Human sperm Toll-like receptor 4 (TLR4) mediates acrosome reaction,oxidative stress markers,and sperm parameters in response to bacterial lipopolysaccharide in infertile men

Purpose

To study the role of Toll-like receptor 4 (TLR4) in human spermatozoa and to assess sperm parameters, oxidative stress markers, and acrosome reaction in response to the stimulation of TLR4 by its ligand, the lipopolysaccharide (LPS), as a major endotoxin of Gram-negative bacteria.

Methods

Our study was carried out in 73 sperm samples from patients undergoing semen analysis for couple infertility investigations. The studied patients were divided into three groups: normozoospermic fertile patients (n = 13), patients with abnormal and leukospermic semen (n = 13), and patients with abnormal and non-leukospermic semen (n = 47). TLR4 expression in human spermatozoa was initially analyzed by western blot. Sperm samples were incubated in the presence of LPS (200 ng/ml) for 18 h. Then, sperm motility and vitality were evaluated by microscopic observation and oxidative stress markers as malondialdehyde (MDA) and carbonyl groups (CG) were spectrophotometrically assessed in neat and selected sperm. A triple-stain technique was also performed to evaluate acrosome reaction in 15 sperm samples from infertile patients.

Results

TLR4 expression was confirmed in human spermatozoa with a molecular weight of 69 kDa. In the normozoospermic group, no significant differences in sperm parameters and oxidative stress markers were shown after incubation with LPS in neat and selected sperms. Regarding samples from the non-leukospermic group, LPS reduced spermatozoa motility and vitality rates in selected sperm (P = 0.003; P = 0.004, respectively). A significant increase of MDA and CG levels was also detected (P = 0.01; P = 0.02, respectively). However, only the MDA levels were significantly increased (P = 0.01) in neat LPS-stimulated sperm. The same results were shown within the leukospermic group. The comparison between the two groups, leukospermic and non-leukospermic, in selected sperms showed a more important LPS effect in the leukospermic group significantly on motility and MDA rates (P = 0.006; P = 0.009, respectively). Furthermore, a significant decrease in reacted spermatozoa rate was detected in response to LPS in selected sperm samples from infertile men (P = 0.03).

Conclusions

These findings indicate that human spermatozoa express TLR4 and respond to LPS stimulation with alterations in viability, motility, and the acrosome reaction implicating reactive oxygen species (ROS) production in sperm samples from infertile patients.

     

The possible meaning of transferrin and its soluble receptors in seminal plasma as markers of the seminiferous epithelium

Transferrin (Tf) and soluble transferrin receptors (S-Tf-R)were measured by enzyme immunoassay in seminal plasma of 130semen samples. The mean concentration of S-Tf-R in cases withnormozoospermia was 10.4 IU/ml (95% confidence interval: 9.5–11.3)and it was significantly lower in patients with oligozoospermia(6.6, 95% CI: 5.8–7.5, P < 0.001), asthenozoospermia(8.5, 95% CI: 5.5–10.7, P < 0.05), azoospermia of primarytesticular origin (7.9, 95% CI: 6.1–9.6, P < 0.05)and post-vasectomy samples (5.9, 95% CI: 5.4–6.9, P<0.001). The concentration of S-Tf-R in post-vasectomy sampleswas lower than that in patients with azoospermia of primarytesticular origin (P< 0.05; positive likelihood ratio= 7at value of 8.3 IU/ml). S-Tf-R was positively correlated withmotile sperm concentration (r= 0.50, P< 0.0001), percentagemotility (r= 0.38, P< 0.001), percentage of normal forms(r = 0.43, P < 0.001), sperm linear velocity (r= 0.42, P<0.001), and ATP concentration (r= 0.67, P< 0.0001). Folliclestimulating hormone (FSH) was found to be negatively correlatedwith the concentrations of both Tf (r= -0.31, P< 0.05) andof S-Tf-R (r= -0.45, P< 0.01). The mean concentration ofTf in seminal plasma was 50.4 µg/ml (35.9–67.2)in samples with normozoospermia (n= 22), and the concentrationwas significantly lower in patients with oligozoospermia (P<0.05), azoospermia of testicular origin (P < 0.001), andpost-vasectomy samples (P< 0.001). Seminal Tf was correlatedwith motile sperm concentration (r = 0.36, P< 0.001), percentageof motile spermatozoa (r= 0.25, P < 0.05), linear velocity(r= 0.24, P< 0.05) and ATP concentration (r= 0.44, P<0.001). The concentration of Tf was positively correlated withthat of S-Tf-R both in cases with spermatozoa present (r= 0.66,P < 0.001), and in cases with azoospermia of testicular origin(r = 0.51, P < 0.05) but not in vasectomy cases. It is concludedthat S-Tf-R in seminal plasma is a marker of spermatogenesisand may give information on the presence or absence of spermatogeneticcells in cases with azoospermia. Further investigations areneeded to assess its usefulness for clinical practice.     

Demonstration of a second rapidly conducting cortico-diaphragmatic pathway in humans

Functional imaging studies in normal humans have shown that the supplementary motor area (SMA) and the primary motor cortex (PMC) are coactivated during various breathing tasks. It is not known whether a direct pathway from the SMA to the diaphragm exists, and if so what properties it has. Using transcranial magnetic stimulation (TMS) a site at the vertex, representing the diaphragm primary motor cortex, has been identified. TMS mapping revealed a second area 3 cm anterior to the vertex overlying the SMA, which had a rapidly conducting pathway to the diaphragm (mean latency 16.7 ± 2.4 ms). In comparison to the vertex, the anterior position was characterized by a higher diaphragm motor threshold, a greater proportional increase in motor-evoked potential (MEP) amplitude with voluntary facilitation and a shorter silent period. Stimulus–response curves did not differ significantly between the vertex and anterior positions. Using paired TMS, we also compared intracortical inhibition/facilitation (ICI/ICF) curves. In comparison to the vertex, the MEP elicited from the anterior position was not inhibited at short interstimulus intervals (1–5 ms) and was more facilitated at long interstimulus intervals (9–20 ms). The patterns of response were identical for the costal and crural diaphragms. We conclude that the two coil positions represent discrete areas that are likely to be the PMC and SMA, with the latter wielding a more excitatory effect on the diaphragm.     

Correlation between recent thymic emigrants and CD31+ (PECAM-1) CD4+ T cells in normal individuals during aging and in lymphopenic children

CD31(+)CD45RA(+)RO(-) lymphocytes contain high numbers of T cell receptor circle (TREC)-bearing T cells; however, the correlation between CD31(+)CD4(+) lymphocytes and TREC during aging and under lymphopenic conditions has not yet been sufficiently investigated. We analyzed TREC, telomere length and telomerase activity within sorted CD31(+) and CD31(-) CD4(+) lymphocytes in healthy individuals from birth to old age. Sorted CD31(+)CD45RA(+)RO(-) naive CD4(+) lymphocytes contained high TREC numbers, whereas CD31(+)CD45RA(-)RO(+) cells (comprising < or =5% of CD4(+) cells during aging) did not contain TREC. CD31(+) overall CD4(+) cells remained TREC rich despite an age-related tenfold reduction from neonatal (100 : 1000) to old age (10 : 1000). Besides a high TREC content, CD31(+)CD45RA(+)RO(-)CD4(+) cells exhibited significantly longer telomeres and higher telomerase activity than CD31(-)CD45RA(+)RO(-)CD4(+) cells, suggesting that CD31(+)CD45RA(+)RO(-)CD4(+) cells represent a distinct population of naive T cells with particularly low replicative history. To analyze the value of CD31 in lymphopenic conditions, we investigated six children after allogeneic hematopoietic stem cell transplantation (HSCT). Reemerging overall CD4(+) as well as naive CD45RA(+)RO(-)CD4(+) cells predominantly expressed CD31 and correlated well with the recurrence of TREC 5-12 months after HSCT. Irrespective of limitations in the elderly, CD31 is an appropriate marker to monitor TREC-rich lymphocytes essentially in lymphopenic children after HSCT.     

Cisplatin-DNA damage in p21WAF1/Cip1 deficient mouse keratinocytes exposed to cisplatin

In response to DNA damage, cell cycle arrest, apoptosis, and DNA repair are mediated by a TP53 pathway that induces p21(WAF1/Cip1). The chemotherapeutic drug cis-diamminedichloroplatinum-II (cisplatin) damages cellular DNA by forming cis-diammineplatinum-N(7)-d[GpG] and cis-diammine-platinum-N(7)-d[ApG] adducts. To investigate the role of p21, skin keratinocytes from p21(WAF1/Cip1) wild-type (+/+), heterozygous (+/-), and null (-/-) mice, cultured in calcium levels designed to maintain a proliferating state, were exposed to 5 microM cisplatin continuously for 0, 8, 24, 48 and 72 h. At all time points the (+/-) cells had the fewest Pt-DNA adducts, and at 24 h mean Pt-DNA adduct levels were 541, 153 and 779 fmol adduct/mug DNA for p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively [P < 0.05 for (+/+) versus (+/-) and (-/-) versus (+/-)]. In order to understand underlying events, we examined p21(WAF1/Cip1) messenger RNA (mRNA), cell cycle arrest, and apoptosis in these cells. At 48 h of cisplatin exposure p21(WAF1/Cip1) mRNA expression was 2-fold higher in the (+/+) cells, compared to the (+/-) cells. At 24 h, the % of cells in S-phase in cisplatin-exposed cultures, compared to unexposed cultures, was decreased by 51, 40 and 11% in p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively (P = 0.04, ANOVA). At 24, 48 and 72 h the % of cisplatin-exposed (+/+) cells in apoptosis was 9.4-10.5%, while the cisplatin-exposed (+/-) and (-/-) cells had 1.2-3.7% of cells in apoptosis. The data support the interpretation that DNA replication arrest and apoptosis do not completely explain the low levels of Pt-DNA adducts in the (+/-) cells, and suggest that p21(WAF1/Cip1) controls activity resulting in either low Pt-DNA adduct formation or enhanced Pt-DNA adduct removal.     

The osteogenic differentiation potentials of umbilical cord blood hematopoietic stem cells

Under specific culture conditions, umbilical cord blood derived mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, and chondrogenic lineages. The purpose of the current study was to assess the differentiation potential of osteogenic umbilical cord blood derived hematopoietic stem cells (HSCs) and to develop an appropriate osteogenic differentiation medium for in vitro differentiation of umbilical cord blood derived HSCs. The study was conducted on 20 cord blood samples. The cells were cultured in osteogenic differentiating medium for 3?weeks. The HSCs differentiated into osteoblasts, which expressed osteoblast-associated genes (osteocalcin and bone sialoprotein), which were detected by RT-PCR. They showed alkaline phosphatase activity and a positive Alizarin red-S (AR-S) stain (calcium phosphate deposition). Umbilical cord blood is a rich source of hematopoietic stem cells that can be differentiated into osteoblasts; thus, it can be used for therapeutic strategies in the context of regenerative therapy.     

Establishment of hybrid cell lines producing monoclonal antibodies to a synthetic peptide from the E1 region of the hepatitis C virus

We aimed at establishing hybridoma cells secreting monoclonal antibodies (mAbs) against E1 synthetic peptide of HCV. BALB/c mice were immunized with HCV E1-synthetic peptide (GHRMAWDMM) and its spleenocytes were fused with the P3NS1 myeloma cell line. Two highly reactive and specific mAbs (10C7 IgG2b mAb, and 10B2 IgG1 mAb) were generated. The target HCV E1 antigen was identified at approximately 38 kDa in serum of infected individuals. A newly developed ELISA detected the target antigen in 90% of sera from HCV RNA infected individuals with a specificity of 84%. So, the generated mAbs may provide promising probes for serodiagnosis of HCV infection.     

Hepatitis C infection, Cognition, and inflammation in an Egyptian sample

Chronic hepatitis C (HCV) infection is associated with cognitive impairments which might be mediated through a secondary inflammatory cascade. Egypt has an unusually high prevalence of HCV monoinfections and is an ideal site for the study of the isolated effects of HCV infection. Therefore, in a hospital‐based cross‐sectional study based in Egypt, this study compared cognitive functioning and serum markers of inflammation in 11 HCV positive cases and 14 HCV negative controls. The Wisconsin Card Sorting Test was used to assess cognitive flexibility and the Brief Visuospatial Memory Test‐Revised was used to assess learning and memory. Circulating levels of soluble tumor necrosis factor receptor II (sTNFR‐II), monocyte chemotactic protein‐1 (MCP‐1/CCL2), and soluble CD14 (sCD14) were determined as indices of inflammation. HCV positive cases had higher levels of sTNFR‐II (t = ?3.5, P = 0.002). HCV positive cases also had significantly worse cognitive flexibility with higher number of total errors (t = ?2.18, P = 0.04), and preservative responses (t = ?2.12, P = 0.05), and lower number of conceptual level responses (t = 1.32, P = 0.04) on the Wisconsin Card Sorting test. In conclusion, results from this pilot study indicate that HCV+ patients have worse cognitive performance and somewhat greater inflammatory activity as compared to controls. The increased inflammation may be associated with the cognitive impairments observed in these HCV+ patients. J. Med. Virol. 83:261–266, 2011. © 2010 Wiley‐Liss, Inc.