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

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.

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Ubiquitin-proteasome system participates in the de-aggregation of spermadhesins and DQH protein during boar sperm capacitation

Reproduction ◽

10.1530/rep-18-0413 ◽

2019 ◽

Vol 157 (3) ◽

pp. 283-295 ◽

Cited By ~ 10

Author(s):

Michal Zigo ◽

Vera Jonakova ◽

Pavla Manaskova-Postlerova ◽

Karl Kerns ◽

Peter Sutovsky

Keyword(s):

Surface Protein ◽

Ubiquitin Proteasome System ◽

Vehicle Control ◽

Sperm Capacitation ◽

Fluorescent Intensity ◽

Sperm Surface ◽

Ubiquitin Proteasome ◽

Sperm Reservoir ◽

Antibody Labeling ◽

Boar Spermatozoa

We studied the participation of the ubiquitin-proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8–16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P < 0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P > 0.99) but showed the accumulation of DQH (P = 0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions. The activity of sperm UPS modulates de-aggregation of boar spermadhesins and DQH sperm surface protein/binder of sperm1 (BSP1) during the sperm capacitation.

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

Biology of Reproduction ◽

10.1093/biolre/ioab225 ◽

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.

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Localization of a 25 kDa human sperm surface protein: its role in in- vitro human sperm capacitation [published erratum appears in Mol Hum Reprod 1997 Jul;3(8):638]

MHR Basic science of reproductive medicine ◽

10.1093/molehr/3.2.109 ◽

1997 ◽

Vol 3 (2) ◽

pp. 109-114 ◽

Cited By ~ 6

Author(s):

M Banerjee

Keyword(s):

Surface Protein ◽

Human Sperm ◽

Sperm Capacitation ◽

Sperm Surface

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Ram seminal plasma and its functional proteomic assessment

Reproduction ◽

10.1530/rep-18-0627 ◽

2019 ◽

Vol 157 (6) ◽

pp. R243-R256 ◽

Cited By ~ 12

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.

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

Reproduction Fertility and Development ◽

10.1071/rd09274 ◽

2010 ◽

Vol 22 (6) ◽

pp. 893 ◽

Cited By ~ 27

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.

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Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm

Animal Reproduction Science ◽

10.1016/j.anireprosci.2016.08.007 ◽

2016 ◽

Vol 173 ◽

pp. 35-41 ◽

Cited By ~ 19

Author(s):

Alba Ledesma ◽

Estela Fernández-Alegre ◽

Adriana Cano ◽

Federico Hozbor ◽

Felipe Martínez-Pastor ◽

...

Keyword(s):

Seminal Plasma ◽

Plasma Proteins ◽

Sperm Surface ◽

Seminal Plasma Proteins ◽

Ram Sperm

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

Veterinary World ◽

10.14202/vetworld.2019.621-628 ◽

2019 ◽

Vol 12 (5) ◽

pp. 621-628 ◽

Cited By ~ 5

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.

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