The hazardous effects of tobacco smoking on male fertility.

The substantial harmful effects of tobacco smoking on fertility and reproduction have become apparent but are not generally appreciated. Tobacco smoke contains more than 4000 kinds of constituents, including nicotine, tar, carbonic monoxide, polycyclic aromatic hydrocarbons, and heavy metals. Because of the complexity of tobacco smoke components, the toxicological mechanism is notably complicated. Most studies have reported reduced semen quality, reproductive hormone system dysfunction and impaired spermatogenesis, sperm maturation, and spermatozoa function in smokers compared with nonsmokers. Underlying these effects, elevated oxidative stress, DNA damage, and cell apoptosis may play important roles collaboratively in the overall effect of tobacco smoking on male fertility. In this review, we strive to focus on both the phenotype of and the molecular mechanism underlying these harmful effects, although current studies regarding the mechanism remain insufficient.

Estradiol pretreatment attenuated nicotine-induced endothelial cell apoptosis via estradiol functional membrane receptor.

Cigarette smoking is highly associated with increased cardiovascular disease complications. The female population, however, manifests reduced cardiovascular morbidity. We define nicotine's effect upon human umbilical vein endothelial cells (HUVECs), determine whether estradiol might ameliorate endothelial dysfunction via its membrane estrogen receptor (mER), and attempt to elucidate the underlying mechanisms. Endothelial cells were pretreated with estradiol-BSA and measured resultant ion flux across the cells via the patch clamp technique to assess mER is functionality. Estradiol-BSA administration was associated with 30% decreased nicotine-induced apoptosis and also attenuated nicotine-activated phosphorylation of p38 and ERK. Pretreatment of estradiol-BSA triggered a low calcium influx, suggesting ahead low influx calcium played a critical role in the underlying protective mechanisms of estradiol. Furthermore, this estradiol-BSA protection against apoptosis remained effective in the presence of tamoxifen, an intracellular estrogen receptor (iER) inhibitor. Additionally, tamoxifen did not abolish estradiol-BSA's inhibitory effect upon p38 and ERK's activation, giving evidence to the obligatory role of p38 and ERK signaling in the estradiol-BSA's anti-apoptotic action via mER. Our study provides evidence that nicotine enhances endothelial cell apoptosis, but estrogen exerts anti-apoptotic effect through its functional membrane estrogen receptor. Clinically, the nicotine in cigarettes might contribute to endothelial dysfunction, whereas ambient estradiol may provide cellular protection against nicotine-induced injury through its functional membrane receptor via MAPK pathway downregulation.

The role of Dby mRNA in early development of male mouse zygotes.

Ejaculated mammalian spermatozoa contain a complex yet specific population of mRNA. However, the possible roles that mRNA has in early zygotic and embryonic development remain unclear. We found that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and is transferred into the oocyte during fertilization by reverse transcription-polymerase chain reaction even though no DBY protein expression is detected. The cellular location of Dby mRNA is seen in the post-acrosome region, and it comprises nearly half of the mouse spermatozoa in in situ hybridization. In contrast, transcripts of the control gene, Smcy, are not detected in capacitated mouse spermatozoa, although the H-Y antigen encoded by Smcy is expressed on the surface of the spermatozoa. In our microinjection experiment, the zygotic development rate of the as-Dby male pronucleus injection group was significantly lower than that of the as-Smcy male pronucleus injection group (35.9% vs. 95%, P = 0.001) and the as-Dby female pronucleus injection group (35.9% vs. 93.8%, P = 0.001). The rate of male-developed zygotes was also lower than that of the as-Smcy male pronucleus injection group (17.4% vs. 57.9%, P = 0.002) and the as-Dby female pronucleus injection group (17.4% vs. 54.1%, P = 0.002). Thus, we conclude that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and it has an important role in the early zygotic development of male mouse zygotes. This might imply that spermatozoa mRNA is involved in early zygotic and embryonic stages of reproduction.

Nicotine induces cyclooxygenase-2 and prostaglandin E(2) expression in human umbilical vein endothelial cells.

Nicotine is a major component of cigarette smoking which may be involved in the progress of atherogenesis. In order to explain the mechanism of nicotine-induced endothelium dysfunction, we investigated the effects of nicotine on cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expression in human umbilical vein endothelial cells (HUVECs). Nicotine treatment increased the expressions of COX-2 at mRNA and protein level in a dose-dependent manner, following prostaglandin E(2) (PGE(2)) release enhancement. Pyrrolidine dithiocarbamate (PDTC, NF-kappaB inhibitor) and alpha-Bungarotoxin (alpha-Btx, nicotinic acetylcholine receptor antagonist) attenuated the nicotine-induced COX-2 expression and PGE(2) production. Furthermore, nicotine-induced ICAM-1 expression was reduced by NS-398 (selective COX-2 inhibitor). Taken together, the present study demonstrated that nicotine-induced COX-2 expression through NF-kappaB activation which mediated by nicotinic acetylcholine receptor and the induction of COX-2 was related to ICAM-1 expression.

Modified PCR methods for 3' end amplification from serial analysis of gene expression (SAGE) tags.

Serial analysis of gene expression (SAGE) is a powerful technique to study gene expression at the genome level. However, a disadvantage of the shortness of SAGE tags is that it prevents further study of SAGE library data, thus limiting extensive application of the SAGE method in gene expression studies. However, this problem can be solved by extension of the SAGE tags to 3' cDNAs. Therefore, several methods based on PCR have been developed to generate a 3' longer fragment cDNA corresponding to a SAGE tag. The list of modified methods is extensive, and includes rapid RT-PCR analysis of unknown SAGE tags (RAST-PCR), generation of longer cDNA fragments from SAGE tags for gene identification (GLGI), a high-throughput GLGI procedure, reverse SAGE (rSAGE), two-step analysis of unknown SAGE tags (TSAT-PCR), etc. These procedures are constantly being updated because they have characteristics and advantages that can be shared. Development of these methods has promoted the widespread use of the SAGE technique, and has accelerated the speed of studies of large-scale gene expression.

Semi-nested PCR analysis of unknown tags on serial analysis of gene expression.

Serial analysis of gene expression (SAGE) is a powerful technique for studying gene expression at the genome level. However, short SAGE tags limit the further study of related data. In this study, in order to identify a gene, we developed a semi-nested PCR-based method called the two-step analysis of unknown SAGE tags (TSAT-PCR) to generate longer 3'-end cDNA fragments from unknown SAGE tags. In the procedure, a modified lock-docking oligo(dT) with two degenerate nucleotide positions at the 3'-end was used as a reverse primer to synthesize cDNAs. Afterwards, the full-length cDNAs were amplified by PCR based on 5'-RACE and 3'-RACE. The amplified cDNAs were then used for the subsequent two-step PCR of the TSAT-PCR process. The first-step PCR was carried out at an appropriately low annealing temperature; a SAGE tag-specific primer was used as the sense primer, and an 18 bp sequence (universal primer I) located at the 5'-reverse primer end was used as the antisense primer. After 15-20 PCR cycles, the 3'-end cDNA fragments containing the tag could be enriched, and the PCR products could be used as templates for the second-step PCR to obtain the specific products. The second-step PCR was performed with a SAGE tag-specific primer and a 22-bp sequence (universal primer II) upstream of universal primer I at the 5'-reverse primer with a high annealing temperature. With our innovative TSAT-PCR method, we could easily obtain specific PCR products covering SAGE from those transcripts, especially low-abundance transcripts. It can be used as a method to identify genes expressed in different cell types.

[Testosterone could induce a rapid rise in intracellular free Ca2+ concentration through binding to the membrane surface of bone marrow-derived macrophages].

OBJECTIVE: To investigate the ways testosterone influences the murine bone marrow-derived macrophages (BMMs) and how testosterone affects the function of BMMs after bound to their membrane surface. METHODS: BMMs were cultured in vitro, their total RNA and proteins isolated, and the expression of intracellular androgen receptor (AR) detected through RT-PCR and Western blotting. The binding site of testosterone (T) to the membrane surface of BMMs was observed by confocal laser scanning microscopy after T-BSA-FITC incubation. Moreover, the intracellular Ca2+ was tested by Fura-2 method, and the influence of ionic currents on BMMs plasma membrane induced by testosterone was examined by the whole cell patch-clamp. RESULTS: RT-PCR and Western blotting failed to detect intracellular ARs in BMMs, but confocal laser scanning microscopy showed testosterone to be bound to the membrane surface of BMMs by impermeable T-BSA-FITC, inducing a rapid rise in the intracellular free Ca2+ concentration ([Ca2+]i) of Fura-2 loaded BMMs, predominantly due to the influx of extracellular Ca2+ through Ni2+ -blockable Ca2+ channels in the plasma membrane. Similarly, the patch-clamp technique revealed T-induced calcium influx in BMMs. CONCLUSION: It is reasonable to assume that the testosterone receptor exists on the plasma membranes, and testosterone act through unconventional plasma membrane receptors, induce Ca2+ influx and a rapid rise in the intracellular Ca2+ concentration, and influence the function of BMMs.

Identification of the proteins related to cytochrome P450 induced by fenvalerate in a Trichoplusia ni cell line.

In order to reveal the metabolic reaction to the presence of fenvalerate mediated by P450 in insects, we used the trypan blue exclusion technique and 3-(4,5-dimethylthiazol)-2,5-diphenyltrazolium bromide (MTT) reduction assay to assess the vitality of Trichoplusia ni (Tn) cells treated with fenvalerate, and observed dose- and time-dependent changes in total cellular P450s. In addition, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify the proteins involved in the fenvalerate reaction process. Finally, the cDNA of P450 fragments was cloned and real-time RT-PCR was performed. Our data showed that at the 0-15 mumol/L challenge concentration of fenvalerate, at which the vitality of Tn cells was not affected (p > 0.05), there was a tendency toward a dose- and time-response of total cellular P450s, which peaked at the 9 h (p < 0.05) and 12 h (p < 0.01) time points following 12.5 mumol/L stimulation with fenvalerate. The 2-DE assay detected more than 1300 protein spots in each two-dimensional gel, of which 33 spots displayed significant differences. Among the changed spots, three isoforms of P450 were identified. One of the three P450 cDNA fragments (CYP4L4) was cloned and sequenced, and its expression in treated Tn cells increased significantly (p < 0.01). It was found that fenvalerate induced the expression of P450s in insect cells. This suggests that fenvalerate could be metabolized by CYP4L4 through a hydroxylation reaction in insect cells.

[Serial analysis of gene expression in parasitological research].

Serial analysis of gene expression (SAGE) is a powerful high-throughput experimental technique that allows rapid, quantitative analysis of global gene expression in eukaryotic organisms. A short sequence taq (10-14 bp), which is defined by an anchoring enzyme site at a fixed distance from polyA tail, contains sufficient information to identify mRNA transcript from which it originates. The taqs are ligated to obtain concatemers that are cloned into a plasmid vector for sequencing. The identification and abundance of mRNA can be observed through bioinformatics and statistical analysis of a given tag. SAGE is not only applied in obtaining global profile of gene expression in a given cell or tissue, but also help identify a set of specific genes to the cellular conditions by comparing the profiles constructed for a pair of cells that are kept at different conditions. This review covers a general introduction of SAGE, its protocol, methodological evolution and applications in parasite biology.

[Characterization of the mRNA profile in ejaculated spermatozoa from healthy fertile men].

OBJECTIVE: To explore the complexity of mRNA in the ejaculated sperm from healthy fertile men. METHODS: Semen samples were collected from 10 healthy fathers. The swim-up method was adopted to purify the sperm from possible contamination of somatic cells and the spermatozoal total RNA extracted by Trizol was used for SAGE library analysis. RESULTS: A totle of 21 052 SAGE raw tags were sequenced from 877 clones and 2 712 unique tags that occurred at least twice in the library were given further analysis. 19.7% of the unique tags had no match in the existing SAGE map, representing novel genes. Molecular function analysis revealed 67% of unique tags related to protein binding or nucleic acid binding categories, 41% to catalytic activity, 13% to message transducer activity, and 10% to transporter, structural and transcription regulator activity, respectively. CONCLUSION: There exists a complex repertoire of mRNAs in the ejaculated spermatozoa from fertile men.

[Study of effects of drugs on myocardial hypertrophy due to overload].

OBJECTIVE: To study the relation of expression change of tumor necrosis factor-alpha (TNF-alpha), angiotensin II (Ang II), and endothelin-1 (ET-1), and the effect of imidapril on myocardial hypertrophy due to overload. METHODS: Sixty-three rats were randomly divided into four groups: sham operation (n=15), overload group (n=16), imidapril group (n=16), and Caweidiluo group (n=16). Hypertrophic myocardium was reproduced in rats by constricting abdominal aorta. Blood samples and heart were harvested 12 weeks after aorta constriction, and myocardial hypertrophy index, the contents of Ang II, ET-1 in the myocardium and plasma were determined by radioimmunoassay and TNF-alpha in the myocardium and plasma were determined by enzyme linked immunoadsorbent assay. RESULTS: Left ventricle showed obvious hypertrophy 12 weeks after operation. The contents of TNF-alpha, Ang II and ET-1 in the myocardium, and the content of TNF-alpha in serum, Ang II and ET-1 in plasma were increased compared with those of controls (all P<0.01). The treatment of imidapril and Caweidiluo could restrain the development of left ventricle hypertrophy after operation, and imidapril decreased the contents of TNF-alpha, Ang II and ET-1 in myocardium compared with overload group (all P<0.01). Imidapril lowered the contents of TNF-alpha in serum, Ang II and ET-1 in plasma, compared with overload group (all P<0.01), but not ET-1. Caweidiluo lowered the contents of TNF-alpha, Ang II and ET-1 in myocardium, the contents of TNF-alpha in serum, Ang II and ET-1 in plasma (all P<0.01) compared with overload group (both P<0.01). CONCLUSION: The activation of rennin-angiotensin system (RAS) by over load results to an elevation of TNF-alpha contents in plasma and myocardium, and it is probably one of the major regulatory pathways of myocardial hypertrophy.

[Ultrastructural study on pharyngeal armature of seven species of sandflies in china by scanning electron microscopy].

OBJECTIVE: To observe the ultrastructure of pharyngeal armature of 7 species of sandflies in China. METHODS: The pharyngeal armature of various sandflies were studied by scanning electron microscopy. RESULTS: The pharyngeal armature of sandfly consisted of pointed-teeth with various shape, number and arrangement among different species. CONCLUSION: Such differences may provide the morphological proof for identification of species.

Effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells. Analysis of the underlying receptor pathways.

This study investigated the effects of testosterone and 17-beta-estradiol on tumor necrosis factor-alpha (TNF-alpha)-induced endothelial expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) and the potential roles of hormone receptors involved in these actions. Human umbilical vein endothelial cells (HUVEC) were stimulated with TNF-alpha in the presence or absence of testosterone or 17-beta-estradiol, and the expression of E-selectin and VCAM-1 was investigated. As shown by Western blot analysis, co-administration with testosterone or 17-beta-estradiol increased the expression of E-selectin and VCAM-1 induced by TNF-alpha at 6 h and 3 h, respectively. Similarly, RT-PCR analysis revealed a significant increase in the amount of mRNA for E-selectin and VCAM-1 after co-administration with testosterone or 17-beta-estradiol in TNF-alpha-stimulated HUVEC. The presence of mRNA and proteins for androgen receptor and estrogen receptor alpha in HUVEC was verified by RT-PCR and Western blot. Flow cytometric analysis showed that preincubation with androgen receptor antagonist cyproterone and estrogen receptor antagonist tamoxifen completely abrogated the upregulating effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression, respectively. Expression of TNF receptors in TNF-alpha-stimulated HUVEC was not influenced by testosterone and 17-beta-estradiol. The data indicate that both testosterone and 17-beta-estradiol increase TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells via a receptor-mediated system, and expression of TNF receptors are not changed in these actions. The implications of these results for the facilitory effects of both sex hormones on immune reactions are discussed.