| As one of the serious public health issues in the world,tobacco smoking reaches a high level of concern.Many researchers found that smoking had hazardous effects on male reproductive functions and even detrimental to the health of the progeny.The mechanisms underlying these effects remain unclear.In order to observe the toxicological effects of smoking on male fertility,our research team established a mouse smoking model mimicking the heavy smokers.Our previous study revealed the alterations of protein profile in mouse testis,epididymis and mature spermatozoa due to tobacco smoking using the proteomic technique.The molecular mechanisms underlying the altered protein profile were also elaborated.In consideration of the variety of the substances in tobacco smoke,it is essential to study the toxicological effects of nicotine,on of the main toxins in tobacco,on male reproductive system per se.In the present study,we observed the protein profile alterations in murine testis and epididymis due to nicotine exposure.Also,the neuropsychiatric effects of nicotine and the paternal transgenerational transmission of these effects were analyzed using epigenetic methods.Firstly,a nicotine exposure mouse model was constructed to mimic the dosage of nicotine exposure of heavy smokers.6-week-old male C57BL/6J mice received total 0.2 mg/100 g free-base nicotine via intraperitoneal injection for 5 weeks.The mice received nicotine treatment 4 times per day(q.q.h.in daytime);thus,a lower dose(0.05 mg/100 g)was received in each injection to avoid malaise or sickness.The control group received a daily equivalent of saline.Subsequently,the differently expressed proteins(DEPs)in mouse testis and after epididymis nicotine exposure were identified using the 2-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ ionization time of flight mass spectrometry.In murine testis,proteomic analysis revealed 17 identified proteins with expressive levels that were altered in the nicotine-treated group compared with the control group.Bioinformatic analyses indicated that most DEPs are related to cell motility by their involvement in cytoskeleton regulation and energy metabolism.Following computer-aided sperm analysis(CASA)indicated that nicotine exposure elevated the motility of mature spermatozoa but inhibited its energy metabolism process.The bioinformatic analyses implied that PFN1 may play a central role in mediating the effect of nicotine on cytoskeleton regulation.The over-expression of PFN1 in the testis and spermatogenic cells of nicotine-treated mice was due to the epigenetic regulation of DNA methylation within the Pfn1 promotor.The over-expression of PFN1 may be an etiology of elevated sperm motility parameters due to nicotine exposure.The attenuated energy metabolism and elevated sperm motility may undermine the mechanism of sperm competition during fertilization process,leading to possible abnormities in offspring.In murine epididymis,the proteomic analysis revealed 15 identified proteins with expression levels that differed between the epididymis of nicotine-treated and control group.Following bioinformatic analyses indicated that most DEPs were related to molecular transportation networks and the polyol pathway indicating an impaired epididymal secretory function.Using immunofluorescence analysis,nicotine was found to reduce the quantity of epididymosomes in the murine epididymal lumen,which can be considered a direct embodiment of impaired epididymal secretory functions.Our study also indicated that nicotine treatment significantly down-regulated the seminal fructose level and inhibited tyrosine phosphorylation level in spermatozoa after in vitro capacitation and this effect may be a marker of impaired sperm capacitation.Reduced expression of SORD in the cauda epididymis,specifically in epididymosomes,has also been observed due to DNA hyper-methylation within the Sord promoter region.Nicotine-induced reduced expression of sorbitol dehydrogenase and impaired secretory functions of the epididymis may outline the etiology of the low capacitation of spermatozoa.The epigenetic information alterations during spermatogenesis due to nicotine exposure can pass down to the next generation causing impaired developmental process.In order to observe the paternal transgenerational transmission of the neuropsychiatric effects of nicotine,several animal behavioral tests were performed to evaluate the neuropsychiatric status of the tobacco smoke / nicotine treated mice and their progeny.Daily tobacco smoke or nicotine exposure induced a depressive phenotype in the F0 generation,resulting in hyperactivity and attenuated depressive level in the F1 generation.Nevertheless,this transgenerational effect was not passed down to the F2 generation.From the gene transcription profile of the sperm of F0 mice,we observed that the nicotine-induced discrepant genes correlated with the neurobehavioral alterations were primarily enhancements in the functions enriched in Wnt4 signaling pathway.Following pathogenetic studies indicated that Wnt4 was the key element in these nicotine-triggered events.Nicotine also induced the down-regulation of mmu-miR-15 b expression due to hyper-methylation in the CpG island shore region of mmu-miR-15 b in both the spermatozoa of F0 mice and the brains of F1 mice.As the target gene of mmu-miR-15 b,Wnt4 expression was elevated in the thalamus of F1 mice due to the inheritance of DNA methylation patterns from the paternal generation.Furthermore,the increased expression of Wnt4 elevated the phosphorylation level of its downstream protein GSK-3 through the canonical Wnt4 pathway,and this elevation might be involved in the behavioral alterations observed in F1 mice.Moreover,in vivo stereotaxic brain injections were used to induce the overexpression of mmu-miR-15 b and WNT4 and confirm the neurobehavioral effects in vitro.The behavioral phenotype of the F1 mice resulting from paternal nicotine exposure could be rescued by viral manipulation of mmu-miR-15 b in the thalamus.These findings suggest that paternal smoking defines different generational neurobehavioral states,providing further insight into the molecular mechanisms underlying this phenomenon.In summary,daily nicotine can cause a series of epigenetic modifications during murine spermatogenesis process.In the testis of nicotine treated mice,Pfn1 was epigenetically up-regulated by DNA hyper-methylation within its promoter region,and thus the turnover of cytoskeleton in spermatogenic cells was elevated,resulting in increased sperm motility parameters.While in the epididymis,nicotine induced DNA hyper-methylation within Sord gene promoter,thereby inhibited the polyol pathway.The semen fructose concentration was significantly lower due to the attenuated polyol pathway,and the in vitro capacitation process of mature spermatozoa was attenuated.In mature spermatozoa,nicotine induced DNA hyper-methylation within the CpG island shore of mmu-miR-15 b gene,and down-regulates the expression level of this regulation miRNA of Wnt4.This epigenetic information then passed down to the next generation through the paternal line and retained in the brain tissue.The Wnt4 pathway was activated in brain tissue of the offspring mice and resulted in a hyperactivity phenotype of the progeny. |
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