response rate obtained with adriamycin alone in another Southwest Oncology Group Study. Major toxicity included nausea, vomiting, myelosuppression, high incidence of pneumocystis carinii pneumonia (5 patients) and congestive heart failure (4 patients). There was 7 drug-associated deaths due to sepsis (1), pneumocystis carinii pneumonia (4), and congestive heart failure (2).     

Interaction of lipids with the plasma membrane of sperm cells. II. Evidence of a membrane thermotropic transition

Cholesterol and phosphatidylcholine uptake from dipalmitoyl-phosphatidylcholine liposomes by rabbit spermatozoa showed a complex dependence on temperature in these experiments. At 5 degrees and 20 degrees C, the rate of lipid uptake correlated with temperature. However, from 20 degrees to 37 degrees C uptake did not evidently increased. The results in interpreted as evidence of a thermotropic transition in the sperm plasma membrane. Data are presented showing incorporation of these lipids, especially that of cholesterol, into sperm plasma membrane.     

Intrinsic signals in the unique domain target p56(lck) to the plasma membrane independently of CD4

In T lymphocytes, the Src-family protein tyrosine kinase p56(lck) (Lck) is mostly associated with the cytoplasmic face of the plasma membrane. To determine how this distribution is achieved, we analyzed the location of Lck in lymphoid and in transfected nonlymphoid cells by immunofluorescence. We found that in T cells Lck was targeted correctly, independently of the cell surface proteins CD4 and CD8 with which it interacts. Similarly, in transfected NIH-3T3 fibroblasts, Lck was localized at the plasma membrane, indicating that T cell-specific proteins are not required for targeting. Some variation in subcellular distribution was observed when Lck was expressed in HeLa and MDCK cells. In these cells, Lck associated with both the plasma membrane and the Golgi apparatus, while subsequent expression of CD4 resulted in the loss of Golgi-associated staining. Together, these data indicate that Lck contains intrinsic signals for targeting to the plasma membrane. Furthermore, delivery to this site may be achieved via association with exocytic transport vesicles. A mutant Lck molecule in which the palmitoylation site at cysteine 5 was changed to lysine (LC2) localized to the plasma membrane and the Golgi region in NIH3T3 cells. However, the localization of a mutant in which the palmitoylation site at cysteine 3 was changed to serine (LC1) was indistinguishable from wild-type Lck. Chimeras composed of only the unique domain of Lck linked to either c-Src or the green fluorescent protein similarly localized to the plasma membrane of NIH-3T3 cells. Thus, the targeting of Lck appears to be determined primarily by its unique domain and may be influenced by the use of different palmitoylation sites.     

The PH domain and the polybasic c domain of cytohesin-1 cooperate specifically in plasma membrane association and cellular function

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in approximately 100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2-3 microM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the beta-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation-isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton's tyrosine kinase with the adjacent Bruton's tyrosine kinase motif, a novel zinc-containing fold.     

Rat sperm surface mannosidase is first expressed on the plasma membrane of testicular germ cells

In previous publications (Tulsiani et al., Biochem J 1993; 290:427-436 and Tulsiani et al., Dev Biol 1995; 167:584-595), we reported that sperm surface mannosidase is present in rat testis and is modified during spermatogenesis and sperm maturation. The present studies were directed towards examining the origin of alpha-D-mannosidase activity present on fertile spermatozoa. Mixed germ cells prepared after sequential enzymatic digestions of rat testis were separated by unit gravity sedimentation using 2-4% linear bovine serum albumin gradient. Fractions enriched in spermatocytes, round spermatids, and condensed/elongated spermatids (> 95% pure cells) were separately pooled and assayed for [3H]Man9-mannosidase activity before (intact) and after lysis with Triton X-100. Interestingly, the cells contained a significant level of alpha-D-mannosidase activity. Approximately 70% of the total [3H]Man9-mannosidase activity present in the detergent-solubilized germ cell extract cross-reacted with anti-rat sperm mannosidase, and 25% of the activity cross-reacted with anti-Golgi mannosidase I. This result indicates that most of the mannosidase activity present in the germ cell extract is antigenically similar to the enzyme present on the cauda spermatozoa. Using cell fractionation techniques, we obtained evidence suggesting that the germ cell-associated mannosidase activity is an integral component of the plasma membranes. Taken together, these results indicate that sperm surface mannosidase is first expressed on the testicular germ cells.     

Glycoproteins of the plasma membrane of Dictyostelium discoideum during development

Polysomal RNA from cultured sublines of baby hamster kidney (BHK) cells directed protein synthesis in an in vitro system derived from wheat germ extract. One product of the in vitro synthesis was dihydrofolate reductase (DHFR), as confirmed by methotrexate-substituted Sepharose affinity chromatography followed by SDS-polyacrylamide slab gel electrophoresis and autoradiography of the proteins labeled with 35S-methionine. The DHFR synthesized in vitro comigrates in the gel with authentic BHK DHFR, indicating that the molecular weights and structures of the in vivo and in vitro enzymes are probably the same. Polysomal RNA obtained from the methotrexate-resistant BHK subline (A5), which possesses some 140 times higher DHFR levels than the methotrexate-sensitive parents subline (B1), directed the synthesis of approximately 70 times more DHFR per unit of total in vitro synthesized protein than did B1 polysomal RNA. Assuming then that the rates of translation of A5 and B1 DHFR mRNAs in the wheat germ cell-free system are the same, our results show that a major part of the high DHFR levels observed in A5 cells is due to the presence of elevated quantities of DHFR mRNA.     

Characterization of the binding of recombinant mouse sperm fertilin alpha subunit to mouse eggs: evidence for function as a cell adhesion molecule in sperm-egg binding

Fertilin (previously known as PH-30) is a sperm protein that is a candidate molecule for mediating the binding and fusion of the sperm and egg plasma membranes. Fertilin is a heterodimer, with a beta subunit that has a region of homology to the disintegrin family of integrin ligands and an alpha subunit that has a region of homology to viral fusion peptides. It has been hypothesized that fertilin beta and alpha subunits mediate the interactions between sperm and egg plasma membranes, namely, binding and fusion, respectively. To address this hypothesis and to examine specifically the role of fertilin alpha in fertilization, we have expressed the predicted extracellular domain of mouse fertilin alpha as a bacterial fusion protein with maltose-binding protein. This fusion protein (hereafter referred to as recombinant fertilin alpha-EC) binds to the microvillar region of zona pellucida (ZP)-free eggs, the region of the membrane to which sperm bind. This binding is reduced in the absence of divalent cations and is supported by Ca2+, Mg2+, or Mn2+. Eggs that have been treated with chymotrypsin bind less recombinant fertilin alpha-EC than do untreated eggs, suggesting that a chymotrypsin-sensitive binding site for recombinant fertilin alpha-EC is present on egg surfaces. Binding to eggs is also affected by the method used to remove the ZP. Finally, recombinant fertilin alpha-EC inhibits the binding of sperm to eggs during in vitro fertilization of ZP-free eggs. These data are the first evidence to suggest that fertilin alpha can function as a cell adhesion molecule during fertilization, mediating the binding of sperm and egg plasma membranes.     

Immunoaffinity localization of the enzyme xanthine oxidase on the outside surface of the endothelial cell plasma membrane

BACKGROUND: Reactive oxygen metabolites generated from endothelial xanthine oxidase (XO) trigger reperfusion injury in many organs. We evaluated the possibility that endothelial XO was localized on the endothelial cell surface, as well as within the cytoplasm. METHODS: Primary cultures of bovine (BAECs) and porcine (PAECs) aortic endothelial cells were grown in media documented to be free of XO. Polyclonal and monoclonal antibodies were developed against XO. These antibodies were used to evaluate BAEC and PAEC for cell surface XO through immunofluorescence staining, hybridoma cell surface labeling, and endothelial cell surface binding. RESULTS: These antibodies bound specifically to the surface of these cells when the membrane was shown to be intact and impermeable (and the cytoplasm inaccessible) to immunoglobulins Moreover, hybridoma cells expressing monoclonal antibody to XO bound specifically to the endothelial cell surface. Finally, intact endothelial cells bound specifically to the anti-XO polyclonal antibodies immobilized to the surface of a Petri dish. The integrity of these endothelial cell plasma membranes was demonstrated by the subsequent growth and replication of these cells in culture. CONCLUSIONS: These findings indicate that XO is present on the outside surface of the endothelial cell plasma membrane. This would not only explain the known in vivo efficacy of intravascularly administered large molecular weight antioxidants (such as superoxide dismutase) but could have important implications for inflammatory signaling.