Jingbo Dai

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.

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.

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.

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.

The alteration of protein profile induced by cigarette smoking via oxidative stress in mice epididymis.

Smoking is associated with a declining quality of semen. The aim of this study was to screen and investigate the differential expression of proteins extracted from the epididymis of mice exposed daily with cigarette smoke. Using MALDI-TOF-MS analysis, we found that the protein profile of the mouse epididymis was altered by cigarette smoking and identified 27 proteins from the most abundant and differentially expressed spots in the 2-DE gels of epididymal samples. These proteins were classified into groups according to their functions such as energy metabolism, reproduction and structural molecule activity. Through pathway analysis, these proteins were associated with the glutathione metabolism and protein processing in the endoplasmic reticulum. These results showed that the epididymis may experience oxidative stress following cigarette smoke exposure, which was confirmed using immunohistochemistry. We determine that cigarette smoking can induce oxidative stress in the mouse epididymis, which may cause protein profile altering, thereby impairing epididymis function, and leading to a decline in semen quality.

Nicotine elevates sperm motility and induces Pfn1 promoter hypomethylation in mouse testis

Many studies have addressed the hazardous role of cigarette smoking on male fertility, but the exact molecular mechanisms involved in the impairments caused by nicotine remain unclear. To evaluate the detrimental effects of nicotine exposure on spermatogenesis, two‐dimensional gel electrophoresis and mass spectrometry analysis were performed to screen and identify differentially expressed proteins from the testes of mice exposed to nicotine daily. Data mining analysis indicated that the 15 identified proteins were mainly involved in actin cytoskeleton regulation and in the tricarboxylic acid cycle, which are related to cell motility. Further investigation of a central regulatory factor in the cytoskeleton regulation, profilin 1 (PFN1), revealed that nicotine‐induced Pfn1 over‐expression in mouse testes, specifically in elongated spermatids, by Pfn1 promoter hypomethylation. Interestingly, elevated sperm motility parameters were observed in nicotine‐treated mice. We assume that nicotine‐induced PFN1 over‐expression in mouse spermatids may promote actin polymerization and ultimately enhance sperm motility.

Alteration of sperm protein profile induced by cigarette smoking

Cigarette smoking is associated with lower semen quality, but how cigarette smoking changes the semen quality remains unclear. The aim of this study was to screen the differentially expressed proteins in the sperm of mice with daily exposure to cigarette smoke. The 2D gel electrophoresis (2DE) and mass spectrometry (MS) analyses results showed that the mouse sperm protein profile was altered by cigarette smoking. And 22 of the most abundant proteins that correspond to differentially expressed spots in 2DE gels of the sperm samples were identified. These proteins were classified into different groups based on their functions, such as energy metabolism, reproduction, and structural molecules. Furthermore, the 2DE and MS results of five proteins (Aldoa, ATP5a1, Gpx4, Cs, and Spatc1) were validated by western blot analysis and reverse transcriptase-polymerase chain reaction. Results showed that except Spatc1 the other four proteins showed statistically significant different protein levels between the smoking group and the control group (P < 0.05). The expressions of three genes (Aldoa, Gpx4, and Spatc1) were significantly different (P < 0.05) at transcription level between the smoking group and the control group. In addition, five proteins (Aldoa, ATP5a1, Spatc1, Cs, and Gpx4) in human sperm samples from 30 male smokers and 30 non-smokers were detected by western blot analysis. Two proteins (Aldoa and Cs) that are associated with energy production were found to be significantly altered, suggesting that these proteins may be potential diagnostic markers for evaluation of smoking risk in sperm. Further study of these proteins may provide insight into the pathogenic mechanisms underlying infertility in smoking persons.

Nicotine Induced Murine Spermatozoa Apoptosis via Up-Regulation of Deubiquitinated RIP1 by Trim27 Promoter Hypomethylation.

ABSTRACT Nicotine significantly promoted apoptosis in stages I, VII, VIII, and XI spermatogonia, stages I, VII, VIII, X, and XI spermatocytes, and stages I–V, VII, and VIII elongating spermatids. To explore the underlying molecular mechanisms, sperm mRNA next-generation sequencing of nicotine-treated mice was conducted. Out of the 86 genes related to apoptosis, Tnf (tumor necrosis factor alpha) was screened to be the most significant varied transcript, and the Onto-pathway analysis indicated that the TNF apoptotic pathway was especially activated by nicotine exposure. The TNF pathway was further studied at the gene and protein levels. The results showed that RIP1, the key component in the TNF apoptotic pathway, was up-expressed in its deubiquitinated form in nicotine-treated mice testis. TRIM27, an E3 ubiquitin ligase that activated TNF apoptotic pathway through up-regulating deubiquitinated RIP1, was also overexpressed in nicotine-treated spermatocytes; moreover, four consecutive CpG sites near the Trim27 transcription start site were less frequently methylated. Finally, in vitro experiments of Trim27 overexpression and RNA interference in GC-1 spermatogonial cells confirmed that the RIP1 deubiquitination and TRIM27 hyopmethylation were both positively correlated with spermatocyte apoptosis. In summary, our study suggests that nicotine may induce murine spermatozoal apoptosis via the TNF apoptotic pathway through up-regulation of deubiquitinated RIP1 by Trim27 promoter hypomethylation.

A novel acrosomal protein, IQCF1, involved in sperm capacitation and the acrosome reaction

On the basis of the unknown tags in the mature human sperm serial analysis of gene expression library constructed by our laboratory, some transcripts were cloned, including Iqcf1 (IQ motif containing F1). To investigate the function of sperm‐retained Iqcf1 in spermatogenesis and fertilization of mice, we investigated the spatial and temporal expression of IQCF1. By using the (transcription activator‐like effector nuclease) strategy, Iqcf1‐knockout mice were produced, and the phenotypes of the Iqcf1−/− mice were analyzed. The results showed that IQCF1 was localized in the acrosome of spermatozoa and spermatids; the expression of IQCF1 in testes was associated with spermatogenic capacity. The Iqcf1−/− mice were significantly less fertile than the wild‐type mice (p = 0.0057) because of reduced sperm motility (p = 0.0094) and the acrosome reaction (AR) (p = 0.0093). In spermatozoa, IQCF1 interacted with calmodulin (CaM) and possibly participated in the tyrosine phosphorylation of sperm proteins during capacitation. In conclusion, a newly identified acrosomal protein, IQCF1, is closely related to sperm capacitation and AR; in particular, it is involved in tyrosine phosphorylation of sperm proteins through interaction with CaM. Research into the function of IQCF1 during fertilization could facilitate the investigation of the molecular mechanism of capacitation, which is unclear.

Maternal nicotine exposure has severe cross-generational effects on offspring behavior.

Our previous studies showed that paternal nicotine exposure can lead to hyperactivity in the offspring. Nevertheless, the cross-generational effects of maternal and biparental nicotine exposure remain unclear. In this study, female and male mice were exposed respectively by nicotine before pregnancy. The maternal pre-pregnancy nicotine exposure led to depression-like behaviors in the F1 offspring. However, after biparental pre-pregnancy nicotine exposure, seventy percentage of the offspring exhibited a depressive phenotype while 20% were hyperactive, and the remaining exhibited no obvious abnormal behavior. The cross-generational effects appeared to be mediated via disruption of the balance between GSK3 and p-GKS3 by nicotine. These results suggested that pre-pregnancy nicotine exposure can induce alterations in the behavior of the offspring, and the cross-generational effects of maternal nicotine exposure were particularly serious.