Effects of the ion-channel blocker quinine on human sperm volume, kinematics and mucus penetration, and the involvement of potassium channels

Sperm defects in the infertile c-ros knockout mouse model have recently highlighted the importance of volume regulation in sperm function. In this study, washed human spermatozoa were shown to change size and shape, as detected by flow cytometry and light microscopy, in response to the ion-channel blocker quinine (minimum effective doses at 20 and 125 micromol/l respectively). The increase in sperm volume was accompanied by reduced straight-line velocity (VSL) and linearity (LIN) of the swim-path but increased lateral head displacement and curvilinear velocity, while percentage motility was unaffected. Spermatozoa in semen and in artificial cervical mucus were similarly affected at 0.2 and 0.5 mmol/l quinine, resulting in marked reduction of mucus penetration and migration. The effects of quinine on sperm volume and kinematics were reduced or abolished by the K(+)-ionophores valinomycin (1 and 5 micromol/l) and gramicidin (0.5 and 1 micromol/l). In Ca(2+)-free medium; however, the quinine effects largely persisted. The K(+)-channel blocker, 4-aminopyridine (1 and 4 mmol/l), mimicked the quinine effects in the reduction of VSL and LIN, while the K(+)-channel blocker, tetraethylammonium chloride (TEA, 2.5-10 mmol/l), did not affect kinematics. The K(+)-channel (Kv1.3)-specific inhibitor, margatoxin, and the Ca(2+)-dependent K(+)-channel blocker, charybdotoxin, also had no effects. This study suggests that volume regulation in human spermatozoa and the linear trajectory of their motion may rely on quinine-sensitive and TEA-insensitive, largely calcium-independent, potassium channels, and possibly volume-sensitive organic anion channels. These channels could be targets for contraception.     

Studies on the origin of redox enzymes in seminal plasma and their relationship with results of in-vitro fertilization

Glutathione (GSH), GSH peroxidase (GPX), GSH reductase (GRD), superoxide dismutase (SOD) and catalase-like enzyme activity were quantified in seminal plasma from normozoospermic patients, men with known distal ductal occlusion, proven fathers and male partners of couples receiving in-vitro fertilization (IVF) treatment for both male and female causes. Glutathione was non-detectable (< 2.5 microM) in seminal plasma. None of the enzyme activities per unit volume were lower in semen from vasectomized men, suggesting that they did not originate substantially from the testis or epididymis. The strongest relationships between enzyme activities and accessory gland markers were between zinc and GRD (r = 0.678), SOD (r = 0.602) and GPX (r = 0.548), suggesting a largely prostatic origin of these enzymes. Only weak relationships between accessory gland markers and catalase-like activity suggested a multi-glandular source of this enzyme. There was no relationship between the activity of any of the enzymes in the IVF patients with their fertilization rates in vitro or the establishment of pregnancy after IVF. Nor was there any correlation of enzyme activity with the morphology and percentage of motile spermatozoa in semen or with the percentage motility of spermatozoa immediately after swim-up or after overnight incubation. These findings suggest that the protective enzymes in the seminal plasma are contributed largely by the prostate and little by the epididymis, and that in most cases of IVF, they have no major influence on the outcome.      

Genetic typing and HIV-1 diagnosis by using 96 capillary array electrophoresis and ultraviolet absorption detection

Current high-throughput approaches to the analysis of PCR products are based primarily on electrophoretic separation and laser-excited fluorescence detection. We show that capillary array electrophoresis can be applied to HIV-1 diagnosis and D1S80 VNTR genetic typing based simply on UV absorption detection. The additive contribution of each base pair to the total absorption signal provides adequate detection sensitivity for analyzing most PCR products. Not only is the use of specialized and potentially toxic fluorescent labels eliminated, but also the complexity and cost of the instrumentation are greatly reduced.     

Glycoproteins present in human follicular fluid that inhibit the zona- binding capacity of spermatozoa

Previous studies have suggested that human follicular fluid contains factors that reduce the zona-binding capacity of spermatozoa. The present study provides further evidence of the existence of such factors. Using the hemizona binding assay (HZA), we have shown that the inhibitory effect of human follicular fluid on the zona-binding capacity of spermatozoa is concentration-dependent, an inhibitory effect being detected when the concentration of human follicular fluid was > or = 10%. A 1% concentration of human follicular fluid did not possess this inhibitory activity. Heating human follicular fluid at 56 degrees C for 30 min did not affect its inhibitory properties; treatment with proteinase-K abolished such inhibition. Human follicular fluid was fractionated sequentially by concanavalin-A affinity chromatography, Mono Q ion-exchange chromatography and Superose-12 gel filtration. The zona binding inhibitory activity resided in the fraction which bound to the lectin and Mono Q column and contained molecules with native molecular weights of 32 and 192 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis suggested that the 192 kDa glycoprotein was a tetramer, while the 32 kDa glycoprotein remained as a single molecular species under denaturing conditions. We conclude that two glycoproteins were responsible for the zona binding inhibitory activity of human follicular fluid. The physiological role of these factors remains unclear.      

A model of corrective gene transfer in X-linked ichthyosis

Single gene recessive genetic skin disorders offer attractive prototypes for the development of therapeutic cutaneous gene delivery. We have utilized X-linked ichthyosis (XLI), characterized by loss of function of the steroid sulfatase arylsulfatase C (STS), to develop a model of corrective gene delivery to human skin in vivo. A new retroviral expression vector was produced and utilized to effect STS gene transfer to primary keratinocytes from XLI patients. Transduction was associated with restoration of full-length STS protein expression as well as steroid sulfatase enzymatic activity in proportion to the number of proviral integrations in XLI cells. Transduced and uncorrected XLI keratinocytes, along with normal controls, were then grafted onto immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with defective skin barrier function, effectively recapitulating the human disease in vivo. Transduced XLI keratinocytes from the same patients, however, regenerated epidermis histologically indistinguishable from that formed by keratinocytes from patients with normal skin. Transduced XLI epidermis demonstrated STS expression in vivo by immunostaining as well as a normalization of histologic appearance at 5 weeks post-grafting. In addition, transduced XLI epidermis demonstrated a return of barrier function parameters to normal. These findings demonstrate corrective gene delivery in human XLI patient skin tissue at both molecular and functional levels and provide a model of human cutaneous gene therapy.      

Clearance and Glomerular Localization of Preformed DNP Anti-DNP Immune Complexes

The in vivo behaviour of well-defined immune complexes in rats was studied using complexes derived from DNP-conjugated bovine thyroglobulin (DNP-BTG) and purified specific goat anti-DNP IgG. Both clearance and glomerular localization were mainly dependent on the nature of the antigen. Soluble immune complexes formed with DNP17-BTG were cleared faster and showed a more marked localization in the glomerular mesangium than complexes formed with DNP3.4-BTG. A slight increase in the antibody to antigen ratio seemed to facilitate mesangial localization of soluble immune complexes. Insoluble immune complexes showed temporary localization as microemboli in the lumina of glomerular and peritubular capillaries. This study thus shows that not only the size and composition of the complexes but also the nature of the antigen within the complex can influence the clearance and organ localization of circulating immune complexes.     

Follicular fluid renin concentration and IVF outcome

Total renin protein concentration (TRC) was measured in stored follicular fluid (FF) samples from 42 women. Samples were selected according to their origin from follicles either without recovered ova ('empty', n = 38) or fertilized but with failed implantation ('failed', n = 36) or successful deliveries ('deliveries', n = 71). Ratios of number of embryos transferred to number of infants delivered were 2:1, 3:1 or 4:2 but 1:1 was not available. Non-parametric testing was applied to FF-TRC, volume and outcome. TRC was significantly higher in the delivery than the failed (P = 0.001) or empty (P = 0.002) categories. Assuming that the range of renin in failed follicles can identify the sub-population of unsuccessful follicles in the delivery category, then elevated FF-TRC was clearly associated with successful outcome. For individual women, the odds of infant delivery increased 17-fold as a function of average FF-TRC between 10,000 and 25,000 microIU/ml. For failed and delivery but not empty follicles, higher renin levels occurred in the smaller follicles, consistent with a burst of renin synthesis associated with the presence of an oocyte. The results suggest that FF-TRC relates to ovum viability with ovarian hyperstimulation and may have predictive use in IVF programmes.     

Endometrial and subendometrial blood flow measured during early luteal phase by three-dimensional power Doppler ultrasound in excessive ovarian responders

BACKGROUND: Impaired implantation in assisted reproduction cycles with high serum estradiol (E(2)) concentrations may be related to suboptimal endometrial perfusion. Endometrial and subendometrial blood flow were compared between excessive responders (serum E(2) on the day of HCG >20 000 pmol/l) and moderate responders (E(2) < or =20 000 pmol/l). METHODS: Three-dimensional (3D) ultrasound examination with power Doppler was performed 2, 4 and 7 days after HCG in 32 patients who did not have embryo transfer in order to measure endometrial thickness, pulsatility index (PI)/resistance index (RI) of uterine vessels, and endometrial volume, vascularization index (VI)/flow index (FI)/vascularization flow index (VFI) of endometrial and subendometrial regions. RESULTS: Excessive responders tended to have lower endometrial and subendometrial VI/VFI on HCG +2 and more absent endometrial/subendometrial blood flow. They had significantly higher endometrial FI and subendometrial VFI than moderate responders on HCG +7. Only in the excessive responder group, uterine PI/RI declined significantly from HCG +2 to HCG +7 and endometrial VI/VFI increased significantly from HCG +4 to HCG +7. CONCLUSION: Changes in uterine Doppler flow indices, and endometrial and subendometrial 3D power Doppler flow indices during the early luteal phase were significantly different between moderate and excessive responders.     

Human oviductal cells produce high molecular weight factor(s) that improves the development of mouse embryo

The cocuhture effects of human oviductal cells on mouse embryodevelopment in vitro were studied. Pronuclear stage mouse zygoteswere cocultured with different cell types, or then culturedeither in medium alone (control), spent medium from oviductalcell culture (conditioned medium) or high molecular weight fractions(>10 and>100 kDa) of the conditioned medium (reconstitutedmedium). Embryotrophic activities were compared between thesegroups in terms of percentage of morula and blastocyst formation,and cell count at the blastocyst stage. The mouse embryos developedbetter in oviductal cell cocutture than in fibroblast cocultureand medium alone culture. Conditioned medium and its reconstitutedmedium also provided a significant enhancement of embryo developmentin vitro when compared with the control medium culture, suggestingthe production of high molecular weight embryotrophic factor(s)by the oviductal cells. The high molecular weight embryotrophicactivity accumulated with the duration of conditioning couldbe serially diluted, and was abolished by heat or trypsin treatment.Replacing bovine serum albumin with polyvinyl alcohol in theculture medium did not affect the production of this high molecularweight embryotrophic activity by oviductal cells. coculture/embryos/embryotrophic factor/oviductal cells     

Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice.

Monoclonal antibodies were raised against a bacterial fusion protein containing amino acids 364 to 623 of the major nonstructural protein, NS-1, of minute virus of mice (MVMp), an autonomous parvovirus. By immunoblot analyses, these antibodies all recognized an 83-kDa protein in MVM-infected mouse fibroblast cells. Indirect immunofluorescence studies showed that five of the six react against a nuclear protein in MVM-infected mouse cells resulting in discrete foci of fluorescence. These foci do not correspond with the nucleoli, the site of MVM DNA replication. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen and showed that four distinct epitopes were recognized by the different antibodies. A 25-amino-acid peptide was used in competition ELISAs to confirm the location of the epitope recognized by two antibodies CE10 and AC6. Preliminary characterization of an NS-1/NS-2 fusion protein synthesized in insect cells using a baculovirus expression vector showed that this fusion protein is also localized within the nucleus; however, in contrast, the full-length NS-1 polypeptide is located within the cytoplasm.