Sperm Proteomics

Currently, there are 20,197 human protein-coding genes in the most expertly curated database (UniProtKB/Swiss-Pro). Big efforts have been made by the international consortium, the Chromosome-Centric Human Proteome Project (C-HPP) and independent researchers, to map human proteome. In brief, anno 2017 the human proteome was outlined. The male factor contributes to 50% of infertility in couples. However, there are limited human spermatozoa proteomic studies. Firstly, the development of the mapping of the human spermatozoa was analyzed. The human spermatozoa have been used as a model for missing proteins. It has been shown that human spermatozoa are excellent sources for finding missing proteins. Y chromosome proteome mapping is led by Iran. However, it seems that it is extremely challenging to map the human spermatozoa Y chromosome proteins based on current mass spectrometry-based proteomics technology. Post-translation modifications (PTMs) of human spermatozoa proteome are the most u...

The human seminal fluid is a complex body fluid. It is not known how many proteins are expressed in the seminal plasma; however in analog with the blood it is possible up to 10,000 proteins are expressed in the seminal plasma. The human seminal fluid is a rich source of potential biomarkers for male infertility and reproduction disorder. In this review, the ongoing list of proteins identified from the human seminal fluid was collected. To date, 4188 redundant proteins of the seminal fluid are identified using different proteomics technology, including 2-DE, SDS-PAGE-LC-MS/MS, MudPIT. However, this was reduced to a database of 2168 non-redundant protein using UniProtKB/Swiss-Prot reviewed database. The core concept of proteome were analyzed including pI, MW, Amino Acids, Chromosome and PTM distribution in the human seminal plasma proteome. Additionally, the biological process, molecular function and KEGG pathway were investigated using DAVID software. Finally, the biomarker identifie...

Precision medicine (PM) is an approach that has the power to create the best effect and safety of medicine and treatment with the least side effects for each person. PM is very helpful as sometimes due to inaccurate or late diagnosis or toxicities of the drugs irreversible side effect for patient’s health are generated. This seemingly new and emerging science is also effective in preventing disease, due to differences in the genes, environment, and lifestyles of any particular person. PM can be a prominent criterion in infertility research. To achieve this goal, there should be information from a healthy human body, including genetic and molecular information. A PM is an evolution in health care, which is very helpful even economically. The guarantor of the PM success is the examination of the molecular profile of the patient, including genes, proteins, metabolites, etc. Therefore, genomics, proteomics, and metabolomics-based techniques are very important in this regard. Unfortunate...

Despite the explosive increase in the use of Assisted Reproductive Technologies (ART) over the last 30 years, their success rates remain suboptimal. Proteomics is a rapidly-evolving technology-driven science that has already been widely applied in the exploration of human reproduction and fertility, providing useful insights into its physiology and leading to the identification of numerous proteins that may be potential biomarkers and/or treatment targets of a successful ART pregnancy. Here we present a brief overview of the techniques used in proteomic analyses and attempt a comprehensive presentation of recent data from mass spectrometry-based proteomic studies in humans, regarding all components of ARTs, including the male and female gamete, the derived zygote and embryo, the endometrium and, finally, the ART offspring both pre- and postnatally.

Validation by sperm motility evaluation The significant higher expression of sperm flagella components ODF protein and Tektin 5 could result in an increase in sperm tail elasticity and strength in KK animals. Furthermore, the energy related enzyme Triose Phosphate Isomerase is significantly down regulated in Mafriwal. Stronger flagella and a greater energy supply in KK animals may have contributed to the increase in motile and fast moving sperm. In addition, more elastic flagella resulted in the higher flagella lateral amplitudes in KK. The Fibrous Sheet component 2, as a homologue of Glutathione S-transferase MU3, is up-regulated in KK and may be required to protect sperm against toxins and hence, increase sperm viability. Further investigation on these proteins will allow us to evaluate their potential as selective markers indicative of highly fertile and more environmentally compatible bulls for breeding purposes in tropical areas.

During epididymal sperm maturation, spermatozoa acquire progressive motility through dynamic protein modifications. However, the relationship between sequential protein modifications during epididymal sperm maturation and sperm motility and fertility has not yet been investigated. This study investigated whether sequential changes in fertility-related protein expression including that of enolase 1 (ENO1), ubiquinol-cytochrome c reductase core protein 1 and 2 (UQCRC1 and UQCRC2), and voltage-dependent anion channel 2 (VDAC2) in spermatozoa during epididymal maturation are related to bovine sperm motility. Moreover, we found that mitochondrial metabolism is closely related to fertility-related proteins. Therefore, we investigated how the sequential modification of mitochondrial proteins during epididymal maturation regulates sperm motility. Materials and Methods: Materials and Methods: To determine the differential protein expression in caput and cauda epididymal spermatozoa from low and high motility bulls, western blot analysis was performed. Moreover, signaling pathways were identified to understand the mechanisms of regulation of sperm motility through the differential protein expression associated with fertilityrelated proteins. Results: Results: We found that ENO1 was substantially higher in the caput spermatozoa from low motility bulls than the caput and cauda spermatozoa from high motility bulls. However, ENO1 expression in low motility bull spermatozoa was downregulated to a level comparable to that in the high motility bull spermatozoa during epididymal maturation. Moreover, there was a lack of modification of mitochondrial proteins, including glutathione peroxidase 4 and NADH:Ubiquinone Oxidoreductase Core Subunit S8, in low motility bull spermatozoa during epididymal maturation, whereas active changes were detected in high motility bull spermatozoa. Conclusions: Conclusions: Irregular modifications of mitochondrial proteins during epididymal sperm maturation may increase excessive ROS production and premature activation of spermatozoa during epididymal maturation. Consequently, spermatozoa may lose their motility by the earlier consumption of their energy source and may be damaged by ROS during epididymal maturation, resulting in a decline in sperm motility and bull fertility.

P oor sperm motility remains predominantly a clinical sign of infertility, rather than a true diagnosis of the cause of infertility. Considering that sperm motility is one of the essential factors for normal fertility, research on proteins or protein functions related to sperm motility is sparse. However, our current understanding of spermatozoa, including their different physiological and pathological aspects, is still limited and not well defined. Therefore, to explore the biological display of the human genome, the physiological functions of each protein should be understood first. Proteomics involves the comprehensive study of proteins with their particular structural and functional aspects. As such, proteomics has the potential to transform our understanding of how mature sperm cells work. Hence, we proposed to identify all the protein molecules that differ in the normal and non-motile conditions of sperm using proteomics methods and validated by immunocytochemistry. The diagnosis of asthenozoospemia was made in the andrology lab of the department of reproductive biology when the sperm motility was observed to be less than 20% as per WHO recommendations. 50 samples each of non-motile and 50 normal samples of semen were used in this study. Proteomics analysis of asthnozoospermia and fertile individuals were done using standard protocol. After identification of proteins were validated by immunocytochemistry. Our results and reports from others suggest that mammalian spermatozoa need local ATP production for flagellar movement and control of the acrosomal reaction. Hence, we demonstrate that glycolysis is very important for human sperm motility. Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH), Enolase and Pyruvate Kinase (PK) can serve as potential targets for the development of biomarkers and can be used as diagnostic indicators and their potential implications to assist fertility or to develop a male contraceptive strategy.

Fertilization is a stepwise process that allows two mature gametes to reach each other, fuse and eventually give rise to a new individual. Despite the tremendous importance of reproduction for species development and maintenance, fertility is decreasing worldwide, with peaks in western countries. It is estimated that about 7% of men experiences problems in conceiving a child because of sperm defects. In such a situation, understanding which are the essential sperm players in each of the steps of the fertilization process is essential for the development of new pharmacological strategies to treat the infertile men, for genetic screening of idiopathic male infertility as well as to produce effective male contraceptive agents. The present review will summarize recent evidence for the identification and characterization of molecular markers of sperm functions with emphasis on post-ejaculatory maturation events and the process of sperm-oocyte interaction.

Background: Male infertility is increasingly becoming a health and demographic problem. While it may originate from congenital or acquired diseases, it can also result from environmental exposure. Hence, the complexity of involved molecular mechanisms often requires a multiparametric approach. This study aimed to associate semen parameters with sperm DNA fragmentation, chromatin maturity and seminal plasma protein N-glycosylation. Methods: The study was conducted with 166 participants, 20–55 y old, 82 normozoospermic and 84 with pathological diagnosis. Sperm was analyzed by Halosperm assay and aniline blue staining, while seminal plasma total protein N-glycans were analyzed by ultra-high-performance liquid chromatography. Results: Sperm DNA fragmentation was significantly increased in the pathological group and was inversely correlated with sperm motility and viability. Seminal plasma total protein N-glycans were chromatographically separated in 37 individual peaks. The pattern of s...

Purpose: Malate Dehydrogenase is an important enzyme of the Krebs cycle, most cells require this enzyme for their metabolic activity. We evaluated the Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates in normozoospermic, fertile and infertile males. Also glucose and fructose concentrations were determined in the seminal plasma samples. Material and Methods: Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates of normozoospermic and infertile males was determined by spectrophotometric method. Semen analysis was considered according to the WHO Criteria. Results: Malat Dehydrogenase-NAD value in seminal plasma (the mean ± SD, mU/ml) of asthenoteratospermic (40.0±25.7) and azospermic (38.0±43.6) groups were significantly lower than normozoospermic, (93.9±52.1) males. Malat Dehydrogenase-NAD value in sperm homogenates (the mean ± SD, mU/ 20x10 6 sperm) of teratospermic group (136.8±61.8) was significantly higher compared to the normozoospermic (87.3±26.5) males. Glucose concentration (mg/dl) in asthenoteratospermic (4.0±1.4) and azospermic (15.4±6.4) groups were significantly higher than fertile (2.0±2.1) males. Also fructose concentration (mg/dl) in asthenoteratospermic (706.6±143.3) and azospermic (338.1±228.2) groups were significantly high compared to the normozoospermic (184.7±124.8) group. Conclusion: Sperm may be some part of the source of Malat Dehydrogenase activity in semen. Malat Dehydrogenase activity in seminal plasma has an important role on energy metabolism of sperm. Intermediate substrates of Krebs cycle might have been produced under the control of Malat Dehydrogenase and these substrates may be important for sperm motility and male infertility.

Background: Bisphenol S (BPS) is increasingly used as a replacement for bisphenol A in the manufacture of products containing polycarbonates and epoxy resins. However, further studies of BPS exposure are needed for the assessment of health risks to humans. In this study we assessed the potential harmfulness of low-dose BPS on reproduction in male mice. Methods: To simulate human exposure under experimental conditions, 8-week-old outbred ICR male mice received 8 weeks of drinking water containing a broad range of BPS doses [0.001, 1.0, or 100 µg/kg body weight (bw)/day, BPS1-3] or vehicle control. Mice were sacrificed and testicular tissue taken for histological analysis and protein identification by nano-liquid chromatography/mass spectrometry (MS) and sperm collected for immunodetection of acetylated lysine and phosphorylated tyrosine followed by protein characterisation using matrix-assisted laser desorption ionisation time-offlight MS (MALDI-TOF MS). Results: The results indicate that compared to vehicle, 100 µg/kg/day exposure (BPS3) leads to 1) significant histopathology in testicular tissue; and, 2) higher levels of the histone protein γH2AX, a reliable marker of DNA damage. There were fewer mature spermatozoa in the germ layer in the experimental group treated with 1 µg/kg bw (BPS2). Finally, Western blot and MALDI-TOF MS studies showed significant alterations in the sperm acetylome and phosphorylome in mice treated with the lowest (BPS1) exposure (0.001 µg/kg/day). Conclusions: In summary, this range of qualitative and quantitative findings in young male mice raise the possibility that relatively low doses of BPS may impair mammalian reproduction through epigenetic modifications of sperm proteins.

Reduced sperm motility, defined as asthenozoospermia, is a frequent cause of male infertility, and is mainly connected with the dysfunction of sperm mitochondria. The aim of this study was to identify the proteins, and thereby the metabolic pathways, responsible for asthenozoospermia, using 2-DE and MALDI-TOF MS, and correlate the results obtained with those of two mitochondrial tests: JC-1 and MitoSox Red. The JC-1 test was performed to test sperm mitochondrial activity, and the MitoSox Red test was performed to check whether the observed sperm poor motility is associated with mitochondrial reactive oxygen species (ROS) production. To identify proteins strictly connected with reduced sperm motility, men with isolated asthenozoospermia (n = 4 versus 10 normozoospermic controls) alone were included in the study. The proteomic analyses resulted in the identification of 25 sperm proteins that are differentially expressed in asthenozoospermic individuals. Most of the identified proteins...

correlation between CMA3 positive cells and DNA damage was observed when damage was greater than 15% in the OAT and T groups. A negative correlation was found between CMA3 positive cells and sperm morphology. CONCLUSION: Decreased protamination positively correlates with DNA damage that exceeds 15% in OAT and T patients. Sperm DNA damage increases with deteriorating semen parameters. Assessment of sperm DNA damage, especially in oligoasthenoteratozoospermic and asthenoteratozoospermic patients, may be beneficial in assisted reproduction.

OBJECTIVE: Sperm functional alterations are considered the main cellular mechanisms of male infertility, observed in 25% of infertile men. We have previously shown, using a shotgun proteomics approach, that Glutathione S-transferase Mu3 (GSTM3), Cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) and Retinoic acid receptor responder protein 1 (RARRES1) seminal proteins are associated with sperm abnormalities. Current limitations in the quantitative aspect of a shotgun proteomics approach demand these results be validated in a larger cohort. In this study, thus, we sought to evaluate, using a confirmatory method, GSTM3, CRISPLD2 and RARRES1 levels in seminal plasma in association with sperm mitochondrial activity and DNA fragmentation. DESIGN: Prospective study. MATERIALS AND METHODS: 197 normozoospermic men were recruited for this study. Semen was collected by masturbation following 2 to 5 days of ejaculatory abstinence. An aliquot was used for semen analysis, another for sperm functional evaluation, and the remaining semen volume was centrifuged for separation of seminal plasma. Sperm functional data were used to separate the experimental groups: High (control, n¼27) and Low (study, n¼27) sperm mitochondrial activity, and Low (control, n¼29) and High (study, n¼29) sperm DNA fragmentation. Seminal plasma of all samples was utilized to evaluate the levels of GSTM3, CRISPLD2 and RARRES1 by Western blotting. Data were normalized using total protein (Ponceau S staining) and multiplied by the ejaculate volume, to obtain the total protein levels in the ejaculate. Groups were compared using an unpaired Student's t test (a¼5%). RESULTS: GSTM3 was observed as two different bands (27 kDa, its expected molecular mass, and 38 kDa). Total levels of both bands were twice as high in samples with sperm mitochondrial alterations. CRISPLD2 was observed as three different bands (17, 28 and 38 kDa, expected molecular mass ¼ 51 kDa), suggesting its digestion by seminal plasma proteases during semen liquefaction. Total seminal levels of the 17 and 28 kDa bands were decreased in samples with high sperm DNA fragmentation (1.6-fold and 2.6-fold decrease, respectively). Likewise, RARRES1 was identified as two bands (50 and 62 kDa, expected molecular mass ¼ 33 kDa), suggesting post-translational modifications and/or dimer formation. The 50 kDa band was 1.9-fold decreased in the seminal plasma of men with sperm DNA fragmentation. CONCLUSIONS: Seminal plasma proteins directly reflect sperm functional alterations and, thus, male infertility. Seminal plasma proteins may be used as markers of functional alterations. In this study, we confirmed that this is the case for GSTM3 (sperm mitochondrial activity), and CRISPLD2 and RARRES1 (sperm DNA fragmentation).

OBJECTIVE: Sperm functional alterations are considered the main cellular mechanisms of male infertility, observed in 25% of infertile men. We have previously shown, using a shotgun proteomics approach, that Glutathione S-transferase Mu3 (GSTM3), Cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) and Retinoic acid receptor responder protein 1 (RARRES1) seminal proteins are associated with sperm abnormalities. Current limitations in the quantitative aspect of a shotgun proteomics approach demand these results be validated in a larger cohort. In this study, thus, we sought to evaluate, using a confirmatory method, GSTM3, CRISPLD2 and RARRES1 levels in seminal plasma in association with sperm mitochondrial activity and DNA fragmentation. DESIGN: Prospective study. MATERIALS AND METHODS: 197 normozoospermic men were recruited for this study. Semen was collected by masturbation following 2 to 5 days of ejaculatory abstinence. An aliquot was used for semen analysis, another for sperm functional evaluation, and the remaining semen volume was centrifuged for separation of seminal plasma. Sperm functional data were used to separate the experimental groups: High (control, n¼27) and Low (study, n¼27) sperm mitochondrial activity, and Low (control, n¼29) and High (study, n¼29) sperm DNA fragmentation. Seminal plasma of all samples was utilized to evaluate the levels of GSTM3, CRISPLD2 and RARRES1 by Western blotting. Data were normalized using total protein (Ponceau S staining) and multiplied by the ejaculate volume, to obtain the total protein levels in the ejaculate. Groups were compared using an unpaired Student's t test (a¼5%). RESULTS: GSTM3 was observed as two different bands (27 kDa, its expected molecular mass, and 38 kDa). Total levels of both bands were twice as high in samples with sperm mitochondrial alterations. CRISPLD2 was observed as three different bands (17, 28 and 38 kDa, expected molecular mass ¼ 51 kDa), suggesting its digestion by seminal plasma proteases during semen liquefaction. Total seminal levels of the 17 and 28 kDa bands were decreased in samples with high sperm DNA fragmentation (1.6-fold and 2.6-fold decrease, respectively). Likewise, RARRES1 was identified as two bands (50 and 62 kDa, expected molecular mass ¼ 33 kDa), suggesting post-translational modifications and/or dimer formation. The 50 kDa band was 1.9-fold decreased in the seminal plasma of men with sperm DNA fragmentation. CONCLUSIONS: Seminal plasma proteins directly reflect sperm functional alterations and, thus, male infertility. Seminal plasma proteins may be used as markers of functional alterations. In this study, we confirmed that this is the case for GSTM3 (sperm mitochondrial activity), and CRISPLD2 and RARRES1 (sperm DNA fragmentation).

Miscarriage is one of the leading obstetric complications that is known to occur in 15% of all clinically recognised pregnancies (Carlini et al., 2017). However, despite exhibiting normal semen parameters and no female factor abnormality, some couples face an unexpected failure of gestation in the first trimester of conception. Thus, leading to a condition termed as idiopathic recurrent pregnancy loss (RPL). RPL has often been defined by two or more consecutive unexplained pregnancy failures (documented by ultrasonography or histopathologic examination), before the completion of 20th week of gestation

Background: Bisphenol S (BPS) is increasingly used as a replacement for bisphenol A in the manufacture of products containing polycarbonates and epoxy resins. However, further studies of BPS exposure are needed for the assessment of health risks to humans. In this study we assessed the potential harmfulness of low-dose BPS on reproduction in male mice. Methods: To simulate human exposure under experimental conditions, 8-week-old outbred ICR male mice received 8 weeks of drinking water containing a broad range of BPS doses [0.001, 1.0, or 100 µg/kg body weight (bw)/day, BPS1-3] or vehicle control. Mice were sacrificed and testicular tissue taken for histological analysis and protein identification by nano-liquid chromatography/mass spectrometry (MS) and sperm collected for immunodetection of acetylated lysine and phosphorylated tyrosine followed by protein characterisation using matrix-assisted laser desorption ionisation time-offlight MS (MALDI-TOF MS). Results: The results indicate that compared to vehicle, 100 µg/kg/day exposure (BPS3) leads to 1) significant histopathology in testicular tissue; and, 2) higher levels of the histone protein γH2AX, a reliable marker of DNA damage. There were fewer mature spermatozoa in the germ layer in the experimental group treated with 1 µg/kg bw (BPS2). Finally, western blot and MALDI-TOF MS studies showed significant alterations in the sperm acetylome and phosphorylome in mice treated with the lowest exposure (0.001 µg/kg/day; BPS1), although the dose is several times lower than what has been published so far. Conclusions: In summary, this range of qualitative and quantitative findings in young male mice raise the possibility that very low doses of BPS may impair mammalian reproduction through epigenetic modifications of sperm proteins.

BackgroundPrevious studies have demonstrated an association between obesity and the decreased male fertility.Objectiveto observe the mechanisms by which obesity affects semen quality.Materials and methodsA prospective study was performed including 47 male volunteers, of which 27 were obese group (body mass index >30 kg/m2) and 20 were eutrophic (body mass index between 18.5 and 25 kg/m2) controls. Sperm functional analysis was performed. The remaining seminal plasma was pooled—four pools per group— and submitted to proteomic analysis by liquid chromatography coupled to tandem mass spectrometry. Groups were compared by an unpaired Student's t‐test. Differentially expressed proteins were submitted to functional enrichment analysis using the online platform PantherDB.ResultsObese men presented decreased non‐progressive motility, morphology, acrosome integrity, mitochondrial activity, and increased sperm DNA fragmentation. In proteomics analysis, 69 proteins were differentially e...

ODF1 has been described as an exclusively expressed testicular protein and is located in the outer dense fibers along the sperm tail. ODF1 has been involved in the sperm motility and in the development of the flagellum, but the function of ODF1 is not already clear. Other ODF proteins, such as ODF2 have been characterized in other tissues like the basal body of the kidney primary cilium, but so far only the mRNA of ODF1 has been described in other tissues. These observations let us to hypothesize that the expression of the protein ODF1 could not be limited to the testis. Therefore, in the present work we proposed to evaluate if the ODF1 protein could also be present in tissues other than the testis. Here we demonstrated through western blot, immunofluorescence, and RT-PCR techniques that the protein and mRNA of ODF1 have been identified in the rat kidney. Finally, the presence of ODF1 in kidney has also been confirmed through proteomic analysis using mass spectrometry. The results derived from these different complementary approaches indicate that, to our knowledge and for the first time, ODF1 is demonstrated to be present in an additional organ different to testis. This results raise new questions about potential other functions and locations of the ODF1 protein.

Objective: Evidence indicates that an imbalance between the production of reactive oxygen species and defense ability of antioxidants has clinical significance in the pathophysiology of male infertility. To investigate the role of seminal prolactin (PRL) in the fertilizing capacity of men, the present study evaluated the associations of seminal PRL levels with semen parameters and heat shock protein 90 (HSP90) transcript abundance in ejaculated spermatozoa.Methods: We assessed seminal PRL levels and the abundance of HSP90 transcripts in ejaculated spermatozoa from normozoospermic donors (n=18) and infertile men (n=18). The transcript content of HSP90 in ejaculated spermatozoa was analyzed using real-time polymerase chain reaction.Results: Seminal PRL concentrations in infertile patients were significantly lower (p=0.004) than in fertile controls. Seminal PRL showed relatively good diagnostic power for discriminating infertile men (area under the curve=0.776; 95% confidence interval,...

The water buffalo (Bubalus bubalis) is an indispensable part of the Indian dairy sector and in several instances, the farmers incur economic losses due to failed pregnancy after artificial insemination (AI). One of the key factors for the failure of conception is the use of semen from the bulls of low fertilizing potential and hence, it becomes important to predict the fertility status before performing AI. In this study, the global proteomic profile of high fertile (HF) and low fertile (LF) buffalo bull spermatozoa was established using a high-throughput LC-MS/MS technique. A total of 1,385 proteins (≥1 high-quality PSM/s, ≥1 unique peptides, p < 0.05, FDR < 0.01) were identified out of which, 1,002 were common between both the HF and LF groups while 288 and 95 proteins were unique to HF and LF groups respectively. We observed 211 and 342 proteins were significantly high (log Fc ≥ 2) and low abundant (log Fc ≤ 0.5) in HF spermatozoa (p < 0.05). Gene ontology analysis revea...

Despite the explosive increase in the use of Assisted Reproductive Technologies (ART) over the last 30 years, their success rates remain suboptimal. Proteomics is a rapidly-evolving technology-driven science that has already been widely applied in the exploration of human reproduction and fertility, providing useful insights into its physiology and leading to the identification of numerous proteins that may be potential biomarkers and/or treatment targets of a successful ART pregnancy. Here we present a brief overview of the techniques used in proteomic analyses and attempt a comprehensive presentation of recent data from mass spectrometry-based proteomic studies in humans, regarding all components of ARTs, including the male and female gamete, the derived zygote and embryo, the endometrium and, finally, the ART offspring both pre- and postnatally.

Aim: Infertility is a global problem affecting both males and females. It is a condition with psychological, economic, and medical implications resulting in trauma and stress, particularly in a society where there is a strong emphasis on childbearing. The study therefore aims to investigate the pattern of some sperm proteins, anti-oxidants, and prostate-specific antigen in the seminal plasma of infertile males with sperm cell deformities. Study Design: The study is a case-control study designed to investigate semen parameters and sperm proteins in infertile males with sperm cell deformities in Port Harcourt, Rivers State. Place and Duration of Study: The study was carried out in the Department of Medical Laboratory Science, Rivers State University, Port Harcourt. However, some of the Laboratory investigations were done in the Chemical Pathology Unit and Medical Microbiology Unit of the University of Port Harcourt Teaching Hospital, Port Harcourt. The Study was between the periods of...

Hyperactive sperm motility is important for successful fertilization. In the present study, a proteome profiling approach was performed to identify the differences between Landrace boars with different levels of hyperactive sperm motility in liquid extended semen. Two contrasts were studied: (i) high versus low levels of sperm hyperactivity at semen collection day and (ii) high versus low change in levels of sperm hyperactivity after 96 h semen storage. Testicular samples were analyzed on a Q Exactive mass spectrometer and more than 6000 proteins were identified in the 13 samples. The most significant differentially expressed proteins were mediator complex subunit 28 (MED28), cell division cycle 37 like 1 (CDC37L1), ubiquitin specific peptidase 10 (USP10), zinc finger FYVE-type containing 26 (ZFYVE26), protein kinase C delta (PRKCD), actinin alpha 4 (ACTN4), N(alpha)-acetyltransferase 30 (NAA30), C1q domain-containing (LOC110258309) and uncharacterized LOC100512926. Of the different...

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma * **

Purpose: Malate Dehydrogenase is an important enzyme of the Krebs cycle, most cells require this enzyme for their metabolic activity. We evaluated the Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates in normozoospermic, fertile and infertile males. Also glucose and fructose concentrations were determined in the seminal plasma samples. Material and Methods: Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates of normozoospermic and infertile males was determined by spectrophotometric method. Semen analysis was considered according to the WHO Criteria. Results: Malat Dehydrogenase-NAD value in seminal plasma (the mean ± SD, mU/ml) of asthenoteratospermic (40.0±25.7) and azospermic (38.0±43.6) groups were significantly lower than normozoospermic, (93.9±52.1) males. Malat Dehydrogenase-NAD value in sperm homogenates (the mean ± SD, mU/ 20x10 6 sperm) of teratospermic group (136.8±61.8) was significantly higher compa...

Testicular angiotensin converting enzyme (ACE) is known to play an essential role in the male reproduction and fertility. Data about tACE in cases of male infertility are quite scarce, and in this respect we aimed to study localization and distribution of tACE protein in the neck and mid-piece of spermatozoa from pathological samples in relation to sperm motility. The enzyme expression during capacitation and acrosome reaction was quantitatively assessed. In human ejaculated spermatozoa tACE is localized on sperm plasma membrane of the head, the neck and mid-piece of the tail. The immunoreactivity becomes stronger in capacitated spermatozoa followed by a decrease in acrosome reacted sperm. In different cases of semen pathology (oligozoospermia, asthenozoospermia and teratozoospermia) fluorescent signals in the neck and mid-piece are in punctate manner whereas in normozoospermia they were uniformly distributed. The expression area of tACE the neck and mid-piece was decreased in ejacu...

Background: Previous studies have demonstrated an association between obesity and the decreased male fertility. Objective: to observe the mechanisms by which obesity affects semen quality. Materials and methods: A prospective study was performed including 47 male volunteers, of which 27 were obese group (body mass index >30 kg/m 2) and 20 were eutrophic (body mass index between 18.5 and 25 kg/m 2) controls. Sperm functional analysis was performed. The remaining seminal plasma was pooled-four pools per group-and submitted to proteomic analysis by liquid chromatography coupled to tandem mass spectrometry. Groups were compared by an unpaired Student's t-test. Differentially expressed proteins were submitted to functional enrichment analysis using the online platform PantherDB. Results: Obese men presented decreased non-progressive motility, morphology, acrosome integrity, mitochondrial activity, and increased sperm DNA fragmentation. In proteomics analysis, 69 proteins were differentially expressed between the two groups. Among them, one protein was absent, 19 were down-regulated, 49 were up-regulated, and one was exclusive in the study group. The main functions enriched were as follows: negative regulation of the intrinsic pathway of apoptosis, activation of immune and inflammatory, antioxidant activity, among others. Conclusion: molecular pathways suggest there is a causative link, and that the effector mechanisms alter sperm metabolic status and defective testicular selection 5 mechanisms.

Seminal plasma is a complex fluid comprised of secretions from the seminal vesicles, the prostate, bulbourethral glands and from the seminiferous tubule lumen / epididymides / vasa deferentia. While it has been established that seminal plasma serves not only as a medium to carry, protect, and nourish sperm after ejaculation up to fertilization, but also as a functional modulator of sperm function, there is still a need to properly characterize the molecular make-up of seminal plasma in fertile men, and to understand how this is altered in different causes of male infertility. The main purpose of this manuscript was to review articles that studied the human seminal plasma proteome, ranging from characterizing a fertile seminal plasma proteomic map to studies comparing seminal plasma from fertile and infertile men, and comparing seminal plasma of fertile or normozoospermic men to a diverse range of biological causes for male infertility. Finally, this review has focused on the association between semen and sperm functional quality and the seminal plasma proteome, in order to demonstrate cellular and molecular mechanisms of male infertility. Due to the untargeted nature of the majority of the studies presented in this review, and to the diverse range of techniques utilized to study the seminal plasma proteomic composition, many differentially expressed proteins were observed. However, in general, it seems that there is a seminal plasma proteome associated to male fertility, and that different biological conditions or cellular phenotypes shift its pathways away from its homeostatic condition to altered energy production pathways. Moreover, it seems there is an inflammatory component to the seminal plasma of infertile men. In conclusion, there are a number of studies focused on the proteomic composition of human seminal plasma; downstream confirmatory studies will help to understand specific pathways of infertility in different biological conditions.

The study of male infertility after spinal cord injury (SCI) has enhanced the understanding of seminal plasma (SP) as an important regulator of spermatozoa function. However, the most important factors leading to the diminished sperm motility and viability observed in semen of men with SCI remained unknown. Thus, to explore SP related molecular mechanisms underlying infertility after SCI we used mass spectrometry-based quantitative proteomics to compare SP retrieved from SCI patients to normal controls. As a result, we present an in-depth characterization of the human SP proteome, identifying ~2,800 unique proteins, and describe, in detail, the differential proteome observed in SCI. Our analysis demonstrates that a hyper-activation of the immune system may influence some seminal processes, which probably are not triggered by microbial infection. Moreover, we show evidence of an important prostate gland functional failure, i.e. diminished abundance of metabolic enzymes related to ATP...

Background Bisphenol S (BPS) is increasingly used as a replacement for bisphenol A in the manufacture of products containing polycarbonates and epoxy resins. However, further studies of BPS exposure are needed for the assessment of health risks to humans. In this study we assessed the potential harmfulness of low-dose BPS on reproduction in male mice. Methods To simulate human exposure under experimental conditions, 8-week-old outbred ICR male mice received 8 weeks of drinking water containing a broad range of BPS doses [0.001, 1.0, or 100 μg/kg body weight (bw)/day, BPS1–3] or vehicle control. Mice were sacrificed and testicular tissue taken for histological analysis and protein identification by nano-liquid chromatography/mass spectrometry (MS) and sperm collected for immunodetection of acetylated lysine and phosphorylated tyrosine followed by protein characterisation using matrix-assisted laser desorption ionisation time-of-flight MS (MALDI-TOF MS). Results The results indicate t...

Background During maturation, spermatozoa acquire motility and fertilizing capacity as they transit through the epididymis. In recent years, two-dimensional gel electrophoresis has been employed in proteomics studies conducted in rat, boar and human. However, there has not been a complete information regarding the proteins associated with sperm maturation in the epididymis. In this study, we employed iTRAQ proteomics to investigate proteins associated with sperm maturation between yak and cattleyak epididymis. Results After a successful sampling and protein extraction, the iTRAQ coupled with LC-MS/MS mass spectrometry and bioinformatics analysis were performed. We identified 288 differentially abundant proteins (DAPs) between yak and cattleyak epididymis; 151 were up-regulated while 137 were down-regulated in cattleyak relative to yak. Gene Ontology analysis identified that down-regulated DAPs in cattleyak were mostly enriched in the acetylation of protein component, along with nega...

Background During maturation, spermatozoa acquire motility and fertilizing capacity as they transit through the epididymis. In recent years, two-dimensional gel electrophoresis has been employed in proteomics studies conducted in rat, boar and human. However, there has not been a complete information regarding the proteins associated with sperm maturation in the epididymis. In this study, we employed iTRAQ proteomics to investigate proteins associated with sperm maturation between yak and cattleyak epididymis. Results After a successful sampling and protein extraction, the iTRAQ coupled with LC-MS/MS mass spectrometry and bioinformatics analysis were performed. We identified 288 differentially abundant proteins (DAPs) between yak and cattleyak epididymis; 151 were up-regulated while 137 were down-regulated in cattleyak relative to yak. Gene Ontology analysis identified that down-regulated DAPs in cattleyak were mostly enriched in the acetylation of protein component, along with nega...

Validation by sperm motility evaluation The significant higher expression of sperm flagella components ODF protein and Tektin 5 could result in an increase in sperm tail elasticity and strength in KK animals. Furthermore, the energy related enzyme Triose Phosphate Isomerase is significantly down regulated in Mafriwal. Stronger flagella and a greater energy supply in KK animals may have contributed to the increase in motile and fast moving sperm. In addition, more elastic flagella resulted in the higher flagella lateral amplitudes in KK. The Fibrous Sheet component 2, as a homologue of Glutathione S-transferase MU3, is up-regulated in KK and may be required to protect sperm against toxins and hence, increase sperm viability. Further investigation on these proteins will allow us to evaluate their potential as selective markers indicative of highly fertile and more environmentally compatible bulls for breeding purposes in tropical areas.

During epididymal sperm maturation, spermatozoa acquire progressive motility through dynamic protein modifications. However, the relationship between sequential protein modifications during epididymal sperm maturation and sperm motility and fertility has not yet been investigated. This study investigated whether sequential changes in fertility-related protein expression including that of enolase 1 (ENO1), ubiquinol-cytochrome c reductase core protein 1 and 2 (UQCRC1 and UQCRC2), and voltage-dependent anion channel 2 (VDAC2) in spermatozoa during epididymal maturation are related to bovine sperm motility. Moreover, we found that mitochondrial metabolism is closely related to fertility-related proteins. Therefore, we investigated how the sequential modification of mitochondrial proteins during epididymal maturation regulates sperm motility. Materials and Methods: Materials and Methods: To determine the differential protein expression in caput and cauda epididymal spermatozoa from low and high motility bulls, western blot analysis was performed. Moreover, signaling pathways were identified to understand the mechanisms of regulation of sperm motility through the differential protein expression associated with fertilityrelated proteins. Results: Results: We found that ENO1 was substantially higher in the caput spermatozoa from low motility bulls than the caput and cauda spermatozoa from high motility bulls. However, ENO1 expression in low motility bull spermatozoa was downregulated to a level comparable to that in the high motility bull spermatozoa during epididymal maturation. Moreover, there was a lack of modification of mitochondrial proteins, including glutathione peroxidase 4 and NADH:Ubiquinone Oxidoreductase Core Subunit S8, in low motility bull spermatozoa during epididymal maturation, whereas active changes were detected in high motility bull spermatozoa. Conclusions: Conclusions: Irregular modifications of mitochondrial proteins during epididymal sperm maturation may increase excessive ROS production and premature activation of spermatozoa during epididymal maturation. Consequently, spermatozoa may lose their motility by the earlier consumption of their energy source and may be damaged by ROS during epididymal maturation, resulting in a decline in sperm motility and bull fertility.

Validation by sperm motility evaluation The significant higher expression of sperm flagella components ODF protein and Tektin 5 could result in an increase in sperm tail elasticity and strength in KK animals. Furthermore, the energy related enzyme Triose Phosphate Isomerase is significantly down regulated in Mafriwal. Stronger flagella and a greater energy supply in KK animals may have contributed to the increase in motile and fast moving sperm. In addition, more elastic flagella resulted in the higher flagella lateral amplitudes in KK. The Fibrous Sheet component 2, as a homologue of Glutathione S-transferase MU3, is up-regulated in KK and may be required to protect sperm against toxins and hence, increase sperm viability. Further investigation on these proteins will allow us to evaluate their potential as selective markers indicative of highly fertile and more environmentally compatible bulls for breeding purposes in tropical areas.

Background: The Co-word analysis has the ability to identify the intellectual structure of knowledge in a research domain and reveal its subsurface research aspects. Objective: This study examines the intellectual structure of knowledge in the field of Andrology during the period 2008-2017 using Co-word analysis. Materials and Methods: In this descriptive-analytical study with a scientometric approach, the WoS database was searched for papers indexed under “Andrology” over the period 2008–2017. The data were analyzed using Co-word, clustering methods, and strategic diagram with the help of SPSS, UcInet, RavarPreMap and VOSviewer software. Results: The highest publication rate in the area of Andrology was seen in countries like the USA, China, Italy, and Iran. The top three journals that published papers on the field were Fertility and Sterility, Andrologia, Human reproduction. The results showed that the keyword “Spermatozoa” and two pairs of frequently used keywords, namely “Azoosp...

Background: The human seminal fluid is a complex body fluid. It is not known how many proteins are expressed in the seminal plasma; however in analog with the blood it is possible up to 10,000 proteins are expressed in the seminal plasma. The human seminal fluid is a rich source of potential biomarkers for male infertility and reproduction disorder. Methods: In this review, the ongoing list of proteins identified from the human sem-inal fluid was collected. To date, 4188 redundant proteins of the seminal fluid are identified using different proteomics technology, including 2-DE, SDS-PAGE-LC-MS/MS, MudPIT. However, this was reduced to a database of 2168 non-redundant protein using UniProtKB/Swiss-Prot reviewed database. Results: The core concept of proteome were analyzed including pI, MW, Amino Acids, Chromosome and PTM distribution in the human seminal plasma proteome. Additionally, the biological process, molecular function and KEGG pathway were investigated using DAVID software. F...

The human seminal fluid is a complex body fluid. It is not known how many proteins are expressed in the seminal plasma; however in analog with the blood it is possible up to 10,000 proteins are expressed in the seminal plasma. The human seminal fluid is a rich source of potential biomarkers for male infertility and reproduction disorder. In this review, the ongoing list of proteins identified from the human seminal fluid was collected. To date, 4188 redundant proteins of the seminal fluid are identified using different proteomics technology, including 2-DE, SDS-PAGE-LC-MS/MS, MudPIT. However, this was reduced to a database of 2168 non-redundant protein using UniProtKB/Swiss-Prot reviewed database. The core concept of proteome were analyzed including pI, MW, Amino Acids, Chromosome and PTM distribution in the human seminal plasma proteome. Additionally, the biological process, molecular function and KEGG pathway were investigated using DAVID software. Finally, the biomarker identifie...

Purpose: Malate Dehydrogenase is an important enzyme of the Krebs cycle, most cells require this enzyme for their metabolic activity. We evaluated the Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates in normozoospermic, fertile and infertile males. Also glucose and fructose concentrations were determined in the seminal plasma samples. Material and Methods: Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates of normozoospermic and infertile males was determined by spectrophotometric method. Semen analysis was considered according to the WHO Criteria. Results: Malat Dehydrogenase-NAD value in seminal plasma (the mean ± SD, mU/ml) of asthenoteratospermic (40.0±25.7) and azospermic (38.0±43.6) groups were significantly lower than normozoospermic, (93.9±52.1) males. Malat Dehydrogenase-NAD value in sperm homogenates (the mean ± SD, mU/ 20x10 6 sperm) of teratospermic group (136.8±61.8) was significantly higher compa...

Post-translational modifications (PTMs) can occur soon after translation or at any stage in the lifecycle of a given protein, and they may help regulate protein folding, stability, cellular localisation, activity, or the interactions proteins have with other proteins or biomolecular species. PTMs are crucial to our functional understanding of biology, and new quantitative mass spectrometry (MS) and bioinformatics workflows are maturing both in labelled multiplexed and label-free techniques, offering increasing coverage and new opportunities to study human health and disease. Techniques such as Data Independent Acquisition (DIA) are emerging as promising approaches due to their re-mining capability. Many bioinformatics tools have been developed to support the analysis of PTMs by mass spectrometry, from prediction and identifying PTM site assignment, open searches enabling better mining of unassigned mass spectra-many of which likely harbour PTMs-through to understanding PTM associati...

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.

Glycan-protein interactions play a key role in mammalian fertilization, but data on the composition and identities of protein complexes involved in fertilization events are scarce, with the added complication that the glycans in such interactions tend to differ among species. In this study we have used a bovine model to detect, characterize and identify sperm lectins relevant in fertilization. Given the complexity of the sperm-towards-egg journey, two important aspects of the process, both primarily mediated by protein-sugar interactions, have been addressed: (i) formation of the sperm reservoir in the oviductal epithelium, and (ii) gamete recognition (oocyte-sperm interaction). Using whole sperm cells and a novel affinity capture method, several groups of proteins with different glycan specificities, including 58 hitherto unreported as lectins, have been identified in sperm surface, underscoring both the efficacy of our selective approach and the complex composition and function of...

The use of extracellular or circulating nucleic acids (cfDNA), as a diagnostic or prognostic tool in oncology, has been widely documented. However, their use in gynecology and obstetrics as non-invasive biomarkers in the management of infertility has become recurrent. Circulating nucleic acids are made up of: free DNA which can be a long or short strand of DNA resulting from apoptotic or necrotic processes, free RNA containing it: micro-RNAs (miRNA) which are ribonucleic acids (RNA) short single-stranded capable of inhibiting the production of proteins from a gene, Piwi interacting RNAs (piRNAs) which are small RNAs expressed in germ cells or even in early embryos, and small RNAs interfering (siRNA) which are small RNAs capable of binding specifically to a messenger RNA sequence and of preventing gene expression by cleaving that RNA. The presence of circulating nucleic acids in many biological fluids such as urine, seminal plasma and serum, the fact that they are easy to detect, the...

OBJECTIVE: Sperm functional alterations are considered the main cellular mechanisms of male infertility, observed in 25% of infertile men. We have previously shown, using a shotgun proteomics approach, that Glutathione S-transferase Mu3 (GSTM3), Cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) and Retinoic acid receptor responder protein 1 (RARRES1) seminal proteins are associated with sperm abnormalities. Current limitations in the quantitative aspect of a shotgun proteomics approach demand these results be validated in a larger cohort. In this study, thus, we sought to evaluate, using a confirmatory method, GSTM3, CRISPLD2 and RARRES1 levels in seminal plasma in association with sperm mitochondrial activity and DNA fragmentation. DESIGN: Prospective study. MATERIALS AND METHODS: 197 normozoospermic men were recruited for this study. Semen was collected by masturbation following 2 to 5 days of ejaculatory abstinence. An aliquot was used for semen analysis, another for sperm functional evaluation, and the remaining semen volume was centrifuged for separation of seminal plasma. Sperm functional data were used to separate the experimental groups: High (control, n¼27) and Low (study, n¼27) sperm mitochondrial activity, and Low (control, n¼29) and High (study, n¼29) sperm DNA fragmentation. Seminal plasma of all samples was utilized to evaluate the levels of GSTM3, CRISPLD2 and RARRES1 by Western blotting. Data were normalized using total protein (Ponceau S staining) and multiplied by the ejaculate volume, to obtain the total protein levels in the ejaculate. Groups were compared using an unpaired Student's t test (a¼5%). RESULTS: GSTM3 was observed as two different bands (27 kDa, its expected molecular mass, and 38 kDa). Total levels of both bands were twice as high in samples with sperm mitochondrial alterations. CRISPLD2 was observed as three different bands (17, 28 and 38 kDa, expected molecular mass ¼ 51 kDa), suggesting its digestion by seminal plasma proteases during semen liquefaction. Total seminal levels of the 17 and 28 kDa bands were decreased in samples with high sperm DNA fragmentation (1.6-fold and 2.6-fold decrease, respectively). Likewise, RARRES1 was identified as two bands (50 and 62 kDa, expected molecular mass ¼ 33 kDa), suggesting post-translational modifications and/or dimer formation. The 50 kDa band was 1.9-fold decreased in the seminal plasma of men with sperm DNA fragmentation. CONCLUSIONS: Seminal plasma proteins directly reflect sperm functional alterations and, thus, male infertility. Seminal plasma proteins may be used as markers of functional alterations. In this study, we confirmed that this is the case for GSTM3 (sperm mitochondrial activity), and CRISPLD2 and RARRES1 (sperm DNA fragmentation).

Mitochondria are structurally and functionally distinct organelles that produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS), to provide energy to spermatozoa. They can also produce reactive oxidation species (ROS). While a moderate concentration of ROS is critical for tyrosine phosphorylation in cholesterol efflux, sperm–egg interaction, and fertilization, excessive ROS generation is associated with male infertility. Moreover, mitochondria participate in diverse processes ranging from spermatogenesis to fertilization to regulate male fertility. This review aimed to summarize the roles of mitochondria in male fertility depending on the sperm developmental stage (from male reproductive tract to female reproductive tract). Moreover, mitochondria are also involved in testosterone production, regulation of proton secretion into the lumen to maintain an acidic condition in the epididymis, and sperm DNA condensation during epididymal maturation. We also establis...