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

2022 ◽  
Author(s):  
Róisín Ann Griffin ◽  
Aleona Swegen ◽  
Mark A Baker ◽  
Rachel Ann Ogle ◽  
Nathan Smith ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Low Fertility ◽  
High Fertility ◽  
Short Term ◽  
Sperm Binding ◽  
Merocyanine 540 ◽  
Seminal Plasma Proteins ◽  
Assessment Strategies

Abstract Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days post-breeding, 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 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 inter-stallion 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 hypothesised 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.

scholarly journals Changes in Bull Semen Metabolome in Relation to Cryopreservation and Fertility

Animals ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 1065
Author(s):  
Valentina Longobardi ◽  
Michal A. Kosior ◽  
Nunzia Pagano ◽  
Gerardo Fatone ◽  
Alessia Staropoli ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Glycine Betaine ◽  
Low Fertility ◽  
High Fertility ◽  
Over Expression ◽  
Bull Semen ◽  
Before And After ◽  
Associated Proteins ◽  
Cellular Compartments

Semen cryopreservation determines several sperm damages, including the loss of fertility-associated proteins. The purpose of the study was to compare the metabolite contents in bovine sperm and seminal plasma before and after cryopreservation, and between high- and low-fertility bulls in vitro. Forty-eight ejaculates, collected from eight bulls (six per bull), were analyzed by liquid chromatography–mass spectrometry. Cryopreservation resulted in an over-expression of lysophosphatidylcholine (0:0/18:2(9Z,12Z)) in seminal plasma. In addition, higher levels of glycine betaine and pyro-l-glutaminyl-l-glutamine were observed in cryopreserved compared to fresh spermatozoa. The fresh seminal plasma of high-fertility bulls showed an over-expression of l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine, and an under-expression of lysophosphatidylcholine and butyrylcarnitine, compared to low-fertility bulls. Higher levels of glycerophosphocholine and lysophosphatidylcholine (16:0/0:0) were recorded in fresh spermatozoa from high-fertility bulls. In high-fertility bulls, a greater content of glycerophosphocholine and lower levels of butyrylcarnitine, glycine betaine and l-carnitine were found in cryopreserved seminal plasma, and lower levels of glycine betaine were detected in cryopreserved spermatozoa. In conclusion, cryopreservation affects bovine semen metabolome at both plasmatic and cellular compartments, and metabolic profile differs between high- and low-fertility bulls.

scholarly journals The Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation

2020 ◽  
Vol 21 (11) ◽  
pp. 4151
Author(s):  
Lucie Tumova ◽  
Michal Zigo ◽  
Peter Sutovsky ◽  
Marketa Sedmikova ◽  
Pavla Postlerova
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Protein Composition ◽  
Surface Protein ◽  
Ubiquitin Proteasome System ◽  
Sperm Capacitation ◽  
Sperm Surface ◽  
Ubiquitin Proteasome ◽  
Seminal Plasma Proteins

Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine β-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.

Flow-sorted ram spermatozoa are highly susceptible to hydrogen peroxide damage but are protected by seminal plasma and catalase

2010 ◽  
Vol 22 (7) ◽  
pp. 1131 ◽  
Author(s):  
T. Leahy ◽  
P. Celi ◽  
R. Bathgate ◽  
G. Evans ◽  
W. M. C. Maxwell ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Seminal Plasma ◽  
Plasma Proteins ◽  
Fluorescein Isothiocyanate ◽  
Oxygen Species ◽  
Merocyanine 540 ◽  
Propidium Iodide Staining ◽  
Flow Sorting ◽  
Seminal Plasma Proteins

To determine whether flow sorting increased the susceptibility of spermatozoa to reactive oxygen species (ROS), ram semen was either diluted with Tris medium (100 × 106 spermatozoa mL–1; D) or highly diluted (106 spermatozoa mL–1) before being centrifuged (DC) at 750g for 7.5 min at 21°C or flow-sorted (S) before cryopreservation. Thawed spermatozoa were resuspended in graded concentrations of hydrogen peroxide to induce oxidative stress. In Experiment 1, following exposure to 30 or 45 μM hydrogen peroxide (H2O2), the total motility (%) of DC (41.0 ± 7.3 or 25.7 ± 6.7, respectively) and S spermatozoa (33.8 ± 6.3 or 20.1 ± 6.3, respectively) was lower (P < 0.001) than that of D spermatozoa (58.7 ± 5.6 or 44.5 ± 6.7, respectively). In Experiment 2, supplementation of samples containing H2O2 with catalase (150 IU mL–1) or seminal plasma proteins (4 mg protein per 108 spermatozoa) negated oxidative stress, resulting in comparable values to samples receiving no H2O2in terms of the proportion of spermatozoa with stable plasmalemma (as determined using merocyanine-540 and Yo-Pro-1) in the D and S groups, the proportion of viable, acrosome-intact spermatozoa (as determined by fluorescein isothiocyanate and propidium iodide staining) in the D group and the motility of control (undiluted) and S spermatozoa. Neither H2O2 nor sperm type (i.e. D, DC or S) had any effect on intracellular concentrations of ROS. These results show that flow sorting increases the susceptibility of spermatozoa to ROS, but the inclusion of anti-oxidants or seminal plasma as part of the sorting protocol improves resistance to oxidative stress.

scholarly journals Ram seminal plasma and its functional proteomic assessment

Reproduction ◽  
2019 ◽  
Vol 157 (6) ◽  
pp. R243-R256 ◽  
Author(s):  
T Leahy ◽  
J P Rickard ◽  
N C Bernecic ◽  
X Druart ◽  
S P de Graaf
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Reproductive Tract ◽  
Reproductive Technologies ◽  
Female Reproductive Tract ◽  
Sperm Function ◽  
Seminal Plasma Proteins ◽  
Ram Sperm

Ejaculation results in the confluence of epididymal spermatozoa with secretions of the accessory sex glands. This interaction is not a prerequisite for fertilisation success, but seminal factors do play a crucial role in prolonging the survival of spermatozoa bothin vitroandin vivoby affording protection from handling induced stress and some selective mechanisms of the female reproductive tract. Reproductive biologists have long sought to identify specific factors in seminal plasma that influence sperm function and fertility in these contexts. Many seminal plasma proteins have been identified as diagnostic predictors of sperm function and have been isolated and appliedin vitroto prevent sperm damage associated with the application of artificial reproductive technologies. Proteomic assessment of the spermatozoon, and its surroundings, has provided considerable advances towards these goals and allowed for greater understanding of their physiological function. In this review, the importance of seminal plasma will be examined through a proteomic lens to provide comprehensive analysis of the ram seminal proteome and detail the use of proteomic studies that correlate seminal plasma proteins with ram sperm function and preservation ability.

Seminal plasma proteins inhibit in vitro- and cooling-induced capacitation in boar spermatozoa

2010 ◽  
Vol 22 (6) ◽  
pp. 893 ◽  
Author(s):  
Melissa L. Vadnais ◽  
Kenneth P. Roberts
Keyword(s):  
Mass Spectrometry ◽  
Seminal Plasma ◽  
Plasma Proteins ◽  
Acrosome Reaction ◽  
Heparin Binding ◽  
Total Proteins ◽  
Binding Properties ◽  
Seminal Plasma Proteins ◽  
Boar Spermatozoa

Dilute boar seminal plasma (SP) has been shown to inhibit in vitro capacitation and cooling-induced capacitation-like changes in boar spermatozoa, as assessed by the ability of the spermatozoa to undergo an ionophore-induced acrosome reaction. We hypothesised that the protein component of SP is responsible for this effect. To test this hypothesis, varying concentrations of total SP protein or SP proteins fractionated by heparin binding were assayed for their ability to inhibit in vitro capacitation, as well as cooling- and cryopreservation-induced capacitation-like changes. In vitro capacitation and cooling-induced capacitation-like changes were prevented by 10% whole SP, as well as by total proteins extracted from SP at concentrations greater than 500 μg mL−1. No amount of SP protein was able to prevent cryopreservation-induced capacitation-like changes. Total SP proteins were fractionated based on their heparin-binding properties and the heparin-binding fraction was shown to possess capacitation inhibitory activity at concentrations as low as 250 µg mL−1. The proteins in the heparin-binding fraction were subjected to mass spectrometry and identified. The predominant proteins were three members of the spermadhesin families, namely AQN-3, AQN-1 and AWN, and SP protein pB1. We conclude that one or more of these heparin-binding SP proteins is able to inhibit in vitro capacitation and cooling-induced capacitation-like changes, but not cryopreservation-induced capacitation-like changes, in boar spermatozoa.

scholarly journals Electrophoretic profile of seminal proteins and their correlation with in vitro sperm characters in Black Bengal buck semen

2019 ◽  
Vol 12 (5) ◽  
pp. 621-628 ◽  
Author(s):  
M. Karunakaran ◽  
Vivek C. Gajare ◽  
Ajoy Mandal ◽  
Mohan Mondal ◽  
S. K. Das ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Seminal Plasma ◽  
Plasma Proteins ◽  
Membrane Integrity ◽  
Sperm Proteins ◽  
Sds Page ◽  
Sperm Characters ◽  
Functional Membrane ◽  
Seminal Plasma Proteins

Aim: This study aimed to study the electrophoretic properties of seminal plasma and sperm proteins of Black Bengal buck semen and their correlation with in vitro sperm characters and freezability. Materials and Methods: Semen ejaculates from nine Black Bengal bucks were collected by artificial vagina (n=20/buck). Ejaculates were evaluated for in vitro sperm characters and electrophoretic profile of seminal protein. In vitro sperm characters were evaluated immediately after collection, after completion of equilibration period, and after freeze-thawing. For seminal protein studies, seminal plasma proteins were precipitated by ice-cold ethanol method, and sperm proteins were extracted by Triton X detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess the molecular weight of seminal proteins. Correlation between in vitro sperm characters and protein bands was determined by Pearson's correlation coefficient, and two-way ANOVA was applied to find the individual buck differences. Results: Significant difference (p<0.01) among the bucks was noticed in the in vitro sperm characters evaluated at all the three stages of semen evaluation such as immediately after collection, after completion of equilibration period, and post-freeze thawing. Progressive loss of sperm motility, membrane integrity, and other in vitro sperm characters were noticed during cryopreservation. A total of ten protein bands in the molecular weight ranging from 17 to 180 kDa were found in the SDS-PAGE of seminal plasma proteins, while nine bands of 17-134 kDa were observed in sperm proteins. Seminal plasma proteins of molecular weight 75, 62-49, 20, and 17 kDa and sperm proteins of 75, 20, and 17 kDa were present in all the nine bucks (100%) screened, and variation among the bucks was noticed for the presence of other proteins. Seminal plasma protein of 180-134 kDa showed a negative correlation with individual motility (−0.716) and functional membrane integrity of sperm cells (−0.724) in post-freeze-thaw analysis and 48 kDa protein had a positive correlation with individual motility (0.649) and functional membrane integrity of sperm cells (0.664) in post-thaw analysis. Sperm proteins of 63 kDa had a negative correlation (−0.616) with sperm concentration in neat semen. Conclusion: Variation among the bucks was noticed in the in vitro sperm characters and semen freezability. Correlation between seminal proteins and in vitro sperm characters and semen freezability had been found which might be useful as a tool to select breeding bucks.

310 SEASON INFLUENCE UPON SEMINAL PLASMA PROTEINS IN BROWN SWISS BULLS

2010 ◽  
Vol 22 (1) ◽  
pp. 311
Author(s):  
M. G. M. Chacur ◽  
F. P. Sirchia ◽  
A. C. L. Ruiz ◽  
M. L. Guaberto
Keyword(s):  
New York ◽  
Seminal Plasma ◽  
Plasma Proteins ◽  
Protein Quality ◽  
Semen Quality ◽  
Reproductive Organs ◽  
Low Fertility ◽  
Significance Level ◽  
Brown Swiss ◽  
Seminal Plasma Proteins

Seminal plasma is a complex of secretions of the male accessory reproductive organs and appears to exert important effects on sperm function (Shivaji et al. 1990 Proteins of Seminal Plasma, Wiley, New York, NY, USA). The protein quality of the seminal plasma may affect positively the bulls’fertility (Killiam et al. 1993 Biol. Reprod. 49, 1202-1207). Peptides of 55 and 66 kDa were present in bulls with excellent spermatic conditions for example motility and vigor. On the other hand, 16- and 36-kDa peptides were observed with unfavorable spermatic conditions (Chacur et al. 2009 Anim. Reprod. 6, 339). The objective was to determine the influence of season on seminal plasma proteins in Brown Swiss bulls. Semen from 33 Brown Swiss bulls 24 months of age was collected by electroejaculation during winter (from June to August) and summer (from December to February) in the southern hemisphere in 2008. Semen samples were collected with 14-day intervals totalizing 196 ejaculates. Samples of semen were centrifuged (1500g/15 min) and the seminal plasma was conditioned in cryotubes and stored at -20°C until further processing. Proteins were extracted from 200 μL of each sample in 2 mL of extraction buffer composed of 0.625 M Tris-HCl, at pH 6.8, in 2% SDS, 5% fi-mercaptoethanol, and 20% of glycerol. Percentages of different plasma proteins by season were statistically compared by the chi-square test with significance level at P < 0.05). Proteins were quantified according to Bradford (1976 Anal. Biochem. 72, 248-254) and electrophoresis was performed according to Laemmili (1970 Nature 227, 680-685). Gels were fixed with isopropanol: acetic acid: water (4:1:5 v/v) for 30 minutes and stained in the same solution with 2% of Coomassie Blue R250. In 26 bulls, the absence of high molecular weight (HMW; 55 kDa, 66 kDa, and 80 kDa) proteins was found in the summer. There was a significant increase (P < 0.05) in total spermatic defects, acrosome defects, and distal cytoplasmatic droplets in these bulls. The 40-kDa protein that reflected low fertility was observed in 10 bulls in the summer with semen quality decreases. The 11 bulls showed presence of HMW (55 kDa) in the winter. In 11 bulls, HMW (55 kDa, 66 kDa, or 80 kDa) proteins were present with a satisfactory semen condition according to Killiam et al. (1993 Biol. Reprod. 49, 1202-1207). In conclusion, the seasons of the year may influence the presence of proteins in seminal plasma. There was a direct relationship of the season with seminal plasma proteins. The presence of the proteins of 20 kDa, 55 kDa, 66 kDa, and 80 kDa suggested an increase of the semen quality during the winter. UNOESTE and Bartira Farm.

Seminal plasma and seminal plasma proteins added to bulk sorted sperm do not alter the mRNA expression of in vitro produced bovine embryos

2012 ◽  
Vol 78 (1) ◽  
pp. 132-139 ◽  
Author(s):  
H. Stinshoff ◽  
M. Krienke ◽  
M. Ekhlasi-Hundrieser ◽  
S. Wilkening ◽  
A. Hanstedt ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Seminal Plasma ◽  
Plasma Proteins ◽  
Bovine Embryos ◽  
Seminal Plasma Proteins

Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates

2017 ◽  
Vol 29 (2) ◽  
pp. 394 ◽  
Author(s):  
Carolina Luna ◽  
Marc Yeste ◽  
María M. Rivera del Alamo ◽  
Juan Domingo ◽  
Adriana Casao ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Principal Component ◽  
Dual Effect ◽  
Epidermal Growth ◽  
Seminal Plasma Proteins ◽  
Motion Characteristics ◽  
Low Amplitude ◽  
Lateral Head

It has been proposed that seminal plasma proteins (SPP) support survival of ram spermatozoa, exerting a dual effect, both capacitating and decapacitating. In this study, changes in motility patterns of ram spermatozoa capacitated in the presence of epidermal growth factor (EGF) were evaluated. Clustering procedures were used to determine the presence of sperm subpopulations with specific motion characteristics. Four sperm subpopulations (SP) were defined after the application of a principal component analysis procedure. Progressive spermatozoa with high straightness (STR) were found in SP1, reflected in the high linearity (LIN) and STR values and low amplitude of lateral head movement (ALH; rapid, non-hyperactivated spermatozoa). SP2 spermatozoa seemed to be starting to acquire hyperactivated motility, while the SP3 group consisted of rapid, hyperactivated spermatozoa. SP4 showed less-vigorous spermatozoa, with non-linear motility. The addition of SPP before in vitro capacitation with EGF induced a decrease in SP1 and an increase in SP3. However, a reduction in the chlortetracycline-capacitated sperm rate and protein tyrosine phosphorylation was found, which corroborates with the hypothesis that the SPP protective effect on spermatozoa is related to their decapacitating role. These findings allow us to deduce that ram spermatozoa are able to undergo capacitation with no hyperactivation and that SPP are able to induce hyperactivation in spermatozoa but maintain them in a decapacitated state.

Boar Seminal Plasma Proteins and Their Binding Properties. A Review

2004 ◽  
Vol 69 (3) ◽  
pp. 461-475 ◽  
Author(s):  
Věra Jonáková ◽  
Marie Tichá
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Sperm Capacitation ◽  
Binding Properties ◽  
Fertilization Process ◽  
Sperm Binding ◽  
Process Formation ◽  
Different Types ◽  
Seminal Plasma Proteins ◽  
Coating Layers

Binding properties of a group of proteins isolated from boar seminal plasma and their role in the fertilization process are discussed. Boar seminal plasma contains different types of proteins: spermadhesins of AQN and AWN family, DQH and PSP proteins belong to the most abundant. Some of these proteins are bound to the sperm surface during ejaculation and thus protein-coating layers are formed. Sperms coated with proteins participate in different types of interactions in the following steps of the fertilization process: formation of oviductal sperm reservoir, sperm capacitation, oocyte recognition and sperm binding. Saccharide-based interactions of boar seminal plasma proteins play role in the binding of sperm to oviductal epithelium, in sperm capacitation and primary binding of sperm to zona pellucida. An interaction with phospholipid components is responsible for the protein adsorption to sperm membrane. Interactions between proteins participate in the arrangement and remodelling of sperm-coating layers. Study of boar seminal plasma proteins, their characterization and elucidation of their interactions will contribute to understanding the fertilization process. A review with 82 references.

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