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Introduction: Polyaromatic hydrocarbons (PAHs) are considered as redox active environmental toxicants inducing oxidative stress (OS) mediated injury to cells. Oxidative predominance is reported in 30%-80% of idiopathic male infertility (IMI) patients. Hence, this work aims to unravel correlation, if any, between seminal PAH exposome and sperm function in IMI patients through a proteomic approach. Methods: Seminal PAH exposome was analyzed in 43 fertile donors and 60 IMI patients by HPLC and receiver operating characteristic (ROC) curve was applied to find out the cut-off limits. Spermatozoa proteome was analyzed by label free liquid chromatography mass spectroscopy (LC-MS/MS) followed by molecular pathway analysis using bioinformatic tools. Validation of key proteins' expression and protein oxidative modifications were analyzed by western blot. Results and discussion: Of the 16 standards toxic PAH, 13 were detected in semen. Impact of the different PAHs on fertility are Anthracene < benzo (a) pyrene < benzo [b] fluoranthene < Fluoranthene < benzo (a) anthracene ROC). Benzo [a] pyrene is invariably present in all infertile patients while naphthalene is present in both groups. Of the total 773 detected proteins (Control: 631 and PAH: 717); 71 were differentially expressed (13 underexpressed, 58 overexpressed) in IMI patients. Enrichment analysis revealed them to be involved in mitochondrial dysfunction and oxidative phosphorylation, DNA damage, Aryl hydrocarbon receptor (AHR) signaling, xenobiotic metabolism and induction of NRF-2 mediated OS response. Increased 4-hydroxynonenal and nitrosylated protein adduct formation, and declined antioxidant defense validates induction of OS. Increased GSH/GSSG ratio in patients may be an adaptive response for PAH metabolism via conjugation as evidenced by over-expression of AHR and Heat shock protein 90 beta (HSP90β) in patients. Seminal PAH concentrations, particularly benzo (a) pyrene can be used as a marker to distinguish IMI from fertile ones with 66.67% sensitivity and 100% specificity (95% confidence interval) along with oxidative protein modification and expression of AHR and HSP90β.

Bi-directional crosstalk between Ca2+ signaling and ROS modulates physiological processes as a part of a regulatory circuit including sperm function. The role of transient receptor potential vanilloid 1 (TRPV1) in this regard cannot be undermined. This is the first report demonstrating the Ca2+-sensitive TRPV1 channel to be under-expressed in spermatozoa of subfertile men, idiopathic infertile men, and normozoospermic infertile males with high ROS (idiopathic infertility and unilateral varicocele). To study the effect of TRPV1 in determining the fertility outcome, we compared the expression profile of TRPV1 in spermatozoa of male partners who achieved pregnancy by natural conception (NC+, n = 10), IVF (IVF+, n = 23), or ICSI (ICSI +, n = 9) and their respective counterparts with failed pregnancy NC (n = 7), IVF (n = 23), or ICSI (n = 10), by both immunocytochemistry and flow-cytometry. Reduced expression of TRPV1 in sperm of IVF ± and ICSI ± men with respect to that NC+ men imply its role in mediating successful fertilization. Unsuccessful pregnancy outcome with an underexpression of TRPV1 in sperm of NC-/IVF-/ICSI-men suggests its role in conception and maintenance of pregnancy. Since ROS is regarded as one of the major contributors to sperm dysfunction, the effect of H2O2 +/- TRPV1 modulators (RTX/iRTX) on acrosomal reaction and calcium influx was evaluated to confirm TRPV1 as a redox sensor in human sperm. A significant increment in the percentage of acrosome reacted spermatozoa along with augmented Ca2+-influx was observed after H2O2 treatment, both in the presence or absence of TRPV1 agonist resiniferatoxin (RTX). The effect was attenuated by the TRPV1 antagonist iodoresiniferatoxin (iRTX), indicating the involvement of TRPV1 in mediating H2O2 response. Enhancement of motility and triggering of acrosomal reaction post TRPV1 activation suggested that disruption of these signaling cascades in vivo, possibly due to down-regulation of TRPV1 in these subfertile males. Bioinformatic analysis of the crosstalk between TRPV1 with fertility candidate proteins (reported to influence IVF outcome) revealed cell death and survival, cellular compromise, and embryonic development to be the primary networks affected by anomalous TRPV1 expression. We therefore postulate that TRPV1 can act as a redox sensor, and its expression in spermatozoa may serve as a fertility marker.

Purpose: Aberrant expression of seminal plasma proteins are associated with altered homeostasis that may affect the fertilizing ability of spermatozoa. However, the precise roles of seminal exosomes on sperm function remain unclear. The objective of this study was to identify the differentially expressed proteins (DEPs) associated with varicocele-mediated infertility by comparing seminal plasma protein profile of unilateral varicocele patients with proven fertile donors. Materials and Methods: Semen samples were obtained from 10 proven fertile donors with normal semen parameters and 33 infertile patients with unilateral varicocele. For proteomic analysis, 5 samples from each group were pooled and run in triplicate. Key DEPs (ANXA2, TF, CD63, KIF5B, SEMG1) associated with the exosome function were selected by bioinformatic tools and validated using Western blotting. Results: A total of 47 seminal plasma proteins were differentially expressed in unilateral varicocele patients compared to fertile donors. Validation of exosome-associated DEPs in unilateral varicocele patients (n=7) and fertile donors (n=7) revealed significant upregulation of ANXA2 (p=0.0016) and downregulation of KIF5B (p=0.009). The main upstream regulators of the DEPs in seminal plasma of unilateral varicocele group were androgen receptor, YB1 and NRF2. Conclusions: This is the first report to identify DEPs in seminal plasma of unilateral varicocele patients compared to fertile donors. Based on the detection of DEPs associated with exosomal function, Western blotting was used to validate the presence of defective exosome machinery in seminal plasma of unilateral varicocele patients. KIF5B and ANXA2 can be utilized as potential biomarkers of infertility in unilateral varicocele patients. [ABSTRACT FROM AUTHOR]

Infertility affects nearly 15 per cent of all couples within the reproductive age worldwide, with about 50 per cent being exhibited in the male, called male factor infertility. Successful reproduction is dependent on sperm chromatin integrity. Spermatozoa are highly specialized cells that aim to transmit the paternal genomic blueprint to the oocyte. The spermatozoon is regulated by redox mechanisms during its epididymal transit to acquire fertilizing ability. While, at physiological levels, the production of reactive oxygen species (ROS) supports the spermatozoon to acquire its fertilizing ability, at high concentrations, it affects sperm function leading to infertility. Emerging proteomic technologies provide an opportunity to address these key issues that may solve many fertility-associated problems resulting from oxidative stress (OS). This review highlights the need for an efficient therapeutic approach to male infertility with the application of high-throughput OS-mediated proteomic technology, and also addresses the question as to whether targeting these altered sperm-specific proteins may help in designing an efficient and reversible male contraceptive. [ABSTRACT FROM AUTHOR]

Purpose Varicocele may disrupt testicular microcirculation and induce hypoxia-ischemia related degenerative changes in testicular cells and spermatozoa. Superoxide production at low oxygen concentration exacerbates oxidative stress in men with varicocele. Therefore, the current study was designed to study the role of mitochondrial redox regulation and its possible involvement in sperm dysfunction in varicocele associated infertility. Materials and Methods We identified differentially expressed mitochondrial proteins in 50 infertile men with varicocele and in 10 fertile controls by secondary liquid chromatography-tandem mass spectroscopy data driven in silico analysis. Identified proteins were validated by Western blot and immunofluorescence. Seminal oxidation-reduction potential was measured. Results We identified 22 differentially expressed proteins related to mitochondrial structure (LETM1, EFHC, MIC60, PGAM5, ISOC2 and import TOM22) and function (NDFSU1, UQCRC2 and COX5B, and the core enzymes of carbohydrate and lipid metabolism). Cluster analysis and 3-dimensional principal component analysis revealed a significant difference between the groups. All proteins studied were under expressed in infertile men with varicocele. Liquid chromatography-tandem mass spectroscopy data were corroborated by Western blot and immunofluorescence. Impaired mitochondrial function was associated with decreased expression of the proteins (ATPase1A4, HSPA2, SPA17 and APOA1) responsible for proper sperm function, concomitant with elevated seminal oxidation-reduction potential in the semen of infertile patients with varicocele. Conclusions Impaired mitochondrial structure and function in varicocele may lead to oxidative stress, reduced ATP synthesis and sperm dysfunction. Mitochondrial differentially expressed proteins should be explored for the development of biomarkers as a predictor of infertility in patients with varicocele. Antioxidant therapy targeting sperm mitochondria may help improve the fertility status of these patients. [ABSTRACT FROM AUTHOR]

Background The spermatozoa undergo a series of changes in the epididymis to mature after their release from the testis and subsequently in the female reproductive tract after ejaculation to get capacitated and achieve fertilization potential. Despite having a silenced protein synthesis machinery, the dynamic change in protein profile of the spermatozoa is attributed either to acquisition of new proteins via vescicular transport or to several post-translational modifications (PTMs) occurring on the already expressed protein complement. Scope of review In this review emphasis is given on the PTMs already reported on the human sperm proteins under normal and pathologic conditions with particular reference to sperm function such as motility and fertilization. An attempt has been made to summarize different protocols and methods used for analysis of PTMs on sperm proteins and the newer trends those were emerging. Major conclusions Deciphering the differential occurrence of PTM on protein at ultrastructural level would give us a better insight of structure-function relationship of the particular protein. Protein with multiple PTMs could be used to generate the complex interaction network involved in a physiological function of a sperm. It can be speculated that crosstalk between different PTMs occurring either on same/ other proteins actually regulate the protein stability and activity both in physiological and pathological states. General significance The analytical prospective of various PTMs reported in human spermatozoa and their relevance to sperm function particularly in various pathophysiological states, would pave way for development of biomarkers for diagnosis, prognosis and therapeutic intervention of male infertility. [ABSTRACT FROM AUTHOR]

Background: Reactive oxygen species (ROS) plays a major role in the pathology of male infertility. It is an independent biomarker of sperm function. Seminal plasma is a natural reservoir of antioxidants responsible for the nourishment, protection, capacitation, and motility of sperm within the female reproductive tract resulting in successful fertilization and implantation of the embryo. A comparative proteomic analysis of seminal plasma proteins from fertile men and infertile men with varying levels of ROS was carried out to identify signature proteins involved in ROS-mediated reproductive dysfunction. Methods: A total of 42 infertile men presenting with infertility and 17 proven fertile donors were enrolled in the study. ROS levels were measured in the seminal ejaculates by chemiluminescence assay. Infertile men were subdivided into Low ROS (0-<93 RLU/s/106 sperm; n = 11), Medium ROS (>93-500 RLU/s/106 sperm; n = 17) and High ROS (>500 RLU/s/106 sperm; n = 14) groups and compared with fertile men (4-50 RLU/s/106 sperm). 4 subjects from fertile group and 4 each from the Low, Medium and High ROS were pooled. 1D gel electrophoresis followed by in-gel digestion and LC/MS-MS in a LTQ-Orbitrap Elite hybrid mass spectrometer system was used for proteome analysis. Identification of differentially expressed proteins (DEPs), their cellular localization and involvement in different pathways were examined utilizing bioinformatics tools. Results: The results indicate that proteins involved in biomolecule metabolism, protein folding and protein degradation are differentially modulated in all three infertile patient groups in comparison to fertile controls. Membrane metallo-endopeptidase (MME) was uniformly overexpressed (>2 fold) in all infertile groups. Pathway involving 35 focus proteins in post-translational modification of proteins, protein folding (heat shock proteins, molecular chaperones) and developmental disorder was overexpressed in the High ROS group compared with fertile control group. MME was one of the key proteins in the pathway. FAM3D was uniquely expressed in fertile group. Conclusion: We have for the first time demonstrated the presence of 35 DEPs of a single pathway that may lead to impairment of sperm function in men with Low, Medium or High ROS levels by altering protein turn over. MME and FAM3D along with ROS levels in the seminal plasma may serve as good markers for diagnosis of male infertility. [ABSTRACT FROM AUTHOR]

Abstract: Oxidative stress impaired sperm function might lead to infertility. The objective of this study was to evaluate the effects of altered thyroid hormone levels on regulation of mitochondrial glutathione redox status and its dependent antioxidant defense system in adult rat testis and their correlation with testicular function. Adult male Wistar rats were rendered hypothyroid by administration of 6-n-propyl-2-thiouracil in drinking water for six weeks. At the end of the treatment period, a subset of the hypothyroid rats was treated with T3 (20μg/100g body weight/day for 3days). Mitochondria were isolated from euthyroid, hypothyroid and hypothyroid+T3-treated rat testes, and sub-fractionated into sub-mitochondrial particles and matrix fractions. Mitochondrial respiration, oxidative stress indices and antioxidant defenses were assayed. The results were correlated with daily testicular sperm production and epididymal sperm viability. Increased pro-oxidant level and reduced antioxidant capacity rendered the hypothyroid mitochondria susceptible to oxidative injury. The extent of damage was more evident in the membrane fraction. This was reflected in higher degree of oxidative damages inflicted upon membrane lipids and proteins. While membrane proteins were more susceptible to carbonylation, thiol residue damage was evident in matrix fraction. Reduced levels of glutathione and ascorbate further weakened the antioxidant defenses and impaired testicular function. Hypothyroid condition disturbed intra-mitochondrial thiol redox status leading to testicular dysfunction. Hypothyroidism-induced oxidative stress condition could not be reversed with T3 treatment. [Copyright &y& Elsevier]