The role of metabolites under the influence of genes and lifestyles in bone density changes.

Purpose: Osteoporosis is a complex bone disease influenced by numerous factors. Previous studies have found that some metabolites are related to bone mineral density (BMD). However, the associations between metabolites and BMD under the influence of genes and lifestyle have not been fully investigated. Methods: We analyzed the effect of metabolites on BMD under the synergistic effect of genes and lifestyle, using the data of 797 participants aged 55-65 years from the Taizhou Imaging Study. The cumulative sum method was used to calculate the polygenic risk score of SNPs, and the healthful plant-based diet index was used to summarize food intake. The effect of metabolites on BMD changes under the influence of genes and lifestyle was analyzed through interaction analysis and mediation analysis. Results: Nineteen metabolites were found significantly different in the osteoporosis, osteopenia, and normal BMD groups. We found two high-density lipoprotein (HDL) subfractions were positively associated with osteopenia, and six very-low-density lipoprotein subfractions were negatively associated with osteopenia or osteoporosis, after adjusting for lifestyles and genetic factors. Tea drinking habits, alcohol consumption, smoking, and polygenic risk score changed BMD by affecting metabolites. Conclusion: With the increased level of HDL subfractions, the risk of bone loss in the population will increase; the risk of bone loss decreases with the increased level of very-low-density lipoprotein subfractions. Genetic factors and lifestyles can modify the effects of metabolites on BMD. Our results show evidence for the precise prevention of osteoporosis.

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.

Immune Infertility Should Be Positively Diagnosed Using an Accurate Method by Monitoring the Level of Anti-ACTL7a Antibody.

Infertility is currently a major public health problem. Anti-sperm antibodies (ASAs) markedly reduce sperm quality, which can subsequently lead to male and/or female infertility. The accurate detection of ASAs derived from specific spermatozoa is, therefore, clinically useful. We have focused on the spermatozoa-specific expression protein ACTL7a for many years and have developed an enzyme-linked immunosorbent assay (ELISA) to detect the concentration of anti-ACTL7a antibodies in fertile sera (n = 267) and infertile sera (n = 193). Infertile sera were collected from the positive sera of tray agglutination tests (TAT), which is a routine ASA screening methodology. We found that the concentration of anti-ACTL7a antibodies was significantly higher in the infertile sera (than in the fertile sera, P < 0.0001) and much higher in the TAT ≥ 16 infertile sera. The ELISA was much better for male sera detection (AUC = 0.9899). If we set the standard at a strongly positive value (calculated by ROC curve), the positive predictive value of the antibody detection reached 100 percent, with a false positive rate of zero. The developed ELISA method for anti-ACTL7a antibody detection is therefore sensitive, accurate, and easy to perform, making it an excellent potential tool for future clinical use.

Anti-GAPDHS antibodies: a biomarker of immune infertility.

Numerous investigations have focused on the detection of antisperm antibodies, which have a naturally occurring impact on male and female fertility. In this study, spermatogenic glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) was considered to be a candidate biomarker of immune infertility. The concentrations of anti-GAPDHS antibodies in the sera of sterile individuals and fertile couples were measured by enzyme-linked immunosorbent assay. Sera were collected from immune infertile (n = 175) and fertile (n = 237) individuals and were screened by tray agglutination tests (TAT). Infertile sera were further divided into two groups according to the serum titers obtained by TAT (titers ≤ 1:8, n = 58; titers > 1:8, n = 117). The concentrations of anti-GAPDHS antibodies were significantly higher in the immune infertile group than in the fertile group and were much higher with regard to the increased degrees of sperm agglutination (titers > 1:8). Surprisingly, we found statistically significantly higher concentrations of antibodies in the sera of infertile men than in those of infertile women, and a similar statistical result was obtained in the sera when primary infertility was compared with secondary infertility. Thus, anti-GAPDHS antibodies seem to be a sensitive parameter in immune infertile detection and might be one of the main factors causing immune infertility. This factor might be valuable as an indicator in the clinical diagnosis and monitoring treatment of infertility.

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.

MiR-489 regulates chemoresistance in breast cancer via epithelial mesenchymal transition pathway.

To investigate the role of microRNAs in the development of chemoresistance and related epithelial-mesenchymal transition (EMT), we examined the effect of miR-489 in adriamycin (ADM)-resistant human breast cancer cells (MCF-7/ADM). MiR-489 was significantly suppressed in MCF-7/ADM cells compared with chemosensitive parental control MCF-7/WT cells. Forced-expression of miR-489 reversed chemoresistance. Furthermore, Smad3 was identified as the target of miR-489 and is highly expressed in MCF-7/ADM cells. Forced expression of miR-489 both inhibited Smad3 expression and Smad3 related EMT properties. Finally, the interactions between Smad3, miR-489 and EMT were confirmed in chemoresistant tumor xenografts and clinical samples, indicating their potential implication for treatment of chemoresistance.

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.

Anti-ACTL7a antibodies: a cause of infertility.

OBJECTIVE: To show that antibodies against ACTL7a, a spermatozoon-specific protein, may be a cause of immunologic infertility. DESIGN: Determine the presence of anti-ACTL7a antibodies in infertile blood, raise antibodies against ACTL7a in rabbits, and demonstrate that the in vitro treatment of mouse spermatozoa with infertile sera markedly reduces their fertilizing capacity. Demonstrate that the active immunization of mice with ACTL7a protein reduces fertility. SETTING: National Research Institute for Family Planning, Beijing, World Health Organization Collaboration Center of Human Reproduction, China. ANIMAL(S): Rabbits, ICR mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Mass spectrometry, indirect immunostaining, spermatozoa agglutination test, and standard fertility assay. RESULT(S): The fertilizing potential of mouse spermatozoa was markedly reduced after in vitro treatment with ACTL7a antibody-containing serum from a vasectomized man. Active immunization with ACTL7a significantly reduced the fertility of mice. Anti-ACTL7a antibodies caused the agglutination of mouse and human spermatozoa in vitro. Furthermore, the antibodies were detected in the sera of additional vasectomized men. CONCLUSION(S): Anti-ACTL7a antibodies may cause infertility in mice because the in vitro treatment of mouse spermatozoa with ACTL7a antibody-containing serum markedly reduced the fertilizing potential of the spermatozoa. In addition, the active immunization of mice with ACTL7a resulted in significant reductions in fertility.

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.

[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).

[Analysis of sperm chromosomal abnormalities and sperm DNA fragmentation in infertile males].

OBJECTIVE: To investigate changes in sperm chromosome and sperm DNA integrity of infertile males. METHODS: The level of DNA fragmentation was determined by Sperm Chromatin Dispersion (SCD) test in infertile males with idiopathic severe oligoasthenozoospermia (ISOA, n= 19), couples with unexplained recurrent miscarriage (URM, n= 38) and adult healthy fertile men (control group, n= 32). Multi-color fluorescence in situ hybridization (FISH) was performed with probes specific for chromosomes 13, 18, 21, X and Y in the control group (n= 5), the ISOA (n= 10) and the URM (n= 12). RESULTS: Patients with ISOA and URM showed a significantly higher abnormality with total rate of 4.02% (n= 19) and 3.91%(n= 38) for chromosomes 13, 18 and 21, and 2.03%, 1.98% for chromosomes X and Y, respectively, in their spermatozoa compared to control (1.29% and 0.61%, P< 0.01). A significantly higher proportion of total sperm DNA fragmentation was detected in patients with ISOA (40.7%+/- 17.8%) and URM (22.1%+/- 10.3%) of sperm compared to the control group (12.1%+/- 5.2%, P< 0.01). Moreover, a positive correlation was found between the rate of sperm chromosomal aberration and the rate of sperm DNA fragmentation (gamma = 0.874, P< 0.01, n= 27). There were significant correlation between sperm DNA fragmentation and sperm density, sperm motility and abnormal sperm (gamma = - 0.571, gamma = - 0.616 and gamma = 0.637, respectively, P< 0.01). CONCLUSION: The result indicates that spermatozoa from patients with ISOA and URM contain greater DNA fragmentation and chromosomal aneuploidy and may lead to male infertility. Screening for sperm DNA damage may provide useful information in the diagnosis of male idiopathic infertility.

Molecular cloning and characterization of Hearm caspase-1 from Helicoverpa armigera.

Members of the caspase family play a central and evolutionary role in programmed cell death (PCD), which removes unwanted, damaged and dangerous cells during development to maintain homeostasis. In this paper, we describe the cloning and characterization of a caspase from the cotton bollworm, Helicoverpa armigera, named Hearm caspase-1. The 1,350 bp full-length cDNA contains an 885 bp open reading frame (ORF) that encodes a Hearm caspase-1 proenzyme of 294 amino acids. The deduced protein is highly homologous to Spodoptera frugiperda Sf caspase-1 and Drosophila melanogaster ICE and has the highly conserved pentapeptide QACQG, the recognized catalytic site of caspases, suggesting that it is an effector caspase of the cotton bollworm. Northern blot and RT-PCR analyses demonstrate that Hearm caspase-1 is expressed in embryos and the fat body, midgut and haemocytes of feeding and wandering larvae. Expression of Hearm caspase-1 in the haemocytes appears to be correlated with the pulse of ecdysone, and it is up-regulated by ecdysone agonist RH-2485, implying that Hearm caspase-1 activation is regulated by ecdysone.

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.

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.

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.

[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.