Seminal Plasma Proteins Research Articles

Addition ofseminal plasma proteins effecting theinvitro kinetic properties ofcanine spermatozoa.

The objective ofthis study isto evaluate the changes inthe motility and kinetic patterns ofcanine spermatozoa, capacitated and decapacitated, after the addition ofseminal plasma protein fractions with different molecular weight. Ithas been proposed that proteins inseminal plasma support the survival ofthe spermatozoa and exert adual effect: capacitation and decapacitation. The seminal plasma from fresh ejaculates was subjected tochromatographic separation and four protein fractions were obtained. Computer-assisted sperm analysis was used todetermine the sperm subpopulations with specific motion and kinetic characteristics after incubation with each ofthe four protein fractions. Two-dimensional electrophoresis ofthe fractions that exhibit asignificant effect onthecapacitation and decapacitation was performed. Bysperm class analyser, capacitation changes were observed inthe sperm subpopulation with ahigh curvilinear velocity and amplitude oflateral head displacement incubatedwith the seminal plasma protein fraction with ahigh molecular weight, which was also reflected inthedecreased linearity, straightness, and progressive motility. The sperm subpopulation incubated with theseminal plasma protein fraction with alow molecular weight seemed toundergo aprocess ofdecapacitation (decreasing ofthe curvilinear velocity, increasing ofthe linearity, straightness and showing progressive motility). Despite their ample panorama ofactions, the role ofseminal plasma proteins regarding capacitation and decapacitation isstill undetermined.

Functional Analysis of KLK3/PSA Protease Activity in Primates

Male reproductive proteins have been the focus of evolutionary biologists due to their rapid evolutionary rates compared to other proteins in humans. However, most of the studies on their accelerated evolution and selective forces behind it rely on DNA sequencing data. The objective of this study is to experimentally compare the function of one of these reproductive proteins in humans and our close evolutionary relatives to analyze the functional consequences of amino acid substitutions, which can then help us understand the possible factors contributing to its selection. To accomplish this aim, one of the most abundant seminal plasma proteins, kallikrein‐related peptidase 3 (KLK3) was used. KLK3 is also known as prostate‐specific antigen (PSA). Its physiological function is to liquefy the seminal coagulum, an important step for fertilization, releasing sperm cells for active motility. The formation of seminal coagulum is considered as a sperm competition strategy. Higher sperm competition was found to be correlated with more nonsynonymous substitutions in many seminal proteins. Therefore, their adaptive evolution is commonly attributed to sexual selection through post‐copulatory sperm competition. Previous studies showed that several codons from KLK3 orthologs have been evolving under positive selection and there is variation in dN/dS ratios among branches of primates, these were also confirmed by our multiple sequence alignment data. Based on the literature, the equivalent of the human KLK3 gene is found as chimeric KLK (cKLK, fusion of KLK2 and KLK3) in gorillas and gibbons. However, it has not yet been clarified whether the cKLK is enzymatically active or not. Considering the role of KLK3 in dissolution of seminal coagulum, its chimeric form might be related to the mating systems of gorillas and gibbons, who likely experience very low sperm competition. We hypothesize that amino acid substitutions have caused changes in KLK3 enzymatic activity across species with varying levels of sperm competition. To test this hypothesis, coding DNA sequences of KLK3/cKLK from human, chimpanzee, their hypothetical ancestor, gorilla, gibbon, and macaque were cloned into a mammalian expression vector with a C‐terminal His6‐tag. All these recombinant proteins were successfully expressed in HEK‐293T cells and purified using Nickel columns. After purification, they were treated with thermolysin to cleave the activation sequence from the core domain of KLK3 and subsequently analyzed for the protease activity on a synthetic fluorescent PSA substrate. Varying levels of enzyme activity were observed between species, supporting our hypothesis such that even a few substitutions were sufficient to cause changes in enzymatic activity. Enzyme kinetics (Kcat/Km) were also calculated for each species’ KLK3 protease activity. However, our preliminary data did not reveal a consistent trend of enzymatic activity in species with similar levels of sperm competition. This suggests that other potential selective pressures such as pathogen response and coevolution with its substrate could also be responsible for KLK3 adaptation along with sexual selection.

High throughput deep proteomic analysis of seminal plasma from stallions with contrasting semen quality

Seminal plasma proteins and pathways associated with sperm motility have not been elucidated in stallions. Therefore, in the current study, using the high throughput LC/MS-MS approach, we profiled stallion seminal plasma proteins and identified the proteins and pathways associated with sperm motility. Seminal plasma from six stallions producing semen with contrasting sperm motility (n = 3 each high-and low-motile group) was utilized for proteomic analysis. We identified a total of 1687 proteins in stallion seminal plasma, of which 1627 and 1496 proteins were expressed in high- (HM) and low- motile (LM) sperm of stallions, respectively. A total number of 1436 proteins were co-expressed in both the groups; 191 (11%) and 60 (3.5%) proteins were exclusively detected in HM and LM groups, respectively. A total of 220 proteins were upregulated (>1-fold change) and 386 proteins were downregulated in SP from LM group stallions as compared to HM group stallions, while 830 proteins were neutrally expressed in both the groups. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed dysregulation of the important proteins related to mitochondrial function, acrosome, and sperm cytoskeleton in the seminal plasma of stallions producing ejaculates with low sperm motility. High abundance of peroxiredoxins and low abundance of seminal Chaperonin Containing TCP1 Complex (CCT) complex and Annexins indicate dysregulated oxidative metabolism, which might be the underlying etiology for poor sperm motility in LM group stallions. In conclusion, the current study identified the seminal plasma proteomic alterations associated with poor sperm motility in stallions; the results indicate that poor sperm motility in stallions could be associated with altered expression of seminal plasma proteins involved in oxidative metabolism.

Baicalin Attenuates Continuous Activation of β-Catenin Induced by Lipopolysaccharide (LPS) and Depression Complicated by Infertility in Male Rats.

Background Baicalin (BA) is a potential candidate drug to inhibit depressive behavior. However, the mechanism of BA's role on depression complicated with male infertility (DCMI) is still unclear. This study aimed to investigate the role of BA in alleviating inflammatory factor-induced DCMI by regulating β-catenin. Methods Firstly, we performed sucrose preference test (SPT), open field test (OFT), tail suspension test (TST), and forced swim test (FST) in the chronic unpredictable mild stress (CUMS) + lipopolysaccharide (LPS) model rats to study the effect of BA on depressive behavior. The levels of neuropeptide Y (NPY), testosterone (T), and IL-1β, IL-6, TNF-α, IL-10, and IL-4 in the peripheral blood plasma of normal people, patients with depression, and patients with DCMI were measured. Then, the levels of IL-1β, IL-6, TNF-α, IL-10, IL-4, β-catenin in rat testis and peripheral blood and ANXA2, APP, SEMG1, and SEMG2 in seminal plasma proteins were examined. Moreover, the level of β-catenin in the testicular tissue was detected. LPS was used to treat Sertoli cells, and the level of β-catenin was detected. Finally, we evaluated the reproductive phenotype and sperm motility of rats. Results BA (especially 100 mg/kg) could notably ameliorate depression-like behavior induced by CUMS + LPS. The levels of IL-4, IL-10, T, and NPY in depression patients, DCMI patients, and CUMS + LPS model rats elevated, while the levels of IL-1β, IL-6, and TNF-α were reduced. However, BA alleviated the changes in these factors. Moreover, BA alleviated male rat depression induced by CUMS + LPS. LPS upregulated β-catenin (NP) but could not adjust β-catenin (TP) level in rat Sertoli cells. BA relieved the symptoms of DCMI by regulating β-catenin. Furthermore, BA ameliorated the reproductive ability of depressed rats. Conclusion BA modulated β-catenin in the relief of inflammatory factor-induced DCMI.

Evaluation of dietary betaine on post-thawed semen quality in mature bulls during summer heat stress.

Heat stress (HS) has caused relative hypoxia, oxidative stress and high level of homocysteine, which contributes significantly to fertility failures in bulls. The aim of present study was to evaluate the role of dietary betaine (BET) in improving dual purpose Simmental (Fleckvieh) post-thawed semen quality especially during the hottest summer days. A total number of 16 mature bulls were randomly assigned to three equal groups including: 1) Control condition (without betaine), 2) BET1: 57.00 mg of betaine kg-1 per day and 3) BET2: 114 mg of betaine kg-1 per day, through daily intakes for 90 days in summer. Plasma levels of homocysteine, seminal plasma antioxidants levels and sperm parameters such as DNA fragmentation, chromatin integrity, motility, viability, morphology and membrane integrity were evaluated. Under maximal HS, serum homocysteine concentrations were reached 16.67 ± 0.09 µmol L-1. Dietary betaine supplementation influenced DNA fragmentation of sperm and was higher in the control group compared to BET2 group. There were significant decreases in seminal plasma superoxide dismutase (SOD), glutathione peroxidase (GPx) activity and sperm viability and motility in bulls treated with betaine. The activity of GPx and SOD in the control group was increased up to 0.08 ± 0.00 U mg-1 protein and 0.52 ± 0.01 U mg-1 protein in seminal plasma. There were no significant differences between groups in the percentage of swollen spermatozoa, membrane integrity, sperm morphology, abnormal head morphology and percentage of spermatozoa stained with aniline blue. In conclusion, BET supplements improved semen parameters in sperm motility, sperm viability and influenced DNA fragmentation during HS with reduction in serum homocysteine concentrations.

What is the relevance of seminal plasma from a functional and preservation perspective?

When preserving sperm in the liquid or cryopreserved state, seminal plasma (SP) components within ejaculates can alter fertilizing capacity of these gametes. Depending on the species or how semen is collected, volume and concentration of SP components varies considerably. The SP contains substances essential for maintenance of sperm viability and fertility; however, these components can be deleterious depending on quantity, or duration of time before there is removal of SP from sperm in semen processing. Substances that impair (e.g., BSP – bull; HSP-1 – stallion; Major seminal plasma protein PSPI - boar) or improve (e.g., spermadhesin PSP-I - boar) spermatozoa fertilizing capacity have been identified. Depending on individual males, species, and semen collection procedures, SP removal may be beneficial before preservation in the liquid or cryopreserved state. In some cases, SP that is removed can be added back to thawing extender with there being positive effects in thawed sperm and for sperm viability in the female reproductive tract. In this review article, there is a focus on different effects of SP in samples of cooled and cryopreserved semen from four domestic species (pigs, horses, cattle, and sheep) with there being emphasis on how SP modulates the function and morphology of sperm cells before, during, and after preservation in the refrigerated or cryopreserved state. The present review is part of the Festschrift in honor of Dr. Duane Garner who made major contributions to the area of focus in this manuscript as evidenced by the many times his research is cited in this manuscript.

Insights into the influence of canine breed on proteomics of the spermatozoa and seminal plasma

This study aimed to characterize the proteome of spermatozoa and seminal plasma of 4 purebred dogs (Golden Retriever, Great Dane, Bernese Mountain Dog, and Maremmano-Abruzzese Sheepdog). The ejaculate of 13 dogs was collected, and sperm characteristics were subjectively evaluated. Seminal plasma and sperm cells were separated and prepared individually for mass spectrometry. Data were evaluated by univariate and multivariate statistical analysis. A total of 162 proteins were identified, 47 in spermatozoa, 109 in seminal plasma, and 6 in both samples. Serum albumin in spermatozoa and tubulin alpha-3E chain, acrosin binding protein, and tubulin alpha-3 chain in plasma seminal were statistically relevant. Serum albumin and acrosin binding protein improve the sperm capacitation, acrosome reaction, and seminal quality. The tubulin family proteins are related to structural cell organization and flagella movement, and their presence in seminal plasma may be related to sample handling. According to cluster formation, a high association was observed among Bernese Mountain Dog and Great Dane, Golden Retriever, and Maremmano-Abruzzese Sheepdog for sperm proteins. For seminal plasma proteins, Bernese Mountain Dog, Great Dane, and Maremmano-Abruzzese Sheepdog were related. Further studies on breed-specific proteins in the semen of purebred dogs need to be performed to clarify its fertility roles. SignificanceFor the first time spermatozoa proteins of dogs are described. The comparison of spermatozoa and seminal plasma proteins of four purebred dogs were performed. These results supporting that differences in semen protein profile of different canine breeds exist, which can improve the biotechnologies of reproduction in this species.

Prostate secretory protein 94 inhibits sterol binding and export by the mammalian CAP protein CRISP2 in a calcium-sensitive manner

Members of the CAP protein superfamily are present in all kingdoms of life and have been implicated in many different processes, including pathogen defense, immune evasion, sperm maturation, and cancer progression. Most CAP proteins are secreted glycoproteins and share a unique conserved αβα sandwich fold. The precise mode of action of this class of proteins, however, has remained elusive. Saccharomyces cerevisiae has three CAP family members, termed pathogen related in yeast (Pry). We have previously shown that Pry1 and Pry2 export sterols in vivo and that they bind sterols in vitro. This sterol binding and export function of yeast Pry proteins is conserved in the mammalian CRISP proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with prostate secretory protein of 94 amino acids (PSP94), another major protein component in the seminal plasma. Here we examine whether the interaction between CRISP proteins and PSP94 affects the sterol binding function of CAP family members. We show that coexpression of PSP94 with CAP proteins in yeast abolished their sterol export function and the interaction between PSP94 and CAP proteins inhibits sterol binding in vitro. In addition, mutations that affect the formation of the PSP94–CRISP2 heteromeric complex restore sterol binding. Of interest, we found the interaction of PSP94 with CRISP2 is sensitive to high calcium concentrations. The observation that PSP94 modulates the sterol binding function of CRISP2 in a calcium-dependent manner has potential implications for the role of PSP94 and CRISP2 in prostate physiology and progression of prostate cancer.

Proteomic analysis of spermatozoa reveals caseins play a pivotal role in preventing short-term periods of subfertility in stallions†.

Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14days postbreeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations and to determine the biological mechanisms governing such differences. Using liquid chromatography-mass spectrometry (LC-MS/MS), we compared the proteomic profile of semen samples collected from commercially "fertile" stallions, during high- and low-fertility periods. A total of 1702 proteins were identified, of which, 38 showed a significant change in abundance (P ≤ 0.05). Assessment of intra- and interstallion variability revealed that caseins (namely κ-, α-S1-, and α-S2-casein) were significantly more abundant during "high-fertility" periods, while several epididymal, and seminal plasma proteins (chiefly, epididymal sperm binding protein 1 [ELSPbP1], horse seminal plasma protein 1 [HSP-1], and clusterin), were significantly more abundant during "low-fertility" periods. We hypothesized that an increased abundance of caseins offers greater protection from potentially harmful seminal plasma proteins, thereby preserving cell functionality and fertility. In vitro exposure of spermatozoa to casein resulted in decreased levels of lipid scrambling (Merocyanine 540), higher abundance of sperm-bound caseins (α-S1-, α-S2-, and κ-casein), and lower abundance of sperm-bound HSP-1 (P ≤ 0.05). This study demonstrates key pathways governing short-term fertility fluctuations in the stallion, thereby providing a platform to develop robust, fertility assessment strategies into the future.

A Novel Approach to Minimising Acute Equine Endometritis That May Help to Prevent the Development of the Chronic State.

One of the most commonly encountered challenges in equine breeding is endometritis, which can be difficult to resolve and causes considerable economic losses to the industry. It is a multifactorial condition, developing as an exaggerated form of the normal physiological response to breeding. Seminal plasma proteins, spermatozoa, bacteria and debris initiate an inflammatory response; the resulting fluid and neutrophils are then cleared from the uterus along with the debris. However, in some mares, the response is prolonged or exaggerated, with much fluid formation and neutrophil infiltration leading to acute endometritis. A bacterial cause has been implicated, although in some cases no pathogenic organisms can be isolated on culture. It has been postulated that any one of a variety of bacteria could be involved, or dysbiosis of the uterine microbiome could be responsible. Repeated episodes of acute endometritis may lead to the pathology associated with chronic endometritis, with mucociliary dysfunction, vascular degeneration and plasma cell infiltration. This review examines the information that is currently available about equine endometritis, particularly about the role of the inseminate in the uterus, and its current treatment. There are some promising lines of research into treatment or prevention that may help to resolve the issue.

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.

Effect of ejaculation frequency on ram semen characteristics, seminal plasma composition and chilled sperm quality

The aim of this study was to see how chilled sperm quality is affected by ejaculation frequency and its correlation to seminal plasma composition in INRA 180 rams. Five rams were collected at high (HFE) and low frequencies (LFE). For the high frequency, the rams were collected three times on the same day every three days for 18 days. In the low frequency collection, three consecutive ejaculates were collected once a week for four weeks. Ejaculates were collected at 20-minute intervals in either HFE or LFE. Semen characteristics, concentration of total protein, lipid, cholesterol and fructose in seminal plasma were assessed. Semen samples were extended in skim milk-based extender at 15 °C, then evaluated at different storage times (0, 8, and 24 h). Fresh sperm quality parameters, seminal plasma composition, and stored sperm quality were shown to be higher in LFE than in HFE and in the first and second ejaculates than in the third one. After 24 h of storage, sperm quality was correlated to seminal plasma components. In conclusion, the frequency of ejaculation has an effect on the fresh and stored semen quality as well as on seminal plasma composition in INRA 180 rams.

Evaluation of Protein Carbonyl and Vitamin C in Seminal Plasma of Infertile Male: A Hospital-based Study in Bengali Population

Introduction: Male infertility has been coupled with the imbalance between production of Reactive Oxygen Species (ROS) and antioxidant (e.g.,: vitamin C) level. Elevated concentrations of ROS in the semen can lead to oxidative protein damage as they counter with the amino acids' side chains in the protein, leading to the production of carbonyl groups. Aim: To assess if there is any difference of seminal plasma Protein Carbonyl (PC) and vitamin C level in male infertile and fertile subjects in the midst of their correlation with other relevant seminal parameters. Materials and Methods: This was a hospital-based case- control study of a Bengali population. Semen samples of 124 males (Group A; 68 infertile males, Group B; 56 fertile males) were tested. Seminal fluid analysis was done with Makler counting chamber. PC and vitamin C were measured by Levin’s and Roe’s photometric methods respectively. To evaluate the differences in the mean ranks of these parameters Mann- Whitney U test was used. Results: Both in group A and group B sperm count was positively correlated with motility and vitamin C but negatively correlated with PC at significant level p<0.05. Statistically significant differences of mean ranks of these parameters (sperm count: 52.82 and 74.26, motility: 52.10 Vs 75.13, PC: 76.57 and 45.41, vitamin C:55.99 and 70.40, Mann Whitney U:1245, 1197, 947 and 1461, respectively) between the two groups were found. Hence, indicate that in infertile subjects the balance between PC and vitamin C is disturbed. Conclusion: Assessment of oxidative status may serve the clinician in additional management of idiopathic male infertility.

LA IMPORTANCIA DE LAS PROTEÍNAS DEL PLASMA SEMINAL BOVINO EN LA FUNCIÓN ESPERMÁTICA Y LA FERTILIDAD

Bovine livestock is one of the most important economic and social sectors for many countries. In this sense, the development of strategies to improve reproductive bull fertility and reproduction rates is relevant. It's highlighted the role of seminal plasma proteins (SPP) in reproductive fertility, so it has found close relationships among studies on the structure and biological activity of SPP, with seminal quality, including viability, sperm motility, and morphology. In addition, they have been found to regulate sperm functions such as capacitation, acrosome reaction, and they are even related to protecting sperm against thermal and oxidative stress. Moreover, the methods of separation and protein identification and their contribution to characterizing the bovine SP proteome should be also highlighted. In this sense, the most recent studies have been directed towards developing supplements with SPP that improve quality sperm subjected to cryopreservation processes. Research has begun and should forward to establish how the networks or sets of proteins are related to the functioning and fertility of sperm, the search for biomarkers of fertility, and the use of proteins in biotechnological processes, to increase efficiency reproductive.

Exploring the Role of Oxidative Stress in Sperm Motility: A Proteomic Network Approach.

Significance: Infertility is a major global health problem, with nearly half of the cases being associated with male factors. Although reactive oxygen species (ROS) are crucial for sperm cell normal physiological processes, an imbalance between ROS production and antioxidants can lead to oxidative stress that can impair sperm function. Indeed, high semen ROS levels are reported in 30%-80% of infertile men. Recent Advances: Male oxidative stress infertility is an uprising classification for idiopathic infertility. Proteomic approaches, including quantitative mass spectrometry (MS)-based proteomics, are being utilized to explore the molecular mechanisms associated with oxidative stress in male infertility. Critical Issues: In this review, proteome data were collected from articles available on PubMed centered on MS-based proteomic studies, performed in seminal plasma and sperm cell samples, and enrolling men with impaired semen parameters. The bioinformatic analysis of proteome data with Cytoscape (ClueGO+CluePedia) and STRING tools allowed the identification of the biological processes more prevalent in asthenozoospermia, with focus on the ones related to oxidative stress. Future Directions: The identification of the antioxidant proteins in seminal plasma and sperm cells that can protect sperm cells from oxidative stress is crucial not only for a better understanding of the molecular mechanisms associated with male infertility but specially to guide new therapeutic possibilities. Antioxid. Redox Signal. 37, 501-520.

Evaluation of seminal plasma HSPA2 protein as a biomarker of human spermatogenesis status

In mammals, testicular Heat shock-related 70 kDa protein 2 (HSPA2) is a chaperon strictly linked to spermatogenesis status, whereas its presence in spermatozoa ensures successful oocyte fertilization. However, there is little information on this protein in seminal plasma in infertile males. Based on our previous two independent studies, we have selected HSPA2 to evaluate this seminal plasma protein is a potential biomarker of correct spermatogenesis.Using immunoblotting and mass spectrometry (MS) we have screened human seminal plasma samples for the presence of HSPA2. Samples were obtained from individuals with normozoospermia, cryptozoospermia, non-obstructive and obstructive azoospermia.Our results showed a lack of HSPA2 in seminal plasma in all azoospermic males however, in cryptozoospermia the results were extremely diversified. Additionally, the application of 2-dimensional gel electrophoresis (2-DE) indicated the presence of additional protein isoforms suggesting possible mechanisms underlying the male infertility.Our findings suggest seminal plasma HSPA2 protein as a possible biomarker not only of spermatogenesis status, especially in cryptozoospermic males, but also as a biomarker predicting the success of reproductive treatment including assisted reproductive techniques (ART).

Identification and Structure Prediction of Human Septin-4 as a Biomarker for Diagnosis of Asthenozoospermic Infertile Patients-Critical Finding Toward Personalized Medicine.

Semen parameters are been found as a key factor to evaluate the count and morphology in the given semen sample. The deep knowledge of male infertility will unravel with semen parameters correlated with molecular and biochemical parameters. The current research study is to identify the motility associated protein and its structure through the in-silico approach. Semen samples were collected and initial analysis including semen parameters was analyzed by using the World Health Organization protocol. Semen biochemical parameters, namely, seminal plasma protein concentration, fructose content, and glucosidase content were calculated and evaluated for correlation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) were carried out for identification of Septin-4 presence in the semen sample. Mascot search was done for protein conformation and in-silico characterization of Septin-4 by structural modeling in Iterative Threading Assembly Refinement (I-TASSER). Twenty-five nanoseconds molecular dynamics (MD) simulations results showed the stable nature of Septin-4 in the dynamic system. Overall, our results showed the presence of motility-associated protein in normospermia and control samples and not in the case of asthenospermia and oligoasthenospermia. Molecular techniques characterized the presence of Septin-4 and as a novel biomarker for infertility diagnosis.

Purification, molecular characterization and ligand binding properties of the major donkey seminal plasma protein DSP-1

Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the protein is heterogeneously glycosylated and also contains multiple acetylations, occurring in the attached glycans. Structural and thermal stability studies on DSP-1 employing CD spectroscopy and differential scanning calorimetry showed that the protein unfolds at ~43 °C and binding to phosphorylcholine (PrC) – the head group moiety of choline phospholipids – increases its thermal stability. Intrinsic fluorescence titrations revealed that DSP-1 recognizes lyso-phosphatidylcholine with over 100-fold higher affinity than PrC. Further, interaction of DSP-1 with erythrocytes, a model cell membrane, revealed that DSP-1 binding is mediated by a specific interaction with choline phospholipids and results in membrane perturbation, suggesting that binding of this protein to sperm plasma membrane could be physiologically significant.

Dissecting EPPIN protease inhibitor domains in sperm motility and fertilizing ability: repercussions for male contraceptive development.

EPPIN (epididymal protease inhibitor) is a mammalian conserved sperm-binding protein displaying an N-terminal WFDC (whey-acidic protein four-disulfide core) and a C-terminal Kunitz protease inhibitor domains. EPPIN plays a key role in regulating sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). EPPIN ligands targeting the SEMG1 binding site in the Kunitz domain are under development as male contraceptive drugs. Nevertheless, the relative contributions of EPPIN WFDC and Kunitz domains to sperm function remain obscure. Here, we evaluated the effects of antibodies targeting specific epitopes in EPPIN's WFDC (Q20E antibody, Gln20-Glu39 epitope) and Kunitz (S21C and F21C antibodies, Ser103-Cys123 and Phe90-C110 epitopes, respectively) domains on mouse sperm motility and fertilizing ability. Computer-assisted sperm analysis showed that sperm co-incubation with S21C antibody (but not F21C antibody) lowered progressive and hyperactivated motilities and impaired kinematic parameters describing progressive (straight-line velocity; VSL, average path velocity; VAP and straightness; STR) and vigorous sperm movements (curvilinear velocity; VCL, amplitude of lateral head movement; ALH, and linearity; LIN) compared with control. Conversely, Q20E antibody-induced milder inhibition of progressive motility and kinematic parameters (VAP, VCL and ALH). Sperm co-incubation with S21C or Q20E antibodies affected in vitro fertilization as revealed by reduced cleavage rates, albeit without changes in capacitation-induced tyrosine phosphorylation. In conclusion, we show that targeting specific epitopes in EPPIN Kunitz and WFDC domains inhibits sperm motility and capacitation-associated events, which decrease their fertilizing ability; nevertheless, similar observations in vivo remain to be demonstrated. Simultaneously targeting residues in S21C and Q20E epitopes is a promising approach for the rational design of EPPIN-based ligands with spermostatic activity.

The seminal plasma proteins Peptidyl arginine deaminase 2, rRNA adenine N (6)-methyltransferase and KIAA0825 are linked to better motility post thaw in stallions

Seminal plasma plays an important role in sperm physiology. Seminal plasma proteins vehiculated in microvesicles, carry RNAs and proteins with a potential role in early embryo development. Additionally, proteins present in seminal plasma participate in redox regulation and energy metabolism. In view of these facts, we hypothesized that differences in protein composition of the seminal plasma among stallions may help to explain differences in freeze-ability seen among them. Three independent ejaculates from 10 different stallions of varying breeds were frozen using standard protocols in our laboratory. Aliquots of the ejaculate were separated and stored at −80 °C until further proteomic analysis. Semen analysis was performed using computer assisted sperm analysis and flow cytometry. Significant differences in proteome composition of seminal plasma were observed in the group of stallions showing better motility post thaw. 3116 proteins were identified, and of these, 34 were differentially expressed in stallions with better motility post thaw, 4 of them were also differentially expressed in stallions with different percentages of linearly motile sperm post thaw and 1 protein, Midasin, was expressed in stallions showing high circular velocity post thaw.Seminal plasma proteins may play a major role in sperm functionality; being vehiculated through extracellular vesicles and participating in sperm physiology. Bioinformatic analysis identifies discriminant proteins able to predict the outcome of cryopreservation, identifying potential new biomarkers to assess ejaculate quality.