The effect of nano hydroxyapatite coating implant surfaces on gene expression and osseointegration.

BACKGROUND: Hierarchical micro-nano structured topography along with surface chemistry modifications of dental implants have been suggested to positively contribute to the osseointegration process. However, the effect of such surface modifications on the molecular response as well as bone formation rate and quality are still unclear, especially in the early healing period. This study aimed to evaluate the effect of coating a double acid etched (DAE) implant surface with nano-sized (20 nm) hydroxyapatite (Nano) with respect to gene expression, histologic parameters, and nanomechanical properties when compared to DAE control at 1 and 2 weeks after implant placement in a rodent femur model. MATERIAL AND METHODS: Expression of bone-related genes was determined by qRT-PCR (Col-I, Runx-2, Osx, Opn, Ocn, Alp). Histomorphometric evaluation of bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO) within implant threads was performed using photomicrographs after histologic processing. Mechanical properties, reduced elastic modulus and hardness, were determined through nanoindentation. RESULTS: At 1 week, the Nano group demonstrated significantly higher expression of Col-I and Ocn compared to the DAE group, indicating upregulation of osteoprogenitor and osteoblast differentiation genes. At 2 weeks, Nano surface further exhibited enhanced gene expression of Col-I and Osx in comparison to the DAE surface, suggesting an increased mineralization of the newly formed bone. Nanoindentation analysis revealed that the Nano group presented no significant difference on the ranks of reduced elastic modulus and hardness compared to DAE for both timepoints. Histomorphometric analysis yielded no significant difference in the percentage of BIC and BAFO between the Nano and DAE surfaces at 1 and 2 weeks. However, Nano implants did present a higher mean value, ~50%, of BIC compared to DAE, ~30%, after 2 weeks in vivo. CONCLUSIONS: While no significant differences were observed in the amount and mechanical properties of newly formed bone, Nano surface positively and significantly increased the expression osteogenic genes compared to DAE surface at early healing periods.

Evaluation of non-Gaussian model-based diffusion-weighted imaging in oral squamous cell carcinoma: comparison with tumour functional information derived from positron-emission tomography.

AIM: To evaluate and compare diffusion-weighted imaging (DWI) parameters derived from a non-Gaussian fitting model and positron-emission tomography (PET) parameters derived from 18F-fluoromisonidazole-PET (FMISO-PET) in patients with oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Primary sites were evaluated prospectively in 18 patients. DWI was performed using six b-values (0-2,500). Diffusion-related parameters of kurtosis value (K), the kurtosis-corrected diffusion coefficient (DK), diffusion heterogeneity (α), distributed diffusion coefficient (DDC), the slow diffusion coefficient (Dslow), and the apparent diffusion coefficient (ADC) were calculated from four diffusion-fitting models. Maximal standardised uptake values (SUVmax), mean standardised uptake values (SUVmean), and the tumour-to-muscle ration (TMR) of the SUV value were calculated for FMISO-PET. Spearman's correlation coefficient was used to evaluate the correlation between each non-Gaussian diffusion model parameters and PET parameter. RESULTS: There was moderate correlation between FMISO-PET SUVmax and Dslow (ρ=-0.45, p=0.06). In addition, there was good correlation between TMRmax and five non-Gaussian diffusion model parameters (K: ρ=0.65, p=0.004, DK: ρ=-0.72, p=0.0008, DDC: ρ=-0.75, p=0.0003, ADC: ρ=-0.74, p=0.0005, and Dslow: ρ= -0.65, p=0.003), and between TMRmean and five non-Gaussian model parameters (K: ρ=0.64, p=0.005, DK: ρ=-0.61, p=0.007, DDC: ρ=-0.63, p=0.005, ADC: ρ=-0.61, p=0.007, and Dslow: ρ=-0.56, p=0.015). CONCLUSION: Non-Gaussian diffusion model parameters can be related to tumour hypoxia.

Diagnostic accuracy of plasma atrial natriuretic peptide concentrations in cats with and without cardiomyopathies.

OBJECTIVES: Plasma atrial natriuretic peptide (ANP) levels have been reported to be elevated in cats with cardiomyopathy. We investigated the diagnostic accuracy of plasma ANP concentration as an indicator of the severity of cardiomyopathies. ANIMALS: This study included 78 control cats and 83 cats with various types of cardiomyopathy. METHODS: This was a prospective multicentre study. Control cats were determined to have a normal heart, and diseased cats were diagnosed by echocardiography. Diseased cats were divided into asymptomatic cats without left atrial dilation (LAD), asymptomatic cats with LAD, and cats with heart failure. Plasma C-terminal ANP concentrations were measured using chemiluminescence. RESULTS: The median plasma ANP concentration in controls was 43.3 (interquartile range, 33.0-56.3) pg/mL. Plasma ANP values were significantly higher in the cardiomyopathic cats with LAD and heart failure, but the values in cats without LAD were comparable to those in control cats. To distinguish cats with cardiomyopathy from controls, a plasma ANP concentration >77.5 pg/mL afforded sensitivity of 66.3% and specificity of 84.6%. Use of plasma ANP concentration >110.9 pg/mL to identify cats with LAD had a sensitivity of 73.6% and specificity of 93.5%. The areas under the receiver-operating characteristic curve were 0.80 and 0.86. CONCLUSIONS: Plasma ANP concentrations were higher in cats with more advanced cardiomyopathy. Although assaying the ANP concentration alone may not help to diagnose cardiac disease, measuring provides additional information that is useful for assessing the severity of cardiomyopathies.

Gastrointestinal dysmotility is associated with altered gut flora and septic mortality in patients with severe systemic inflammatory response syndrome: a preliminary study.

BACKGROUND: The gut is an important target organ for injury after severe insult, and resolution of feeding intolerance is crucial for critically ill patients. We investigated gut flora and motility to evaluate the impact of gastrointestinal dysmotility on septic complications in patients with severe systemic inflammatory response syndrome (SIRS). METHODS: Sixty-three ICU patients with severe SIRS were divided into two groups depending on their intestinal condition. Patients with feeding intolerance comprised patients who had feeding intolerance, defined as ≥ 300 mL reflux from nasal gastric feeding tube in 24 h, and patients without feeding intolerance comprised patients with no feeding intolerance. We compared fecal microflora, incidences of bacteremia, and mortality between these groups. KEY RESULTS: Analysis of feces showed that patients with feeding intolerance had significantly lower numbers of total obligate anaerobes including Bacteroidaceae and Bifidobacterium, higher numbers of Staphylococcus, lower concentrations of acetic acid and propionic acid, and higher concentrations of succinic acid and lactic acid than those in patients without feeding intolerance (P ≤ 0.05). Patients with feeding intolerance had higher incidences of bacteremia (86%vs 18%) and mortality (64%vs 20%) than did patients without feeding intolerance (P ≤ 0.05). CONCLUSIONS & INFERENCES: Gut flora and organic acids were significantly altered in patients with severe SIRS complicated by gastrointestinal dysmotility, which was associated with higher septic mortality in SIRS patients.

Evaluation of plasma C-terminal atrial natriuretic peptide in healthy cats and cats with heart disease.

BACKGROUND: The clinical implications of evaluating C-terminal atrial natriuretic peptide (ANP) concentration in cats are still controversial. HYPOTHESIS: The objective of this study was to investigate the relationship between plasma C-terminal ANP concentration and left atrial pressure (LAP) in healthy cats with volume overload (study 1), and to compare plasma C-terminal ANP in normal cats and cats with cardiomyopathy (study 2). ANIMALS: Five healthy adult cats were used in study 1, and clinically healthy cats (n=8) and cats with cardiomyopathy (n=14) were used in study 2. METHODS: In study 1, cats were anesthetized and given acetated Ringer's solution (100 mL/kg/h for 60 minute) via the cephalic vein. Hemodynamic measurements and blood samples, collected from the jugular vein, were performed at 10-min intervals. In study 2, blood samples from normal cats and cats with cardiomyopathy were collected from the cephalic vein. The plasma C-terminal ANP concentration was determined by radioimmunoassay for human alpha-ANP. RESULTS: In study 1, volume overload significantly increased the C-terminal ANP concentration and LAP from baseline. The C-terminal ANP concentration was strongly correlated with the mean LAP. In study 2, age, E wave velocity, and the ratios of the left atrium to aorta were significantly higher in the cats with cardiomyopathy compared with the normal cats. The C-terminal ANP concentration was significantly higher in the cats with cardiomyopathy compared with the normal cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Our results suggest that the measurement of plasma C-terminal ANP in cats may provide additional information for the diagnosis of heart disease.

Immunohistochemical examination of epiphyseal growth plates of Japanese Brown cattle with chondrodysplasia.

A new type of inherited chondrodysplasia is described in Japanese Brown cattle, but the basic defects of the epiphyseal growth plate (EGP) in the limb long bones, and proliferation and differentiation of the chondrocytes in the EGP, are not yet understood. In the present study, the EGPs of the limb long bones in eight cases of chondrodysplasia and four normal (control) cattle were examined histologically and immunohistochemically. In the control cattle, proliferative chondrocytes (PCs) and hypertrophic chondrocytes (HCs) were arranged in columns parallel to the long axis of the bone, and HCs were situated on the metaphyseal side of the EGP. In all the affected cattle, many chondrocytes with a hypertrophic appearance were detected in the inner areas of the central portion of the EGP. The PC columns were short and arranged irregularly. Bone tissue and small blood vessels were found frequently in these areas. Six affected cattle showed complete EGP-closure. Backscattered electron (BSE) imaging showed that the calcified cartilage matrix was restricted to the lower region of the hypertrophic zone (HZ) of the EGP in the control cattle, while the calcified cartilage matrix and bone tissue were scattered in the inner areas of the EGP in all the chondrodysplastic cattle. Immunohistochemistry revealed type X collagen in the HCs and cartilage matrix of the HZ in the control cattle. In all the affected cattle, type X collagen was detected in apparently hypertrophic chondrocytes in the inner areas of the EGP. Type II collagen was detected in the entire EGP in all the affected cattle, as in the controls. BrdU (5-bromo-2'-deoxyuridine), injected intravenously 1h before euthanasia was detected in many PCs in the EGP in the control cattle; none, however, was detected in the central portion of the EGP in any affected animal. These observations indicate that differentiation into HCs and calcification of cartilage matrix occur in the inner areas of the central portion of the EGP in chondrodysplasia of Japanese Brown cattle. Differentiation into the HCs at this abnormal site may be caused by the inadequate proliferation and disorganization of the PCs. Premature EGP-closure, observed commonly in chondrodysplasia of Japanese Brown cattle, was thought to be caused by replacement of the calcified cartilage in the inner areas of the EGP by bone tissue.

Enhanced transduction of mouse salivary glands with AAV5-based vectors.

We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4 and 5 are distinct from rAAV2 and from each other, suggesting that they may direct binding and entry into different cell types. In the present study, we investigated the tropisms, transduction efficiencies, and antibody response to AAV vectors based on AAV serotypes 2, 4, and 5. Administration of rAAV2beta-galactosidase (betagal), rAAV4betagal, or rAAV5betagal to murine submandibular salivary glands by retrograde ductal instillation resulted in efficient transduction of salivary epithelial cells, with AAV4 and AAV5 producing 2.3 and 7.3 times more betagal activity compared with AAV2. Improved transduction with AAV5 was confirmed by QPCR of DNA extracted from glands and immunohistochemical staining for transgene expression. Like AAV2, AAV5 primarily transduced striated and intercalated ductal cells. AAV4 transduction was evident in striated, intercalated, and excretory ductal cells, as well as in convoluted granular tubules. In keeping with the encapsulated nature of the salivary gland, the majority of persistent viral genomes were found in the gland and not in other tissues. Neutralizing antibodies (NABs) found in the serum of virus-infused animals were serotype specific and there was no crossreactivity between serotypes. No NABs were detected in saliva but sialic acid conjugates present in saliva could neutralize AAV4 at low dilutions. Together our data suggest that because of differences in receptor binding and transduction pathways, other serotypes may have improved utility as gene transfer vectors in the salivary gland and these differences could be exploited in gene therapy applications.

Recruitment pattern of muscle fibre type during high intensity exercise (60-100% VO2max) in thoroughbred horses.

To consider the optimal training programme for Thoroughbred horses, we examined the recruitment pattern of muscle fibres including hybrid muscle fibres in well-trained Thoroughbred horses. The horses performed exercise at three different intensities and durations; i.e., 100% VO2max for 4 min, 80% and 60% VO2max for 8 min on a treadmill with 10% incline. Muscle samples were obtained from the middle gluteal muscle before, during (4 min at 80% and 60% VO2max), and after exercise. Four muscle fibre types (types I, IIA, IIA/IIX, and IIX) were immunohistochemically identified, and optical density of periodic acid Schiff staining (OD-PAS) in each fibre type, and the glycogen content of the muscle sample, were determined by quantitative histochemical and biochemical procedures. The changes in OD-PAS showed that the recruitment of all fibre types were identical at the final time stage of each exercise bout, i.e., 4 min running at 100% VO2max, and 8 min running at 80% and 60% VO2max. The changes in OD-PAS of type IIA/IIX fibre were very similar to those of type IIX fibre. The recruitment of these fibres were obviously more facilitated by 4 min running at 100% VO2max than by 4 min running at 80% or 60% VO2max. Short duration with high intensity exercise, such as 4 min running at 100% VO2max or 8 min running at 80% or 60% VO2max, is effective to stimulate type IIX fibre and IIA/IIX fibres that have the fastest speed of contraction.

Recruitment pattern of muscle fibre type during flat and sloped treadmill running in thoroughbred horses.

REASONS FOR PERFORMING THE STUDY: There is little information about the muscle fibre recruitment pattern during sloped and flat track running in Thoroughbred horses. OBJECTIVES: To examine the glycogen depletion pattern of each muscle fibre type during running on a flat and sloped treadmill. METHODS: Thirteen Thoroughbred horses (3-9 years old) were used. They were initially subjected to incremental exercise tests on a treadmill at 10 and 0% inclines in each horse to determine running speed at 90 and 60% VO2max. Needle biopsy samples were obtained from the middle gluteal muscle immediately after the running at 90% VO2max for 4 min and 60% VO2max for 12 min on 10% and 0% inclines treadmill. Four muscle fibre types (Types I, IIA, IIA/IIX, and IIX) were immunohistochemically identified, and optical density of Periodic Acid Schiff staining (OD-PAS) in each fibre type and the glycogen content of the muscle sample were determined by quantitative histochemical and biochemical procedures. RESULTS: The changes in OD-PAS showed that the recruitment of all fibre types were identical after each exercise bout, i.e., 4 min running at 90% VO2max (8.4-9.4 m/sec on 10%, 13.9-14.1 m/sec on 0%), and 12 min running at 60% VO2max (5.4-6.0 m/sec on 10%, 7.9-11.2 m/sec on 0%). No significant differences were found in the recruitment patterns of each muscle fibre type between 10 and 0% inclined exercise bouts at the same exercise intensity. CONCLUSIONS: The recruitment pattern of muscle fibre type is mainly determined by exercise intensity (%VO2max) and duration, but not by running speed. POTENTIAL RELEVANCE: The results of this study indicate the possibility that up-hill running results in the same training effect as faster running on a flat track.

Effect of high intensity training on anaerobic capacity of middle gluteal muscle in Thoroughbred horses.

We hypothesize that high intensity training for Thoroughbred horses that have been subjected to conventional training could further improve the metabolic properties of the middle gluteal muscle. Nine well-trained horses were subjected to high intensity (80-100% Vdot;O(2)max, 5 minx2) training for 12 weeks. Biopsy samples were obtained from the muscle before and after 4 and 12 weeks of training. Three of the 9 horses did not complete the training programme. In the remaining 6 horses, activities of succinic dehydrogenase (SDH), phosphofructokinase (PFK) and 3-hydroxy acyl CoA dehydrogenase (HAD), and the composition of myosin heavy chain isoforms were analyzed by biochemical techniques. After 12 weeks of training, a significant increase was found in PFK activity but not in the SDH and HAD activities. There were no significant changes in the composition of myosin heavy chain isoforms. The high intensity training in this study was effective at increasing glycolytic enzyme activity, indicating the possibility to improve anaerobic capacity, which potentially could contribute greatly to performance in Thoroughbred horses. This study also highlighted a fact that high intensity training should be given with the great care to prevent the skeletal muscle injuries.

Effect of controlled exercise on middle gluteal muscle fibre composition in Thoroughbred foals.

REASONS FOR PERFORMING STUDY: Most racehorses are trained regularly from about age 18 months; therefore, little information is available on the effect of training in Thoroughbred foals. HYPOTHESIS: Well-controlled exercise could improve muscle potential ability for endurance running. METHODS: Thoroughbred foals at age 2 months were separated into control and training (treadmill exercise) groups and samples obtained from the middle gluteal muscle at 2 and 12 months post partum. Muscle fibre compositions were determined by histochemical and electrophoretical techniques and succinic dehydrogenase (SDH) activity was analysed in each fibre type. RESULTS: All fibre types were hypertrophied with growth and type I and IIA fibres were significantly larger in the training than the control group at age 12 months. A significant increase of SDH activity was found in type IIX muscle fibres in the training group. CONCLUSIONS: Training in young Thoroughbred horses can facilitate muscle fibre hypertrophy and increase the oxidative capacity of type IIX fibres, which could potentially enhance stamina at high speeds. POTENTIAL RELEVANCE: To apply this result to practical training, further studies are needed to determine more effective and safe intensities of controlled exercise.

The scheduled ovarian hyperstimulation method makes it easy to perform ICSI with fresh testicular sperm (ICSI/TESE).

The authors evaluated whether scheduled ovarian stimulation makes it easy to perform ICSI with fresh testicular sperm. Scheduled ovarian hyperstimulation was applied for testicular sperm extraction and ICSI with fresh testicular spermatozoa. Fifteen cycles in 10 couples were included in the present study; all couples were azoospermic, 5 were obstructive, and the remaining 5 were nonobstructive. No cycles were canceled, and all oocyte retrievals were performed on the scheduled day. Testicular sperm were obtained in 14 treatment cycles (93%). The mean numbers of retrieved and injected oocytes were 9.4 and 6.4, respectively. The fertilization and cleavage rates were 47 and 91%, respectively. Embryo transfers were performed in 12 cycles except 2 cycles that had no embryos. The number of transferred embryos was 2.3. Two clinical pregnancies were obtained. This scheduled ovarian hyperstimulation was applicable for ICSI with fresh testicular sperm.

Tissue compatibility of two biodegradable tubular scaffolds implanted adjacent to skin or buccal mucosa in mice.

Radiation therapy for cancer in the head and neck region leads to a marked loss of salivary gland parenchyma, resulting in a severe reduction of salivary secretions. Currently, there is no satisfactory treatment for these patients. To address this problem, we are using both tissue engineering and gene transfer principles to develop an orally implantable, artificial fluid-secreting device. In the present study, we examined the tissue compatibility of two biodegradable substrata potentially useful in fabricating such a device. We implanted in Balb/c mice tubular scaffolds of poly-L-lactic acid (PLLA), poly-glycolic acid coated with PLLA (PGA/PLLA), or nothing (sham-operated controls) either beneath the skin on the back, a site widely used in earlier toxicity and biocompatibility studies, or adjacent to the buccal mucosa, a site quite different functionally and immunologically. At 1, 3, 7, 14, and 28 days postimplantation, implant sites were examined histologically, and systemic responses were assessed by conventional clinical chemistry and hematology analyses. Inflammatory responses in the connective tissue were similar regardless of site or type of polymer implant used. However, inflammatory reactions were shorter and without epithelioid and giant cells in sham-operated controls. Also, biodegradation proceeded more slowly with the PLLA tubules than with the PGA/PLLA tubules. No significant changes in clinical chemistry and hematology were seen due to the implantation of tubular scaffolds. These results indicate that the tissue responses to PLLA and PGA/PLLA scaffolds are generally similar in areas subjacent to skin in the back and oral cavity. However, these studies also identified several potentially significant concerns that must be addressed prior to initiating any clinical applications of this device.

A human RNA polymerase II subunit is encoded by a recently generated multigene family.

BACKGROUND: The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes. RESULTS: While those characterized so far for the human (h) RPB are also unique, we show that hRPB subunit 11 (hRPB11) is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22. We focused on two members of this family, hRPB11a and hRPB11b: the first encodes subunit hRPB11a, which represents the major RPB11 component of the mammalian RPB complex; the second generates polypeptides hRPB11balpha and hRPB11bbeta through differential splicing of its transcript and shares homologies with components of the hPMS2L multigene family related to genes involved in mismatch-repair functions (MMR). Both hRPB11a and b genes are transcribed in all human tissues tested. Using an inter-species complementation assay, we show that only hRPB11balpha is functional in yeast. In marked contrast, we found that the unique murine homolog of RPB11 gene maps on chromosome 5 (band G), and encodes a single polypeptide which is identical to subunit hRPB11a. CONCLUSIONS: The type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus.

Protection from experimental endotoxemia by a recombinant adeno-associated virus encoding interleukin 10.

BACKGROUND: Interleukin 10 (IL-10) is a homodimeric cytokine that shows considerable clinical promise. Adeno-associated virus (AAV) vectors appear increasingly useful for in vivo gene-transfer applications. METHODS: A recombinant AAV type 2 vector encoding human IL-10 (rAAVhIL10) was constructed by using an adenoviral-free, three-plasmid co-transfection. Cytokine production was measured by using an enzyme-linked immunosorbent assay. Endotoxic shock was induced by lipopolysaccharide (LPS) injection. RESULTS: As media from rAAVhIL10-infected COS cells caused a dose-dependent blockade of IL-12 secretion from spleen cells of IL-10 knockout (KO) mice challenged with Brucella abortus, it was clear that vector-derived hIL-10 was biologically active in vitro. Intravenous or intramuscular administration of relatively modest levels of rAAVhIL10 (10(10) genomes) to IL-10 KO mice resulted in hIL-10 secretion into the bloodstream, which, at 8 weeks, gave median serum levels of 0.9 and 0.45 pg/ml, respectively. Acute endotoxic shock led to a 33% mortality rate, and severe morbidity, in control IL-10 KO mice, whereas no mortality and little morbidity were seen in IL-10 KO mice given rAAVhIL10 7 weeks earlier. CONCLUSIONS: The findings demonstrate that a modest dose of rAAVhIL10 administered in vivo provides long-term protection against LPS-induced endotoxic shock in a murine model. Thus, this vector may be useful for clinical applications requiring sustained IL-10 expression, for example in the treatment of several autoimmune diseases.

Transient increase in the levels of T-helper 1 cytokines in postmenopausal women and the effects of hormone replacement therapy.

The aim of this study was to determine, at least in part, T-cell function in postmenopausal women and the effects of hormone replacement therapy (HRT). Levels of T-helper 1 (Th1) cytokines (IL-2 and IFN-gamma) and T-helper 2 (Th2) cytokines (IL-4 and IL-10) produced by phytohemagglutinin-stimulated whole blood cells from 72 untreated and 44 HRT-treated women were measured by ELISA. Thirteen of the 44 HRT-treated women were examined before and during HRT. The production of IL-2 increased gradually with advance of the postmenopausal period. The levels of IL-2 in women in the early (< or =10 years) and mid (>10 and <30 years) postmenopausal stages were significantly higher than those in women in their second, third and fourth decades. The level in women in the late (> or =30 years) postmenopausal stage, however, was significantly lower than those in women in the early and mid postmenopausal stages. The level of IFN-gamma was highest in women in the mid postmenopausal stage. On the other hand, the levels of Th2 cytokines did not change with age or after menopause until the mid postmenopausal period but were significantly lower in women in the late postmenopausal stage. IFN-gamma levels in women on HRT were significantly lower than those in untreated postmenopausal women at all postmenopausal stages. HRT induced a significant decrease in the production of IL-2 and IL-4. In conclusion, production of Th1 cytokines is augmented in women after menopause. HRT prevents this increase, thereby improving the aberration of Th1/Th2 balance that is implicated in an inadequate immune response and pathological conditions.

Progesterone accelerates the onset of capacitation in mouse sperm via T-type calcium channels.

This study was undertaken to evaluate whether progesterone induces capacitation of mouse spermatozoa. When sperm were evaluated by chlortetracycline staining, addition of progesterone significantly increased the proportion of spermatozoa exhibiting the B pattern at 60 minutes of incubation, compared with that before incubation (23 +/- 6.2% vs. 13 +/- 2.9%, p < 0.01) and that in hTF medium without progesterone (23 +/- 6.2% vs. 13 +/- 4.2%, p < 0.01). If the redistribution of proteins in sperm plasma membrane such as protein binding calcium ion were defined as capacitation, it could be said that progesterone promoted capacitation of mouse sperm. This progesterone-induced capacitation was prevented by depletion of extracellular calcium ion and addition of NiCl2, a T-type calcium channel blocker, although thapsigargin, an inhibitor of Ca2+-ATPase, did not increase the number of capacitated sperm (B pattern; progesterone vs. progesterone + depletion of calcium ion, 18 +/- 3.5% vs. 8 +/- 2.5%, p < 0.05, progesterone vs. progesterone + NiCl2, 20 +/- 3.8% vs. 6 +/- 5.2%, p < 01). Furthermore, genistein, a protein tyrosine phosphorylation inhibitor, inhibited progesterone-induced capacitation (B pattern; progesterone vs. progesterone + genistein, 20 +/- 3.8% vs. 11 +/- 2.4%, p < 01). In conclusion, progesterone induces capacitation in mouse sperm and this capacitation may be associated with calcium influx and tyrosine phosphorylation.

Sperm-immobilizing antibodies block capacitation in human spermatozoa.

Sperm-immobilizing antibodies block human fertilization by interfering with the acrosome reaction (AR). To clarify the mechanism of blockage of AR by sperm-immobilizing antibodies, the authors examined their effects on the increase of intracellular free Ca2+ concentration induced by follicular fluids (Ca2+ influx) in spermatozoa and on their capacitation. Sperm-immobilizing antibodies did not suppress Ca2+ influx induced by follicular fluid, but they inhibited capacitation of human spermatozoa. Namely delta%AR (%AR after addition of an AR inducer--%AR before treatment) induced by progesterone was significantly (p < .0001) lower when spermatozoa were incubated in human tubal fluid medium cotaining antibody-positive serum (1.2%), compared to that when incubated in control medium (19.2%). Furthermore, the proportion of both spermatozoa that became capacitated and ones that had become capacitated decreased significantly (p < .0001) after 2, 4, and 6 h of incubation in medium containing antisperm antibody-positive serum, compared to those of spermatozoa incubated in control medium. In conclusion, sperm-immobilizing antibodies may be closely related to their blockage of capacitation.

Specific cytotoxicity of antibody to YAL-198, a sperm antigen peptide, to murine zygote.

Active immunization with the peptide segments rSMP-230 and YAL-198, corresponding to the hydrophilic extracellular domain of two human sperm antigens (rSMP-B and YWK-II, respectively), reduced fertility in female rats by different mechanisms. The anti-rSMP-230 antibody interferes with human and murine fertilization, and the anti-YAL-198 antibody blocks the development of mouse embryos. The authors examined in vitro at which stage the antibodies to rSMP-230 and YAL-198 were cytotoxic to murine embryos up to morula/blastocyst stage. Anti-rSMP-230 antibody was not cytotoxic to any stages. On the other hand, the anti-YAL-198 antibody arrested the growth of embryos at the 2-cell stage but not at more advanced developmental stages. When the anti-YAL-198 antibody was used, spotty staining was observed only on the surfaces of embryos that had arrested at the 2-cell stage. Unstained embryos, however, continued to develop normally. In contrast, the anti-rSMP-230 antibody stained murine sperm but failed to stain murine ova and embryos. The present results suggest that the human sperm components rSMP-B and YWK-II play important roles in sperm-egg interaction and early development of the embryo, respectively.

Altered sensitivity to a novel vasoconstrictor endothelin-1 (1-31) in myometrium and umbilical artery of women with severe preeclampsia.

We have suggested that a novel endothelin-1 with 31 amino acids [ET-1 (1-31)] plays an important role in fetal circulation, owing to a strong contractile activity on the umbilical artery. To clarify the pathophysiological significance of ET-1 (1-31) in the development of severe preeclampsia, its contractile activities on human umbilical arteries and uterine smooth muscle from patients with preeclampsia were studied. The contraction by ET-1 (1-31) was stronger in uterine smooth muscle of the patients with severe preeclampsia than that of normal subjects. On the contrary, the constriction of umbilical artery of the patients with eclampsia was significantly weaker than that of normal pregnant women. The stronger contraction of myometrium by ET-1 (1-31) in patients with severe preeclampsia observed for the first time in the present study suggests that ET-1 (1-31) might be involved in the development of preeclampsia.