Wangjie Xu

Proteomic characteristics of spermatozoa in normozoospermic patients with infertility.

Male infertility is a vexing yet common problem for men all over the world. However, its etiology remains unknown in most cases. The aim of this study was to screen and investigate the differentially expressed proteins in the sperm of infertile patients, whose sperm clinical parameters met the WHO guidelines. Using MALDI-TOF/TOF analysis, we identified 24 differentially expressed proteins from the 31 most abundant different protein spots in 2D gels of sperm samples, then verified and analyzed localization in sperm of the proteins. Following data mining analysis showed that these 24 proteins were categorized into five functional clustering groups: sexual reproduction, response to wounding, metabolic process, cell growth and/or maintenance, not clear. Additionally, 9 of the 24 differentially expressed proteins are involved in a main pathway network including TGF-β1, MYC, β-estradiol, MYCN, and TP53, which are known to be involved in cell communication, proliferation and differentiation. The observed differences in signaling and metabolic pathways between the infertile sperm and the normal fertile spermatozoa have implications in sperm motility, capacitation, acrosomal reaction and sperm-oocyte communication. These proteins are potential diagnostic markers, and the study of these proteins could help gain further insight into the pathogenic mechanisms in infertility.

Astragalus membranaceus Inhibits Inflammation via Phospho-P38 Mitogen-Activated Protein Kinase (MAPK) and Nuclear Factor (NF)-κB Pathways in Advanced Glycation End Product-Stimulated Macrophages

Advanced glycation end products (AGEs) and inflammation contribute to the development of diabetic complications. Astragalus membranaceus has properties of immunological regulation in many diseases. The aim of this study was to determine the function of A. membranaceus extract (AME) on the AGE-induced inflammatory response in Ana-1 macrophages. The viability of cells treated with AME or AGEs was evaluated with the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] method. The secretion and mRNA levels of IL-1β and TNF-α were measured by ELISA and RT-PCR, respectively. The activity of NF-κB was assayed by EMSA. The phosphorylation of p38 MAPK was assessed by western blotting. The results showed that AME was not toxic to macrophages. The treatment of macrophages with AME effectively inhibited AGE-induced IL-1β and TNF-α secretion and mRNA expression in macrophages. These effects may be mediated by p38 MAPK and the NF-κB pathway. The results suggest that AME can inhibit AGE-induced inflammatory cytokine production to down-regulate macrophage-mediated inflammation via p38 MAPK and NF-κB signaling pathways and indicate that AME could be an immunoregulatory agent against AGE-induced inflammation in diabetes.

Estimated Diversity of Messenger RNAs in Each Murine Spermatozoa and Their Potential Function During Early Zygotic Development

ABSTRACT To study the diversity of mRNAs in murine spermatozoa and their potential function during zygotic development, total RNAs in murine spermatozoa were sequenced via RNA-Seq and analyzed through bioinformatics techniques. The delivery and translation of sperm-borne mRNA in fertilized oocyte were detected using RT-PCR (reverse transcription-polymerase chain reaction), Western blot, and immunofluorescence. A total of 35 288 825 reads matching 33 039 transcripts, including 27 310 coding transcripts, were obtained. Based on our analyses, we hypothesized that the transcripts with RPKM (reads per kilobase of exon model per million mapped reads) higher than six may exist in each sperm cell as consistently retained transcripts. There were 4885 consistent transcripts in each sperm, and the remainder were randomly retained. If the baseline RPKM increased, the remaining coding transcripts were more likely related to reproduction and development. The sperm-borne transcripts Wnt4 and Foxg1 were delivered into fertilized oocytes on fertilization. Furthermore, Wnt4 was translated into protein in zygotes, whereas Foxg1 was not translated. In conclusion, approximately 4885 mRNAs were present in each murine spermatozoon, and the spermatozoal mRNAs related to reproduction and development were more likely retained. The sperm-borne mRNA Wnt4 was delivered into the fertilized oocyte and translated, evidence of a paternal effect on zygotic development.

Heparanase induced by advanced glycation end products (AGEs) promotes macrophage migration involving RAGE and PI3K/AKT pathway

BackgroundAdvanced glycation end products (AGEs), inflammatory-associated macrophage migration and accumulation are crucial for initiation and progression of diabetic vascular complication. Enzymatic activity of heparanase (HPA) is implicated strongly in dissemination of metastatic tumor cells and cells of the immune system. In addition, HPA enhances the phosphorylation of selected signaling molecules including AKT pathway independent of enzymatic activity. However, virtually nothing is presently known the role of HPA during macrophage migration exposed to AGEs involving signal pathway.MethodsThese studies were carried out in Ana-1 macrophages. Macrophage viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. HPA and AKT protein expression in macrophages are analysed by Western blotting and HPA mRNA expression by real time quantitative RT-PCR. Release of HPA was determined by ELISA. Macrophage migration was assessed by Transwell assays.ResultsHPA protein and mRNA were found to be increased significantly in AGEs-treated macrophages. Pretreatment with anti-HPA antibody which recognizes the nonenzymatic terminal of HPA prevented AGEs-induced AKT phosphorylation and macrophage migration. LY294002 (PI3k/AKT inhibitor) inhibited AGEs-induced macrophage migration. Furthermore, pretreatment with anti-receptor for advanced glycation end products (RAGE) antibody attenuated AGEs-induced HPA expression, AKT phosphorylation and macrophage migration.ConclusionsThese data indicate that AGEs-induced macrophage migration is dependent on HPA involving RAGE-HPA-PI3K/AKT pathway. The nonenzymatic activity of HPA may play a key role in AGEs-induced macrophage migration associated with inflammation in diabetic vascular complication.

Apoptosis caused by Hsp90 inhibitor geldanamycin in Leishmania donovani during promastigote-to-amastigote transformation stage

The role of heat shock protein 90 inhibitor geldanamycin (GA) during Leishmania donovani promastigote-to-amastigote transformation in axenic conditions was investigated. Promastigotes exhibited apoptotic morphologic changes after GA treatment at a high temperature, and the effect is in a dose- and time-dependant manner. Meanwhile, cell cycle analysis showed a significant increase at the expense of cells in the G0/G1 phase and a decrease in the S and G2/M phases after GA treatment. In addition, cellular glutathione level was reduced and reactive oxygen species content was increased afterwards. Pretreatment with antioxidants reduced the percentage of GA-induced cell apoptosis. After treatment, cultures in pH 5.5 showed a lower percentage of apoptosis than those in pH 7.4. The present study showed that GA could cause apoptosis in L. donovani but could not cause stage differentiation in high temperature and that acidic conditions were likely to be crucial for the transformation and survival of the parasite within its human host.

Comparison of the expression profiles of promastigotes and axenic amastigotes in Leishmania donovani using serial analysis of gene expression

In this study, we examined the transcriptome of Leishmania donovani promastigotes and axenic amastigotes to identify differentially regulated mRNAs utilizing the serial analysis of gene expression (SAGE). The axenic culture of amastigotes was initiated from stationary phase promastigotes. Transformation from promastigote to amastigote occurred when cultures in Medium 199 (pH 5.5), supplemented with 20% (v/v) fetal bovine serum, were transferred from 26°C to 37°C. A total of 20,299 and 20,132 tags were generated from promastigote and amastigote libraries, respectively. The containing unique genes identified in these two SAGE libraries were 8,615 and 7,835, respectively. Characteristics of the expressed genes’ frequency distribution were remarkably similar in both libraries: the most abundant tags (frequency ≥20), whose levels were equal to or >1.3% of the identified tags, constituted >23% of the total sequenced tags. Correspondingly, 75.72% or 71.65% of the genes accounted for those tags present at low abundance (frequency = 1) contributed only 32.13% or 27.89% of the total tags. A total of 968 genes (11.2% of the total genes in promastigotes and 12.4% in amastigotes) were recorded to have statistically different transcript levels between promastigotes and axenic amastigotes. Of the 968 distinct total genes, there are 326 promastigote-enriched transcripts and 642 amastigote-enriched mRNAs.

A Novel Drug Candidate for Alzheimer's Disease Treatment: gx-50 Derived from Zanthoxylum Bungeanum

This study focused on a promising drug candidate, N-[2-(3,4-dimethoxyphenyl)ethyl]-3-phenyl-acrylamide (gx-50), a compound extracted from Sichuan pepper (Zanthoxylum Bungeanum), to determine whether it would be an effective therapeutic for Alzheimers disease (AD) via biological experiments. In vivo, we determined the pharmacokinetic profile of gx-50 and evaluated the effect of gx-50 on the cognitive abilities of amyloid-β protein precursor transgenic (AβPP-Tg) mice by Morris water maze testing. In addition, we examined the effects of gx-50 on amyloid-β (Aβ) oligomers in the brains of AβPP-Tg mice by immunohistochemistry. In vitro, we observed a direct effect of gx-50 on Aβ oligomers by atomic force microscopy, detected the neuroprotective effects of gx-50 by western blotting and cell apoptosis assays, and measured its effects on intracellular calcium currents by laser confocal microscopy. Experiments in vivo showed that gx-50 could penetrate the blood brain barrier and improve the cognitive abilities of mice. Moreover, gx-50 treatment decreased the accumulation of Aβ oligomers in the cerebral cortex. The results in vitro demonstrated that gx-50 could disassemble Aβ oligomers, inhibit Aβ-induced neuronal apoptosis and apoptotic gene expression, and reduce neuronal calcium toxicity. These results strongly suggest that gx-50 is a potential candidate drug for treating AD.

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.

Cigarette Smoking Exposure Alters Pebp1 DNA Methylation and Protein Profile Involved in MAPK Signaling Pathway in Mice Testis

ABSTRACT Many studies have addressed the role of cigarette smoking on semen quality, but the exact mechanisms remain inconclusive. To evaluate the detrimental effects of smoking on the spermatogenesis process, we initially screened and investigated 31 differentially expressed proteins extracted from the testes of mice exposed daily to cigarette smoke using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis. Data mining analysis showed that these 31 proteins were categorized into five functional clustering groups: metabolic process, cell growth and/or maintenance, RNA and protein processing, stress response, and spermatogenesis. Additionally, 23 of 31 proteins were involved in a main pathway network, including Pkc (s), ERK1/2, Akt, and NF-kappaB, which are known to be involved in cell communication, proliferation, and differentiation. Interestingly, among the 31 proteins, a spermatogenesis-associated protein, phosphatidylethanolamine-binding protein 1 (PEBP1), was especially expressed in serial sections of spermatids of spermiogenesis and interacted with ERKs. The bisulfite sequencing result showed four CpGs near the Pebp1 transcriptional start site were largely methylated in the treated group. A 5-aza-2′-deoxycytidine treatment on GC-1 spg cells reversed the hypermethylation status and elevated both Pebp1 mRNA and protein expression levels. ERK1/2 phosphorylation levels were also increased with upregulation of Pebp1 expression in GC-1 spg cells. In conclusion, protein profile in testes could be altered by cigarette smoking. Moreover, we also suggest that epigenetic Pebp1 inactivation may affect activation of ERK, and it could impair spermatogenesis of mice. Our data could provide further insight into the mechanisms of spermatogenesis.

A Study of Y Chromosome Gene mRNA in Human Ejaculated Spermatozoa

We asked if messenger ribonucleic acid (mRNA) of Y chromosome genes was selectively retained in human ejaculated spermatozoa. A panel of genes on the nonrecombining region of Y chromosome (NRY) and their X homologues were selected and screened in human testis and ejaculated spermatozoa by reverse transcription‐polymerase chain reaction (RT‐PCR). Then, the cellular localization of DBY, RPS4Y and SMCY mRNA in testis and matured spermatozoa were investigated by in situ hybridization. The expression pattern of these three genes in human ejaculated spermatozoa was also tested by Western blot and Immunofluorescence. All 11 Y chromosome gene mRNAs were found in the testis, but the transcripts of only three genes DBY, SRY, and RPS4Y were detected in ejaculated sperm by RT‐PCR. Further investigation showed that DBY and RPS4Y mRNA were present in both human testis and the post‐acrosome region comprising nearly 50% of spermatozoa, while the encoded proteins were not detected in spermatozoa. In contrast, although SMCY mRNA was not detected in mature spermatozoa, the H‐Y antigen encoded by SMCY was expressed on the surface of spermatozoa. We therefore conclude that the spermatozoa mRNA could be selectively reserved in mature ejaculated spermatozoa. They might be useful in early zygotic and embryonic development. Mol. Reprod. Dev. 77: 158–166, 2010.