Sperm Protein Profile Research Articles

Cryoprotectant-specific alterations in the proteome of Siberian sturgeon spermatozoa induced by cryopreservation

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods—liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability.

Bisphenol A Negatively Impacts Human Sperm MicroRNA and Protein Profiles

AbstractBisphenol A (BPA), a widely prevalent endocrine disruptor, has detrimental effects on human health and male reproduction. Elevated BPA levels have been linked to reduced sperm quality and production, while paternal exposure may result in poor reproductive outcomes and offspring health. For many years, the effects of BPA exposure in human reproduction were mainly attributed to the deregulation of the hypothalamic–pituitary–gonadal (HPG) axis. In the past decades, other mechanisms of action have been proposed, and studies highlighting epigenetic alterations linked to BPA in sperm emerged. Nevertheless, the impact of BPA on human sperm miRNA and protein profiles and its implications for fertilization and post-fertilization events remain unexplored. This study aimed to investigate the association between BPA concentration in seminal plasma and seminal quality and to examine the alterations in human sperm miRNA and protein expression among men with varying BPA levels in seminal plasma. BPA levels were measured in seminal plasma from 102 Portuguese men, and it was detected in 88% of the samples. No correlation was observed between BPA levels and age or seminal parameters. The small RNA content of 15 normozoospermic samples was evaluated using small RNA sequencing, revealing that 15 miRNAs exhibited a correlation with BPA levels. Gene ontology analysis of their target genes indicated their involvement in embryonic development and response to stress. The proteome of 20 normozoospermic human sperm samples, categorized into four groups based on BPA levels, was accessed by liquid chromatography–mass spectrometry. Data are available via ProteomeXchange with the identifier PXD047280. Sixty-two differentially expressed proteins were identified across groups. The biological processes most affected in samples with higher BPA levels were “protein sumoylation” and “cytoplasmic translation”. The altered miRNAs and proteins identified in spermatozoa from men environmentally exposed to BPA represent potential biological markers of exposure to this endocrine disruptor and may help elucidate cases of idiopathic male infertility, fertilization failure, and abnormal embryo development.

Identification and validation of ram sperm proteins associated with cryoinjuries caused by the cryopreservation process

The change of sperm protein profile after the cryopreservation process may influence fertilization and early embryonic development. The purpose of the present study was to identify ram sperm proteomic modifications induced by the cryopreservation process using the isobaric tags for relative and absolute quantification labeling technology (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. Semen samples were collected from five Yunnan semi-fine wool rams using an electroejaculator. Sperm motility (CASA), plasma membrane (HOST test), and acrosome integrity (FITC-PSA) were evaluated after freeze-thawing. The total proteins of fresh and frozen-thawed sperm were extracted and purified, followed by identifying ram sperm proteomic modifications using the isobaric tags for relative and absolute quantification labeling technique (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. The results showed a significant reduction (P < 0.05) in all sperm parameters after thawing. 126 differentially abundant proteins (DAPs) were identified through comparison of the proteomes between fresh and frozen-thawed sperm. Among them, 90 proteins were down-regulated after the cryopreservation process. The remaining 36 proteins were up-regulated in frozen-thawed sperm. The results of functional annotation demonstrated the potential relationship of 10 DAPs with oxidoreductase activity. 18 and 15 DAPs may be involved in the stress and carbohydrate metabolic process, respectively. Furthermore, 8 DAPs may be functionally associated with reproduction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results demonstrated the primary enrichment of these identified DAPs in metabolic activities, disease, and oxidative phosphorylation. In order to confirm the reliability of the iTRAQ results, the changing trends of 10 proteins analyzed by PRM were similar to those of the corresponding proteins identified by iTRAQ. In conclusion, the cryopreservation process modifies the proteome of ram sperm, possibly leading to compromised fertility of post-thaw sperm. Additionally, the identified DAPs in this study may function as potential biomarkers for assessing the post-thaw quality of ram semen.

Association of bull semen protein estimates and SDS-PAGE profiles on semen freezability

It is vital to identify the ejaculate with good freezability by determining the biochemical makeup of the ejaculate at the pre-freeze stage. The present study targeted to assess the use of the protein estimates and profiles at the pre-freeze stage as markers of freezability in Frieswal populations. Storing the proteins for proteomic studies is always tricky in the case of animal studies, where accessibility to liquid nitrogen is limited. Hence alternative storing approaches need to be optimized. The second part of this study examined the protein concentration and protein profiles of RNALater and frozen stored sperm cells to assess the use of RNALater preservation in sperm proteomic studies. Sperm and seminal plasma protein concentrations were quantified using Bradford assay, and total protein quantities were derived. The seminal plasma and sperm protein profiles were generated with SDS-PAGE. The protein estimates and SDS-PAGE profiles of good and poor freeze-groups were similar. Also, sperm and seminal plasma protein concentration were not correlated with the semen volume and sperm count. Even though the yield was comparatively less, the protein profiles of sperm preserved by RNALater were similar to that of frozen sperms. The present study results indicate that the protein estimates and qualitative profiles of sperm and seminal plasma proteins may not be sufficient to reveal the differences in the proteome of good and poor freezable bulls at the macro level. Hence, the protein estimates and profiles of neat semen may not be helpful for the prediction of freezability at the pre-freeze stage. Secondly, this study indicates that RNALater preservation helps store sperms for proteome analysis studies.

Morphometry and molecular characterisation of semen in four carp species

Present study aimed to compare the morphometric features of sperm and seminal protein profile of four cyprinid species viz. Labeo rohita (Hamilton, 1822), Labeo fimbriatus (Bloch, 1795), Cyprinus carpio (Linnaeus, 1758) and Puntius carnaticus (Jerdon, 1849) in order to elucidate quantitative characteristics of sperm morphology and seminal protein profiles within the family. Morphological studies revealed that carps have uniflagellate, acrosome less sperm with an ovoid head and irregular shaped mid piece. Size of sperm in terms of length and breadth showed significant variation among the four species studied. The sperm and seminal protein profiles of Puntius carnaticus differed from other three groups with the presence of proteins with molecular mass of 15 and 49 kDa and absence of four proteins equivalent to molecular mass of 13, 20, 21 and 23 kDa. Two dimensional gel electrophoresis (2D-GE) analysis of sperm protein profile between Puntius carnaticus and Labe orohita revealed presence of 237 protein spots. Keywords: Cyprinid, Morphometry, Protein profiling, SDS-PAGE, Sperm

Acquiring the potential for motility is accompanied by profound changes in the testicular sperm proteome of sex-reversed female and normal male rainbow trout

Salmonid fish spermatozoa acquire the potential for motility during the transition from the testis to the spermatic duct. Due to the lack of a spermatic duct in sex-reversed females (srf) of rainbow trout, sperm resemble testicular semen and must be collected directly from the testes; consequently, they are immature with no or low potential for motility. Such potential can be acquired through artificial in vitro maturation-incubation of testicular sperm suspensions in buffered-saline solutions mimicking seminal plasma with high pH and containing bicarbonate (artificial seminal plasma (ASP)). The objective of this study was to investigate the effect of the in vitro maturation of sperm on the sperm quality parameters and sperm proteome, for both srf and normal males of rainbow trout. The incubation of sperm in ASP resulted in an increase in sperm motility parameters (from the mean values of 26 to 66% and 28 to 56% for srf and normal males, respectively), a decrease in the proportion of ROS-positive cells (from the mean values 12.9% to 9.1% for srf and from 17.9% to 12.2% for normal male sperm) and no effect on sperm viability maturation, resulting in profound changes in the sperm protein profile in srf and normal male sperm. The application of a 2D-DIGE approach revealed a slightly higher number of differentially abundant protein spots (126 protein spots) after the sperm motility acquisition of normal male sperm than that of srf sperm (116 protein spots). Most of the proteins that change in abundance after sperm incubation in ASP were similar for both srf and normal male sperm and were involved in a variety of pathways and functions including cytoskeleton and cell movement, metabolism and energy production, protein folding, turnover and molecule binding. The number of significant changes in the sperm proteome is related to the cytoskeleton, flagella and cell movement, and metabolic proteins demonstrate changes in the sperm motility apparatus during the short period of artificial in vitro maturation, indicating similarities to the protein changes that occur during the longer process of natural sperm maturation. The lack of differences in sperm quality between srf and normal males after incubation in ASP indicates the similar modes of sperm motility acquisition. The alterations in proteins after artificial maturation partially overlapped with the changes noticed after trout sperm motility activation, strongly suggesting the participation, at least partially, of the same proteins, both in the process of acquiring the spermatozoa ability to move and sperm movement.

Sperm proteomic changes associated with early embryo quality after ICSI

Do alterations of human sperm protein profile affect embryo quality? Sperm proteins from 27 infertile couples undergoing intracytoplasmic sperm injection (ICSI) were extracted and digested. The resulting peptides were labelled using tandem mass tags, separated by two-dimensional liquid chromatography, and identified and quantified using tandem mass spectrometry. Subsequently, sperm protein and peptide abundance were statistically analysed for correlation with ICSI-derived embryo quality in the subset of idiopathic infertile couples. Detected correlations were further assessed in the subset of infertile patients with a known factor. The abundance of 18 individual sperm proteins was found to correlate with embryo quality after ICSI. Of note, a high percentage of poor-quality ICSI-derived embryos was associated with alterations in several components of the eight-membered chaperonin-containing T-complex, which plays an important role in the folding of many essential proteins. Additionally, the abundance of sperm proteins with known functions in embryogenesis, such as RUBVL1, also correlated with early embryo quality (r = -0.547; P = 0.028). Some of the correlations found in this study were validated using either proteomic data from infertile patients with a known factor or data from similar published studies. Analysis at the peptide level revealed the association of some correlations with specific post-translational modifications or isoforms. Our results support the hypothesis that the sperm proteome plays a role in early embryogenesis. Moreover, several sperm proteins have emerged as potential biomarkers that could predict the outcome of in-vitro assisted reproductive technologies, leading to the possibility of improved diagnosis of couples with idiopathic infertility.

Proteomics analysis of asthenozoospermia and identification of glucose-6-phosphate isomerase as an important enzyme for sperm motility

Asthenozoospermia, in which sperm motility is affected, is one of the primary causes of male infertility. However, the exact mechanism responsible for the defective motility remains unknown. It is important to identify the precise proteins or pathways involved in sperm motility. The present study analyzed five asthenozoospermic sperm samples and five healthy controls using TMT-based quantitative method and identified 152 differentially expressed proteins, with 84 upregulated and 68 downregulated in asthenozoospermia. Four proteins (GPI, MDH1, PGAM1 and PGAM2) were found in several over-represented energy metabolism pathways using bioinformatics analysis. Glucose-6-phosphate isomerase (GPI), a rate-limiting enzyme converting glucose-6-phosphate to fructose-6-phosphate, was found to be significantly decreased in asthenozoospermia by Western blotting and ELISA on an extended sample size. Furthermore, substitution of glucose with fructose-6-phosphate significantly promoted asthenozoospermic sperm motility in vitro. Taken together, our results suggest that the poor motility of sperm in asthenozoospermia may partly result from defects in GPI-associated energy metabolism. SignificanceTo identify the key proteins or pathways involved in sperm motility, the accurate TMT-based quantitative method was applied to characterize protein profiles of asthenozoospermic sperm. GPI, an enzyme involved in energy metabolism, was found to be differentially abundant, and validated by extended sample analysis. The supplement of the product of GPI, fructose-6-phosphate, could significantly improve sperm motility. Our study could provide new insights into the molecular basis of sperm motility and the improvement of motility in asthenozoospermia.

Maladaptation of trout spermatozoa to fresh water is related to oxidative stress and proteome changes.

Rainbow trout sperm are 'maladapted' to freshwater spawning, resulting in shorter duration of sperm motility in fresh water compared to buffered saline solution. We hypothesized that different sperm motility-activating media have various effects on sperm motility characteristics and oxidative stress, as well as on the protein profiles of rainbow trout sperm. We designed an experimental model for activation of rainbow trout sperm motility in different osmotic conditions: (i) isosmotic and (ii) hypoosmotic. Spermatozoa activation with hypoosmotic solution was associated with lower values for sperm motility parameters (52%) and an induced increase in ROS level (19.4%) in comparison to isosmotic activation with isosmotic solution (67 and 9.5% for sperm motility and ROS, respectively). Hypoosmotic activation resulted in a higher number of differentially abundant sperm proteins (out of which 50 were identified) compared to isosmotic conditions, where only two spots of protein disulfide-isomerase 6 were changed in abundance. The proteins are mainly involved in the TCA cycle, tight and gap junction signaling, Sertoli cell-Sertoli cell junction signaling and asparagine degradation. Our results, for the first time, indicate that during hypoosmotic activation of sperm motility, osmotic stress triggers oxidative stress and disturbances mostly to structural proteins and metabolic enzymes. Our results strongly suggest that comparative physiological and biochemical analysis of rainbow trout sperm characteristics in isosmotic and hypoosmotic conditions could be a useful model for studying the mechanism of sperm activation in salmonid fish.

Psychiatric symptoms depend on tinnitus-related characteristics and clinical variables in tinnitus patients

Asthenozoospermia, in which sperm motility is affected, is one of the primary causes of male infertility. However, the exact mechanism responsible for the defective motility remains unknown. It is important to identify the precise proteins or pathways involved in sperm motility. The present study analyzed five asthenozoospermic sperm samples and five healthy controls using TMT-based quantitative method and identified 152 differentially expressed proteins, with 84 upregulated and 68 downregulated in asthenozoospermia. Four proteins (GPI, MDH1, PGAM1 and PGAM2) were found in several over-represented energy metabolism pathways using bioinformatics analysis. Glucose-6-phosphate isomerase (GPI), a rate-limiting enzyme converting glucose-6-phosphate to fructose-6-phosphate, was found to be significantly decreased in asthenozoospermia by Western blotting and ELISA on an extended sample size. Furthermore, substitution of glucose with fructose-6-phosphate significantly promoted asthenozoospermic sperm motility in vitro. Taken together, our results suggest that the poor motility of sperm in asthenozoospermia may partly result from defects in GPI-associated energy metabolism.To identify the key proteins or pathways involved in sperm motility, the accurate TMT-based quantitative method was applied to characterize protein profiles of asthenozoospermic sperm. GPI, an enzyme involved in energy metabolism, was found to be differentially abundant, and validated by extended sample analysis. The supplement of the product of GPI, fructose-6-phosphate, could significantly improve sperm motility. Our study could provide new insights into the molecular basis of sperm motility and the improvement of motility in asthenozoospermia.

Boar sperm protein tyrosine phosphorylation in the presence of egg yolk soluble and low density lipoprotein fractions during cooling

Increased protein tyrosine phosphorylation and the appearance of a phosphorylated protein of 32 kD (p32) are reported among the capacitation-like changes in cryopreserved boar sperm. Egg yolk freezing extenders are composed by two fractions: insoluble granules and soluble plasma, which contains the low density lipoproteins (LDL) proposed as responsible for the egg yolk cryoprotective action. The aim of this work was to analyze the effects of complete egg yolk and its insoluble, soluble and LDL fractions on boar sperm quality and protein tyrosine phosphorylation after the first stage of a standard cryopreservation protocol. Semen samples in Androstar® Plus diluent were centrifuged and resuspended in the different egg yolk extenders. Temperature was decreased from 17 °C to 5 °C and sperm quality, protein tyrosine phosphorylation and protein pattern were analyzed. Results showed that complete egg yolk as well as soluble and LDL egg yolk fractions maintained sperm quality after temperature decrease. Cooling without any lipid component or in the presence of the insoluble fraction, significantly reduced sperm motility. About sperm protein tyrosine phosphorylation analysis, the p32 band appeared before treatments or after cooling in Androstar® Plus diluent. Complete egg yolk and its insoluble fraction interfered with sperm tyrosine phosphorylation even after cells were extensively washed. Analysis of extenders revealed a high amount of tyrosine phosphorylated proteins in the insoluble fraction, which may have co-precipitate with sperm in experiments. Samples submitted to temperature decrease from 17 °C to 5 °C in the presence of soluble and LDL egg yolk fractions in Androstar® Plus diluent did not show any change in the p32 band associated with sperm capacitation. However, a tyrosine-phosphorylated protein of 33 kD present in clarified egg yolk was also observed in sperm treated with this extender. Protein transference from plasma and LDL egg yolk extenders was also observed in sperm protein profile. Results suggested that soluble and LDL fractions might have a protective action preventing sperm protein tyrosine phosphorylation during cooling from 17 °C to 5 °C. Further studies are needed to expand the knowledge of the LDL protection mechanism as well as to determine the possible benefits of clarified egg yolk in freezing protocols.

Impact of cryopreservation on sterlet, Acipenser ruthenus sperm motility and proteome

Fish sperm cryopreservation is a well-established technique allowing for artificial insemination on a commercial scale. The extent of proteome alterations in seminal plasma and sperm due to cryopreservation, however, is not known. This study was conducted to evaluate the effect of cryopreservation on motility variables of sterlet Acipenser ruthenus sperm and to detect the differences in protein profiles of fresh and cryopreserved sterlet sperm and seminal plasma. Fresh sperm had 89 ± 3% motility and 160 ± 14 μm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ± 5%) was less at 15 s post-activation. No difference (ANOVA; P > 0.05) in mean curvilinear velocity of fresh and cryopreserved sperm was detected. The protein profiles of seminal plasma and sperm were characterized using comparative proteomics to determine the influence of cryopreservation. Six altered protein spots in seminal plasma and thirteen altered spots in sperm were detected in fresh and thawed sperm. Subsequent protein characterization suggested that the proteins identified were involved in sperm metabolism, cytoskeleton, and stress response. The results broaden the understanding of the effects of cryopreservation and identify the proteins associated with cryo-injury. These data may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation.

A comparative study of effects of different freezing methods on sperm quality, DNA integrity and membrane protein of cryopreserved boar semen*

The present study determined the effects of conventional, and controlled freezing method adopting three freezing rates, viz. 20°C, 40°C and 60°C/min on quality of sperm (motility, viability and plasma membrane integrity), DNA integrity and plasma membrane protein profile of cryopreserved boar semen. Sixty sperm-rich fractions of ejaculates from six boars were utilized for freezing of semen with different freezing methods in lactose-egg yolk glycerol extender. Semen samples were evaluated for sperm motility, viability (Propidium Iodide assay), functional integrity of plasma membrane (HOST), DNA integrity (Acridine Orange stain) and plasma membrane protein profile (SDSPAGE) after equilibration and after freezing. The results revealed that the post thaw sperm motility, sperm viability, and plasma membrane integrity (HOST-reacted) were significantly higher in all the three controlled freezing methods (20°C, 40°C and 60°C/min) as compared to that in conventional method. In addition, the number of sperm plasma membrane protein loss was less in controlled freezing methods as compared to that in conventional freezing. However, the post thaw sperm DNA integrity did not influence by difference in freezing methods. No significant difference on the post thaw sperm characteristics was recorded among the three controlled freezing rates. All the sperm parameters assessed declined significantly after freezing as compared to that after equilibration irrespective of freezing method employed. In conclusion, controlled freezing methods conferred better post thaw sperm quality as compared to conventional method, and thus the freezing rates of either 20, 40 or 60°C/min could provide better freezability of boar semen.

Effects of Lunasia amara Blanco (Sanrego) on Male Fertility: A Preliminary Study on Sperm Proteomic Analysis

Lunasia amara Blanco or sanrego has long been applied as a pro-fertility agent in folk medicine and it has also been scientifically proven to improve sperm quality in rats. Nevertheless, little is known about the underlying molecular mechanism of improved sperm function upon treatment with sanrego. As such, this study looked into the potential proteomic changes in sperm treated with 60 mg/kg body weight sanrego aqueous extract, in comparison to control. The sperm proteins were subjected to proteomic analysis using 2-D SDS PAGE and MALDI-TOF. The comparison of the sperm protein profiles between treated and control rats revealed two downregulated proteins (cenexin-1 and GLUL) and 11 upregulated proteins (Eno1, PGK2, F1-ATP synthase, FO-ATP synthase, LDHC, GLUL1, ODF2, Sp17, asparaginase, DNAJB13, and tubulin) in treated rats. These proteins have been identified to be mainly involved in energy metabolism, sperm motility, amino acid metabolism, and cell signalling. This improved sperm function in sanrego-treated rats may, thus, be a direct result of proteomic changes, as discovered in this present study.

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.

Differential Proteomic Profiling of Spermatozoal Proteins of Infertile Men With Unilateral or Bilateral Varicocele

To compare the sperm protein profile between infertile men with unilateral varicocele and infertile men with bilateral varicocele. This prospective study investigated 50 infertile patients with clinical varicocele (33 unilateral and 17 bilateral) seeking fertility workup between March 2012 and April 2014. Routine sperm parameters, reactive oxygen species, total antioxidant capacity, and sperm deoxyribonucleic acid fragmentation were assessed in their semen. Sperm protein profile was characterized only in pooled samples of 5 unilateral and 3 bilateral varicocele samples, respectively, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Linear Trap Quadrupole-Orbitrap Elite hybrid mass spectrophotometer system. Differences in protein expression were analyzed using gel analysis software, followed by protein identification using mass spectroscopy analysis. Differentially expressed proteins and their abundance were quantified by comparing spectral counts, followed by bioinformatics analysis. Unique expression of 64 proteins in the bilateral group and 31 proteins in the unilateral group was obtained. Core functions of the top protein interaction networks were post-translational modification (∼122 proteins associated with acetylation), protein folding, free-radical scavenging, cell death, and survival. The top molecular and cellular functions were protein degradation, free radical scavenging, and post-translational modifications, whereas the top pathways were protein ubiquitination and mitochondrial dysfunction. Major biological pathways for the 253 differentially expressed proteins were metabolism, apoptosis, and signal transduction. Functional proteomic profiling helps identify the differential processes or pathways that are affected based on the nature of varicocele (bilateral or unilateral) and provide insights into the mechanistic implications of varicocele-associated male infertility.

Evaluation of potential protein biomarkers in patients with high sperm DNA damage

The laboratory evaluation of male infertility remains an essential area of research as 40-60% of infertility cases are attributable to male-related factors. Current sperm analysis methods add only partial information on sperm quality and fertility outcomes. The specific underlying cause of infertility in most cases is unknown, while a proportion of male infertility could be caused by molecular factors such as the absence or abnormal expression of some essential sperm proteins. The objective of this study was to screen for associations between sperm protein profiles and sperm concentration, motility, and DNA fragmentation index in patients undergoing fertility evaluation in a clinical setting. Based on those parameters, semen samples were categorized as either normal or abnormal. We screened 34 semen samples with various abnormal parameters and compared them to 24 normal control samples by using one dimensional (1-D) gel electrophoresis and mass-spectrometry. In this study, we anticipated to establish a normal sperm parameter profile which would be compared to abnormal sperm samples and reveal candidate proteins. Our preliminary results indicate that no normal uniform profile could be established, which affirms the complexity of male fertility and confirms the limitations of standard semen analysis. Four main protein groups were identified in correlation with abnormal DNA fragmentation and/or motility. The first group included sperm nuclear proteins such as the SPANX (sperm protein associated with the nucleus on the X chromosome) isoforms and several types of histones. The second group contained mitochondria-related functions and oxidative stress proteins including Mitochondrial Ferritin, Mitochondrial Single-Stranded DNA Binding Protein, and several isoforms of Peroxiredoxins. Two other protein groups were related to sperm motility such as microtubule-based flagellum and spindle microtubule as well as proteins related to the ubiquitin-proteasome pathway. Further research is required in order to characterize these potential biomarkers of male fertility potential.

Study of Sperm Protein Profile in Men With and Without Varicocele Using Two-Dimensional Gel Electrophoresis

To compare the sperm protein profile between men with and without varicocele. The present study was designed as a case-control study. The research patients were recruited from the Infertility Unit of the Royan Institute in 2009. We included 20 sperm samples from normozoospermic men without varicocele (control group) and 20 sperm samples from oligozoospermic patients with varicocele, grade 3 (varicocele group) in the present study. The sperm protein profile in the 2 groups was characterized using 2-dimensional gel electrophoresis. Differences in protein expression were established using gel analysis software, and protein identification was performed using mass spectroscopy analysis. In the varicocele group, we noted 15 consistent differences in protein expression (1, spots missing; 12, less abundant; and 2, more abundant) compared with the control group (P < .01). The findings revealed that heat shock proteins, mitochondrial proteins, and cytoskeleton proteins are the proteins mainly affected by varicocele disease. To our knowledge, the present study is a novel study, with few studies describing the correlation between sperm protein in men with and without varicocele obtained using a 2-dimensional proteomic approach. It could be an important prerequisite to the development of diagnostic tests to predict varicocelectomy outcomes in patients with varicocele and abnormal findings on a spermogram in the clinical environment.

Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele

What are the effects of smoking on the functional aspects of the sperm, the levels of lipid peroxidation and the protein profile of seminal plasma in patients with varicocele? In men with varicocele, smoking is associated with altered semen quality, decreased sperm functional integrity and seminal oxidative stress. Alterations in seminal plasma protein profiles are also present and may explain the altered semen phenotype. Varicocele is a major cause of male infertility. It reduces testicular blood renewal with a consequent accumulation of toxic substances. Thus, it can potentiate the toxic effects of environmental exposure to genotoxic substances such as those found in cigarette smoke. A cross-sectional study was performed in 110 patients presenting with variococele to the Human Reproduction Section of the Sao Paulo Federal University (2006-2010). The patients were divided into a control group of non-smokers, a moderate smokers group and a heavy smokers group. Semen parameters were analysed by standard methods. Sperm DNA integrity and mitochondrial activity were assessed by Comet assays and by 3,3'-diaminobenzidine deposition, respectively. The level of lipid peroxidation in semen was determined by malondialdehyde quantification. Proteomic studies were performed by 2D-electrophoresis and mass spectrometry. Both groups of smokers showed reduced semen quality in comparison with the control group. In the groups of smokers, sperm DNA integrity and mitochondrial activity were also decreased and lipid peroxidation levels were increased. Proteomic analyses revealed 20 proteins differentially expressed between the study groups. A study including smokers without varicocele is still warranted as these results apply only to smokers who present varicocele. Patients with varicocele who are exposed to tobacco smoking present more important alterations to semen quality and sperm functional integrity and show changes in the seminal plasma proteome. This suggests testicular, and possibly systemic, adverse effects of smoking. Funding for the study was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) (2007/59423-7) and by the Division of Urology, Human Reproduction Section at the São Paulo Federal University.

Evaluation of Sperm Proteins in Infertile Men: A Proteomic Approach

In this study, the sperm protein profile was compared between fertile and infertile men using 2-dimensional gel electrophoresis, liquid chromatography mass spectrometer analysis, and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry. Four unique proteins, semenogelin II precursor, prolactin-induced protein, clusterin isoform 1, and prostate-specific antigen isoform 1 preproprotein, were predominantly present in the semen of healthy men; however, semenogelin II precursor and clusterin isoform 1 were not seen in the semen of infertile men, suggesting unique differences in the spermatozoa protein profiles of fertile and infertile men.