Cigarette Smoking Exposure Alters Pebp1 DNA Methylation and Protein Profile Involved in MAPK Signaling Pathway in Mice Testis1

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.

Smoking-induced genetic and epigenetic alterations in infertile men

“…PEBP1 is an essential protein involved in spermatogenesis through its interaction with ERKs. Mice exposed to cigarette smoke were shown to have elevated methylation level near Pebp1 transcriptional start site, which has been suggested to decrease Pebp1 expression and in turn cause the inactivation of ERK pathway resulting in spermatogenetic defects (Xu et al, ). Apart from hypermethylation, smoking may also promote DNA hypomethylation.…”

Sperm DNA Methylation, Infertility and Transgenerational Epigenetics

“…However, the role of paternal smoking in DNA methylation in offspring is less understood. Xu et al, found significant differences in gen-specific methylation in sperm DNA of mice exposed to cigarette smoke [112]. However, in humans paternal smoking was not found to be linked to altered H19 or IGF2 DMR methylation in umbilical cord DNA of newborns [113].…”

Understanding perspectives of signalling mechanisms regulating PEBP1 function

“…52 This indicates that PEBP1 function is essential for normal development of sperm. Recent studies on cigarette smoking showed that the protein profile in the cells could be altered during spermiogenesis.…”

“…Spermiogenesis in mice was altered as a result of cigarette smoking, as the CpG sites in PEBP1 transcription sites were methylated excessively, thereby altering the pathways important for the normal sperm development in mice. 52 This indicates that PEBP1 function is essential for normal development of sperm. 53…”