Translocation of PKG1α acts on TRPV4-C1 heteromeric channels to inhibit endothelial Ca(2+) entry.

AIM: TRPV4-C1 heteromeric channels contribute to store-operated Ca(2+) entry in vascular endothelial cells. However, the negative regulation of these channels is not fully understood. This study was conducted to investigate the inhibitory effect of PKG1α on TRPV4-C1 heteromeric channels. METHODS: Immuno-fluorescence resonance energy transfer (FRET) was used to explore the spatial proximity of PKG1α and TRPC1. Phosphorylation of endogenous TRPC1 was tested by phosphorylation assay. [Ca(2+)]i transients and cation current in MAECs were assessed with Fura-2 fluorescence and whole-cell recording, respectively. In addition, rat mesenteric arteries segments were prepared, and vascular relaxation was examined with wire myography. RESULTS: In immuno-FRET experiments, after exposure of these cells to 8-Br-cGMP, more PKG1α was observed in the plasma membrane, and PKG1α and TRPC1 were observed to be in closer proximity. TAT-TRPC1(S172) and TAT-TRPC1(T313) peptide fragments, which contain the PKG targeted residues Ser172 and Thr313, respectively, were introduced into isolated endothelial cells to abrogate the translocation of PKG1α. Furthermore, a phosphorylation assay demonstrated that PKG directly phosphorylates TRPC1 at Ser172 and Thr313 in endothelial cells. In addition, PKG activator 8-Br-cGMP markedly reduced the magnitude of the 4αPDD-induced and 11,12-EET-induced [Ca(2+)]i transients, the cation current and vascular relaxation. CONCLUSION: This study uncovers a novel mechanism by which PKG negatively regulates endothelial heteromeric TRPV4-C1 channels through increasing the spatial proximity of TRPV4-C1 to PKG1α via translocation and through phosphorylating Ser172 and Thr313 of TRPC1.

[Mechanism for sperm immobilization activity of extract from Platycodon grandiflorum in vitro].

OBJECTIVE: To explore the mechanism of spermicidal effect of crude extract and platycodin-D from Platycodon grandiflorum (PG) root in vitro. METHODS: Between February 2006 and December 2009, 38 fertile and healthy adult males were selected as donors. PG root was extracted and platycodin-D purified. Grouping was as follows: crude extract from PG root, platycodin-D, nonoxynol-9 (N-9, as a reference standard) and semen-added physiological saline (as control). Spermicidal experiments were carried out in vitro (Sander-Cramer test). The hypo-osmotic swelling (HOS) test and modified Eosin-Giemsa (EG) staining were used to detect the integrity of sperm membrane. Four types of sperm morphology were divided through HOS-EG test: Type A: spermatozoa with swelling in tails and head white staining HOS(+)-EG(-) (membrane intact); Type B: spermatozoa with no swelling in tails (membrane-damaged) and head white staining HOS(-)-EG(-); Type C: spermatozoa with tail swelling and head red HOS(+)-EG(+); Type D: spermatozoa with no swelling in tails and head red HOS(-)-EG(+). Sperm chromatin dispersion (SCD) test was performed to determine the integrity of sperm DNA. RESULTS: The crude extract from PG root could immobilize and kill 100% spermatozoa within 20 s in vitro at the concentrations of 50.0 g/L and 20.0 g/L (v:v = 1:1 in semen). When the semen sample was exposed to the concentrations of 2.0 g/L and 1.0 g/L of platycodin-D, all spermatozoa were immobilized within 20 s. In the control group, the mean percentage of Types A, B, C and D was (69.0 ± 8.3)%, (3.4 ± 0.5)%, (10.2 ± 1.7)% and (17.4 ± 2.1)% respectively. In the groups of platycodin-D and N-9 solution, the rate of Types A and B was 0. The rate of Types C [(65.3 ± 3.8)%] and D [(34.7 ± 7.1)%] significantly increased versus control in the platycodin-D group (P < 0.01). Sperm DNA fragmentation had no change upon an exposure to the extract from PG root, platycodin-D and N-9 solution. And the sperm revival test showed none of the spermatozoa recovered their motility. CONCLUSION: The extract and platycodin-D from PG root have a quick sperm-killing effect in a short time in vitro by disrupting the integrity of sperm membrane (main head).

Molecular cloning and characterization of a C-type lectin from the cotton bollworm, Helicoverpa armigera.

C-type lectins participate in pathogen recognition and other defense responses in innate immunity as well as in cell-cell interactions. A new cDNA encoding a 335-residue polypeptide containing two tandem C-type lectin domains was cloned from the haemocytes of Helicoverpa armigera (Ha-lectin). Northern hybridizations revealed that the mRNA of Ha-lectin was expressed constitutively in haemocytes, and was up-regulated following injections of bacteria, yeast, or virus. Ha-lectin expression was also induced in the fat body when larvae were injected with bacteria, yeast or 20-hydroxyecdysone and a non-steroidal ecdysone agonist, RH-2485. The Ha-lectin was detected in granular haemocytes. The recombinant protein (rHa-lectin) expressed in Escherichia coli had hemagglutinating and sugar-binding activities. The native Ha-lectin protein was identified in haemocytes and plasma using a polyclonal antiserum raised against rHa-lectin by immunoblotting techniques. All together, our results suggest that the Ha-lectin gene is involved in innate immunity, and may also participate in the molting process.

Percutaneous vasal sperm aspiration and intrauterine insemination for infertile males with anejaculation.

OBJECTIVE: To explore the effectiveness of percutaneous vasal sperm aspiration (PVSA) in combination with intrauterine insemination (IUI) to treat infertile men with anejaculation. DESIGN: Clinical study. SETTING: Department of reproductive endocrinology and andrology of a family planning research clinic. PATIENT(S): Twenty-six anejaculatory infertile men. INTERVENTION(S): Spermatozoa obtained from the vas deferens by percutaneous aspiration were incubated in sperm preparation medium. MAIN OUTCOME MEASURE(S): Sperm quality by PVSA and pregnancy outcome. RESULT(S): Thirty-four PVSA-IUI procedures were performed in 26 men with anejaculation. Nineteen pregnancies were achieved (pregnancy rate, 73.1%). Mean (+/-SD) values for sperm variables were as follows: motility, 78.6% +/- 14.2%; progressive motility, 60.4% +/- 11.2%; density, 37.6 +/- 13.2 x 10(6) cells/mL; total count, 35.2 +/- 13.2 x 10(6) cells; and abnormal sperm, 18.6% +/- 7.6%. CONCLUSION(S): Percutaneous vasal sperm aspiration may obtain high-motility sperm, and PVSA plus IUI is an effective treatment for male infertility with anejaculation.

Restoration of fertility in vasectomized men using percutaneous vasal or epididymal sperm aspiration.

OBJECTIVE: To restore fertility of vasectomized men using percutaneous epididymal sperm aspiration (PESA) and percutaneous vasal sperm aspiration (PVSA) via intrauterine insemination (IUI). PATIENTS: Twenty-eight vasectomized men who required restoration of their fertility with PESA, PVSA and IUI. RESULTS: Of 28 vasectomy reversal subjects, 16 cycles of IUI using vasal sperm by percutaneous aspiration were performed in 16 subjects and 6 pregnancies were achieved. IUIs with epididymal sperm by percutaneous aspiration were carried out in 12 subjects with epididymal obstruction due to vasovasostomy for vasectomy reversal, and 2 pregnancies were achieved using caudal and epididymal sperm by percutaneous aspiration, respectively. CONCLUSION: The PESA-IUI and PVSA-IUI techniques are attractive, economical and effective for vasectomy reversal. The pregnancy by IUI using PESA and PVSA reveals that the caput epididymal sperm possess fertilization capacity in female reproductive tract and provides a new approach for the restorative fertility of vasectomized men.

Successful pregnancy and birth after intrauterine insemination using caput epididymal sperm by percutaneous aspiration.

AIM: To manage male infertility with obstructive azoospermia by means of percutaneous epididymal sperm aspiration (PESA) and intrauterine insemination (IUI). METHODS: Ninety azoospermic patients with congenital bilateral absence of the vas deferens (BAVD, n=58) or bilateral caudal epididymal obstruction (BCEO, n=32) requesting for fine needle aspiration (FNA), PESA and IUI were recruited. The obstruction was diagnosed by vasography and determination of the fructose, carnitine and alpha-glucosidase levels in the seminal fluid. RESULTS: The mean sperm motility, density, abnormal sperm and total sperm count of the caput epdidymis were 16 %+/-22 %, (12+/-31) x 10(6)/mL, 55 %+/-36 % and (16+/-14) x 10(6), respectively. In the 90 couples, a total of 74 PESA procedures and 66 cycles of IUI were performed. Three pregnancies resulted, including one twin pregnancy giving birth to two healthy boys, one single pregnancy with a healthy girl and another single pregnancy aborted at week 6 of conception. The pregnancy rate per IUI cycle was 4.5 %. CONCLUSION: The birth of normal, healthy infants by IUI using PESA indicates that the caput epididymal sperm possess fertilization capacity. The PESA-IUI programme is a practical and economical procedure for the management of patients with obstructive azoospermia.

[Study on the sexual development of adolescent male].

OBJECTIVES: The investigation of the testicular volume, the penis length and the T, FSH, LH, PRL levels in serum were taken in 289 adolescent males to provide the valuable data for andrology. METHODS: The adolescent males were grouped according to their age. The testicular volume was measured with testicular model and the T, FSH, LH, PRL levels in serum were determined by immunoenzymetric assay. RESULTS: The male sexual development was rapid from age 11 to 16 and close to that of adult at age 18. Serum PRL of adolescent males was higher than that of adult males. CONCLUSIONS: The age 11 to 16 is a period of rapid growth in sexual maturation. PRL may play an important role in sexual maturation.

Microvesicles mediate transfer of P-glycoprotein to paclitaxel-sensitive A2780 human ovarian cancer cells, conferring paclitaxel-resistance.

The overexpression of P-glycoprotein (P-gp) causes resistance to chemotherapy in human ovarian cancer. However, the underlying mechanism remains unclear. In the present study, we showed that, at membrane-bound protein level, P-gp was 'shared' between human ovarian cancer cells by the intercellular transfer of microvesicles (MVs). Paclitaxel-resistant human ovarian cancer cells (A2780/PTX) readily formed and released P-gp-containing MVs into the extracellular space compared with the wild-type parental line (A2780/WT). Shedding MVs bound to the chemosensitive A2780/WT cells in a time- and dose-dependent manner, transferring P-gp via the microenvironment. MV-mediated transfer of P-gp led to redistribution of the chemotherapeutic drug adriamycin in recipient cells (A2780/WT), which displayed 5- and 5-fold higher resistance to adriamycin and paclitaxel, respectively. Thus, these findings demonstrate a new mechanism of drug-resistance acquisition via MVs.

A new experimental three-dimensional, reticular intrauterine device (3-DRIUD) composed of nitinol and silicone rubber.

BACKGROUND: The aim of this study was to explore a new three-dimensional, reticular intrauterine device (3-DRIUD) composed of nitinol and silicone rubber and to observe the contraceptive effect of the device in rats. STUDY DESIGN: Two contraceptive experiments were performed. In the first, female rats underwent bilateral placement of a 20.0-35.0-mm 3-DRUID (experimental group, n=30) via an abdominal incision or a sham operation with no IUD (control group, n=30). Two weeks after the operation was performed, the rats from either group were caged together with male rats. The contraceptive effects of the 3-DRIUD were observed at 1 to 3 months postoperation, after which the 3-DRIUDs were removed. One month after this second operation, the rats from the two groups were again coupled with fertile male rats. In a second experiment, female rats underwent bilateral placement of a 10.0-mm 3-DRUID (n=5) via an abdominal incision or a two-dimensional IUD (2-DIUD, n=20) and mated 1 month after surgery. The single-pipeline IUD was placed in 10 rats, while the enfolded-pipeline IUD was placed in 10 different rats. RESULTS: In the first experiment, none of the females in the experimental 3-DRIUD group became pregnant (0/30, 0%) after 3 months, compared to 28/30 (93.3%, p<.0001) rats in the control group. After the 3-DRIUDs were removed from the experimental group after 3 months, 27/30 (90%) became pregnant, compared with 29/30 (97%, p>.05). The litter size (mean±SD) did not differ between groups (10.9±1.5 3-DRUID, 11.2±1.1 control, p>.05). In the second experiment, five rats had a 10.0-mm 3-DRUID (which was one third the length of one uterine horn) inserted into the bilateral uterine horns, and three of the five rats became pregnant. All 20 rats were pregnant 1 month after the insertion of the 2-DIUD. Thus, the contraceptive rate for the 2-DIUD group was 0. CONCLUSIONS: The primary contraceptive mechanism effect of the new 3-DRIUD in rodents appears to be a result of occupying physical space in the uterus.

Effects of the crude extract of Polygala tenuifolia Willd on human sperm in vitro.

The aim of the present study is to analyze sperm membrane changes and the spermicidal effect in treatment with the crude extract from Polygala tenuifolia Willd (PTW) in vitro. The root of PTW was extracted in distilled water. Normal human spermatozoa were used to assess the spermicidal activity (Sander-Cramer assay) of the extract from the PTW root. The hypo-osmotic swelling (HOS) test and the eosin Y (EY) staining were used to detect the integrity of sperm membrane and vitality. The sperm chromatin dispersion (SCD) test was performed to determine sperm DNA integrity. N-9 was used as a reference standard and semen added to physiological saline was used as the control. Semen samples were donated by 42 healthy fertile men. The crude extract from the root of PTW could immobilize and kill 100% spermatozoa within 20 s in vitro at the concentrations of 20.0 and 10.0 mg/ml; at the concentration of 5.0 mg/ml, spermatozoa were immobilized in (39.5±3.2) s. In the groups of the crude extract from the root of PTW and N-9 solution, the rate of the normal HOS (tails swollen) and the white head (unstained) was 0%, and the rate of the abnormal HOS (tails unswollen) and red head (stained) was 100%. Sperm DNA fragmentation showed no change in exposure to the crude extract from the root of PTW and N-9 solution. The sperm revival test did not show any spermatozoa that recovered their motilities. The rapid spermicidal activity of the crude extract from the root of PTW in vitro may occur by the disruption of the sperm membrane integrity.

Hsc70 binds to ultraspiracle resulting in the upregulation of 20-hydroxyecdsone-responsive genes in Helicoverpa armigera.

To probe the specific functions of the chaperone protein Hsc70 in 20-hydroxyecdysone signaling, we report on the roles of the Hsc70 from Helicoverpa armigera. RT-PCR analysis revealed that the genes for HaEcRB1 and HaUSP1 were upregulated in 5th molting and metamorphic molting larvae, whereas HaHsc70 maintained a constitutive expression level throughout larval development. Silencing HaEcRB1, HaUSP1 or HaHsc70 by RNAi inhibited the expression of a set of 20E-responsive genes. Immunocytochemical assay demonstrated that HaHsc70 is located predominantly in the cytoplasm of unstimulated cells and partially translocated to the nucleus after stimulation by 20E. Knockdown of HaHsc70 by RNAi decreased the amount of both HaEcRB1 and HaUSP1 in the nucleus. HaHsc70 was capable of binding to HaUSP1 in pull-down assays. These data suggest that Hsc70 participates in the 20E signal transduction pathway via binding to USP1 and mediating the expression of EcRB1, USP1 and then a set of 20E-responsive genes.