Unbiased label-free quantitative proteomic profiling and enriched proteomic pathways in seminal plasma of adult men before and after varicocelectomy

Camargo, Mariana; Intasqui, Paula; Giudice, P.T. Del; Carvalho, Valdemir Melechco; Cardozo, Karina Helena Morais; Andreoni, Cássio; Fraietta, Renato; Bertolla, Ricardo Pimenta

doi:10.1093/humrep/des357

Cited by 48 publications

(52 citation statements)

References 88 publications

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“…This result differs from that of another study that found no significant differences between the two isoforms of semenogelin I and II in asthenozoospermic groups compared to healthy donors under oxidative stress conditions [12]. However, the overexpression of both semenogelin I and II was reported in post-varicocelectomy patients largely due to oxidative stress [46]. …”

Section: Discussioncontrasting

confidence: 89%

Proteomic analysis of seminal fluid from men exhibiting oxidative stress

Sharma

Agarwal

Mohanty

et al. 2013

Reprod Biol Endocrinol

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BackgroundSeminal plasma serves as a natural reservoir of antioxidants. It helps to remove excessive formation of reactive oxygen species (ROS) and consequently, reduce oxidative stress. Proteomic profiling of seminal plasma proteins is important to understand the molecular mechanisms underlying oxidative stress and sperm dysfunction in infertile men.MethodsThis prospective study consisted of 52 subjects: 32 infertile men and 20 healthy donors. Once semen and oxidative stress parameters were assessed (ROS, antioxidant concentration and DNA damage), the subjects were categorized into ROS positive (ROS+) or ROS negative (ROS-). Seminal plasma from each group was pooled and subjected to proteomics analysis. In-solution digestion and protein identification with liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by bioinformatics analyses was used to identify and characterize potential biomarker proteins.ResultsA total of 14 proteins were identified in this analysis with 7 of these common and unique proteins were identified in both the ROS+ and ROS- groups through MASCOT and SEQUEST analyses, respectively. Prolactin-induced protein was found to be more abundantly present in men with increased levels of ROS. Gene ontology annotations showed extracellular distribution of proteins with a major role in antioxidative activity and regulatory processes.ConclusionsWe have identified proteins that help protect against oxidative stress and are uniquely present in the seminal plasma of the ROS- men. Men exhibiting high levels of ROS in their seminal ejaculate are likely to exhibit proteins that are either downregulated or oxidatively modified, and these could potentially contribute to male infertility.

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Section: Discussioncontrasting

confidence: 89%

Proteomic analysis of seminal fluid from men exhibiting oxidative stress

Sharma

Agarwal

Mohanty

et al. 2013

Reprod Biol Endocrinol

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“…The remaining semen volume (aliquot 3) was centrifuged at 800×g for 30 min to separate the seminal plasma supernatant, used in other studies [31,32], and the cellular fraction. The resulting pellet containing the cellular fraction was submitted to discontinuous density gradient centrifugation.…”

Section: Aliquot 3: Sperm Proteomic Analysismentioning

confidence: 99%

“…Next, 10 μL of 5 % trifluoroacetic acid was added to the digestion mixture in order to hydrolyze the RapiGest used in the sample preparation step and the samples were incubated at 37°C for 90 min. The tryptic peptide solution was then centrifuged at 16,000×g for 30 min at 6°C and the supernatant was combined with 10 μL of the internal standard (Yeast Alcohol Dehydrogenase at 1 pmol/μL) [31,32].…”

Section: Aliquot 3: Sperm Proteomic Analysismentioning

confidence: 99%

Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation

Intasqui

Camargo

Giudice

et al. 2013

J Assist Reprod Genet

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Purpose Sperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation. Methods Sperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MS E ). Differentially expressed proteins were used for a functional enrichment study. Results Two hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells. Conclusions Sperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.

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“…Moreover, few studies have used proteomics to assess the quality of human semen . Recently, we have shown through a strategy of liquid chromatography (LC)‐mass spectrometry (MS) E proteomics that there is a shift back to homeostasis in the seminal plasma after varicocelectomy . The use of proteomics analysis may expand clinical diagnostic testing for sperm infertility, an analysis currently limited to sperm count, motility and morphology.…”

Section: Introductionmentioning

confidence: 99%

Sperm nuclear DNA fragmentation rate is associated with differential protein expression and enriched functions in human seminal plasma

Intasqui¹,

Camargo²,

Giudice³

et al. 2013

BJU International

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Objective• To analyse the proteomic profile of seminal plasma with the aim of identifying the proteins and post-genomic pathways associated with sperm DNA fragmentation. Materials and Methods• A cross-sectional study including 89 subjects from a human reproduction service was carried out.• All semen samples were assessed for sperm DNA fragmentation using a comet assay.• Results from 60 sperm were analysed using Komet 6.0.1 software and the 'Olive tail moment' variable was used to stratify these into low and high sperm DNA fragmentation groups.• Seminal plasma proteins from the two groups were pooled and used for proteomic analysis.• Quantitative data were used for functional enrichment studies. Results• Seventy-two proteins were identified or quantified in seminal plasma. Of these, nine were differentially expressed in the low group and 21 in the high group. Forty-two proteins were conserved between these groups.• Functional enrichment analysis indicated that sperm DNA fragmentation was related to functions such as lipoprotein particle remodelling and regulation, fatty acid binding and immune response.• Proteins found exclusively in the low group may be involved in correcting spermatogenesis and/or improving sperm function. Proteins in the high group were associated with increased innate immune response, sperm motility and/or maturation and inhibition of mitochondrial apoptosis. Conclusion• Protein expression and post-genomic pathways of seminal plasma differ according to the rate of sperm DNA integrity.

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Unbiased label-free quantitative proteomic profiling and enriched proteomic pathways in seminal plasma of adult men before and after varicocelectomy

Abstract: The funding for this project was received from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) as a scholarship for Ms Camargo. There was no conflict of interest.

Cited by 48 publications

References 88 publications

Proteomic analysis of seminal fluid from men exhibiting oxidative stress

Proteomic analysis of seminal fluid from men exhibiting oxidative stress

Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation

Sperm nuclear DNA fragmentation rate is associated with differential protein expression and enriched functions in human seminal plasma

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