178. THE CHAPERONIN CONTAINING TCP-1 (CCT/TRiC) MULTISUBUNIT COMPLEX IS INVOLVED IN MEDIATING SPERM - OOCYTE INTERACTIONS

2010 ◽  
Vol 22 (9) ◽  
pp. 96
Author(s):  
M. D. Dun ◽  
R. Aitken ◽  
B. Nixon
Keyword(s):  
Zona Pellucida ◽  
Reproductive Tract ◽  
Protein Complexes ◽  
Female Reproductive Tract ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Sperm Surface ◽  
Collective Data ◽  
Concomitant Reduction ◽  
Future Work

Mammalian spermatozoa only express their capacity for fertilization following capacitation, a process characterized by a suite of biophysical and biochemical changes that occurs as the cells ascend the female reproductive tract. A key event associated with the attainment of a capacitated state is a dramatic reorganization of the sperm surface architecture to render these cells competent to bind to the protective matrix of the oocyte, the zona pellucida. Our previous analysis of these remodeling events has provided compelling evidence that they include the assembly and/or presentation of multimeric protein complexes on the sperm surface. In addition, we have demonstrated that at least two of these complexes possess strong affinity for solubilized zona pellucida. In our current study we have utilised mass spectrometry analysis to reveal that one of these complexes comprises the eight subunits that form a composite, multimeric structure known as the chaperonin containing TCP-1 (CCT/TRiC) complex. Our collective data suggest that this complex participates indirectly in zona pellucida interaction, possibly through the conveyance of key zona adhesion molecules to the sperm surface during capacitation. Consistent with this notion, we were able to demonstrate that the sperm CCT/TRiC complex releases its bound substrates upon exposure to ATP, and this treatment induced a significant, concomitant reduction in the ability of capacitated sperm to bind to the zona pellucida. Furthermore, the use of immunoprecipitation assays confirmed the interaction of the CCT/TRiC complex with at least one putative zona pellucida receptor candidate, namely zona pellucida binding protein 2 (ZPBP2). Future work is now aimed at identifying additional zona receptors that may reside within this complex and the pathways that regulate its functional assembly.

168. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA

2009 ◽  
Vol 21 (9) ◽  
pp. 86
Author(s):  
K. A. Redgrove ◽  
E. A. McLaughlin ◽  
M. K. O'Bryan ◽  
R. J. Aitken ◽  
B. Nixon
Keyword(s):  
Zona Pellucida ◽  
Reproductive Tract ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Sperm ◽  
Female Reproductive Tract ◽  
Human Spermatozoa ◽  
20S Proteasome ◽  
Sperm Surface ◽  
Final Maturation

Upon leaving the testis mammalian spermatozoa are functionally incompetent and are thus unable to fertilize an oocyte. As the spermatozoa ascend the female reproductive tract, functional maturity is achieved through a complex cascade of biophysical and biochemical changes known as capacitation. An important aspect of this final maturation phase is the remodelling of the sperm surface architecture to enable it to interact with the zona pellucida, a glycoprotein matrix that surrounds the oocyte, and initiate fertilisation. While originally thought to be underpinned by a simple lock and key mechanism, emerging evidence has suggested that this interaction may instead be mediated by a multimeric recognition complex that is formed on the sperm surface during capacitation. However, to date the presence and composition of such a complex has yet to be described. Through the application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have provided evidence that human spermatozoa express a number of high molecular weight protein complexes on their surface. Furthermore, the affinity of these surface expressed complexes for the zona pellucida was assessed utilising solubilised human zona pellucida and the technique of Far Western Blotting. Among the complexes that showed affinity for the zona pellucida we identified one comprising 14 subunits of the 20S proteasome. Interestingly, the 20S proteasome has previously been implicated in various aspects of mammalian fertilisation, including zona pellucida penetration and the acrosome reaction, although its precise role in these events has yet to be elucidated. Collectively, these results demonstrate the presence of multimeric protein complexes on the surface of human spermatozoa, and support their putative role in the initial interaction between the sperm and the zona pellucida. Our current research is focused on elucidation of the role of the 20S proteasome in human sperm-zona binding and further investigation of surface expressed protein complexes.

119. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA

2010 ◽  
Vol 22 (9) ◽  
pp. 37 ◽  
Author(s):  
K. A. Redgrove ◽  
B. Nixon ◽  
E. A. McLaughlin ◽  
M. K. O'Bryan ◽  
R. J. Aitken
Keyword(s):  
Zona Pellucida ◽  
Reproductive Tract ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Sperm ◽  
Female Reproductive Tract ◽  
Human Spermatozoa ◽  
Apical Region ◽  
Post Translational Modification ◽  
Cellular Mechanisms

A unique characteristic of mammalian spermatozoa is that upon ejaculation, they are unable to recognise and bind to an ovulated oocyte. These functional attributes are only realised following the sperms ascent of the female reproductive tract whereupon they undergo a myriad of biochemical and biophysical changes collectively referred to as ‘capacitation’. Since spermatozoa are both transcriptionally and translationally quiescent cells, this functional transformation must be engineered by a combination of post-translational modification and spatial reorganisation of existing sperm proteins. Indeed, evidence from our laboratory suggests that a key attribute of capacitation is the remodeling of the sperm surface architecture leading to the assembly and / or presentation of multimeric sperm-oocyte receptor complex(es). Through the novel application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have secured the first direct evidence that human spermatozoa express a number of these protein complexes on their surface. Furthermore, we have demonstrated that a subset of these complexes harbour putative zona adhesion proteins and display strong affinity for solubilised zona pellucidae. In this study, we have extended our findings through the characterisation of one such complex containing arylsulfatase A (ASA), a protein with recognised affinity for sulfated ligands present within the zona pellucida. Through the application of immunohistochemistry and flow cytometry we revealed that ASA undergoes a capacitation-associated translocation to become expressed on the apical region of the human sperm head, a location compatible with a role in the mediation of sperm-zona pellucida interactions. This dramatic relocation was completely abolished by incubation of capacitating spermatozoa in exogenous cholesterol, suggesting that it may be driven in part by alteration in the membrane fluidity characteristics. Our current research is focused on confirming the role of ASA in human sperm-zona pellucida adhesion and elucidating the precise cellular mechanisms that underpin the proteins translocation to the cell surface.

scholarly journals LYPD4, mouse homolog of a human acrosome protein, is essential for sperm fertilizing ability and male fertility†

2020 ◽  
Vol 102 (5) ◽  
pp. 1033-1044
Author(s):  
Dan Wang ◽  
Liping Cheng ◽  
Wenjuan Xia ◽  
Xiaofei Liu ◽  
Yueshuai Guo ◽  
...  
Keyword(s):  
Male Fertility ◽  
Reproductive Tract ◽  
Female Reproductive Tract ◽  
Mass Spectrometry Analysis ◽  
Small Subset ◽  
Vitro Fertilization ◽  
Spectrometry Analysis ◽  
Human Fertilization ◽  
Sperm Migration

Abstract Fertilization is one of the fundamental biological processes, but so far, we still do not have a full understanding of the underlying molecular mechanism. We have identified a human acrosome protein, LY6/PLAUR domain containing 4 (LYPD4), expressed specifically in human testes and sperm, and conserved within mammals. Mouse Lypd4, also specific to the testis and sperm, is essential for male fertility. LYPD4 protein first appeared in round spermatids during acrosome biogenesis and became part of acrosomes during spermatogenesis and in mature sperm. Lypd4 knockout mice are infertile with normal sperm number and motility. Mutant sperm, however, failed to reach oviduct during sperm migration inside the female reproductive tract, leading to fertilization failure and infertility. In addition, Lypd4 mutant sperms were unable to fertilize denuded egg via IVF (in vitro fertilization) but could fertilize eggs within intact Cumulus-Oocyte Complex, supporting an additional role in sperm-zona interaction. Out of more than five thousand spermatozoa proteins identified by mass spectrometry analysis, only a small subset of proteins (26 proteins) was changed in the absence of LYPD4, revealing a whole proteome picture of mutant sperm defective in sperm migration and sperm-zona binding. ADAM3, a key component of fertilization complex, as well as other sperm ADAM proteins are significantly reduced. We hence propose that LYPD4 plays an essential role in mammalian fertilization, and further investigation of its function and its interaction with other sperm membrane complexes may yield insights into human fertilization and novel strategy to improve IVF success.

226. Characterisation of a putative mouse sperm - zona pellucida receptor complex

2008 ◽  
Vol 20 (9) ◽  
pp. 26
Author(s):  
M. D. Dun ◽  
B. Nixon ◽  
R. J. Aitken
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Zona Pellucida ◽  
Molecular Chaperone ◽  
Reproductive Tract ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Female Reproductive Tract ◽  
Receptor Complex ◽  
High Molecular Weight Complex

Mammalian spermatozoa acquire the ability to fertilise an oocyte as they ascend the female reproductive tract. This process is characterised by a complex cascade of biophysical and biochemical changes collectively known as capacitation. The attainment of a capacitated state is accompanied by a dramatic reorganisation of the surface architecture which renders the spermatozoa competent to recognise the heterogenetic matrix of the zona pellucida surrounding the oocyte and initiate fertilisation. Emerging evidence from our laboratory indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, to date the presence and composition of such a complex has yet to be described. Through the novel application of blue native polyacrylamide gel electrophoresis (BN-PAGE), we have provided the first evidence that capacitated mouse spermatozoa express high molecular weight, multimeric protein complexes on their surface. Interestingly, at least two of these complexes contain heat shock protein 1 (HSPD1), a molecular chaperone that has previously been implicated in sperm-zona pellucida interaction. Furthermore, we were able to demonstrate that one of these complexes also possessed an affinity for solubilised zona pellucida as determined by Far-western blotting. 2D BN-PAGE was employed to further delineate the individual constituents of this high molecular weight complex, with several other chaperonin proteins not previously reported in functional sperm indentified. Collectively, these results support the notion the sperm-zona pellucida interaction are mediated by a multimeric receptor complex. Our current work is focussed on the identification of the key zona adhesion molecules that comprise this complex.

103 MASS SPECTROMETRY ANALYSIS OF UTERUS ENDOMETRIUM DURING PREGNANCY IN THE PIG

2009 ◽  
Vol 21 (1) ◽  
pp. 152
Author(s):  
J.-I. Chae ◽  
Y. K. Cho ◽  
S.-K. Cho ◽  
K.-K. Lee ◽  
D.-B. Koo
Keyword(s):  
Growth Factor ◽  
Western Blotting ◽  
Reproductive Tract ◽  
Female Reproductive Tract ◽  
Late Gestation ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Molecular Events ◽  
Associated Proteins ◽  
Endothelial Growth Factor

In general, many important molecular events occur within the female reproductive tract, especially within the uterine endometrium, during periods of pregnancy. The endometrium includes a mucosal lining of the uterus, which functions to provide a suitable site for implantation and development of a fertilized egg and fetus. To date, developmental integrity involves molecular cascades whose interrelationships are not fully understood within the endometrium during pregnancy in pigs. In this study, we explored the functional regulated proteins in endometrium during periods of pregnancy periods (Day 40, n = 6; Day 70, n = 7; Day 90, n = 6 of pregnancy; and nonpregnancy, n = 1) using 2-dimensional gel electrophoresis (2-DE) and Western blotting. The functional regulated proteins were identified and discovered from differentially expressed proteins in the endometrium during pregnancy. In the proteomic analysis of the endometrium, 820 protein spots were matched on 2-DE gels. With 98 proteins regulated differentially among nonpregnant and pregnant tissues (matched and unmatched spots), 63 up- or down-regulated proteins have been identified. Interestingly, 6 of these 63 proteins were endothelial growth factor-associated proteins such as transgelin, transferrin, galectin-1, tropomyosin alpha, protein DJ-1, and beta-defensin. We also confirmed the expression levels of these proteins in the endometrium during pregnancy by using Western blotting. Our results suggest that the expressions of these genes involved in endometrial function and development from early to late gestation are associated with the regulation of endothelial growth factor.

scholarly journals Getting more out of co-immunoprecipitation mass spectrometry experiments by reducing interference using FAIMS.

2021 ◽  
Author(s):  
Ching-Seng Ang ◽  
Joanna Sacharz ◽  
Michael G Leeming ◽  
Shuai Nie ◽  
Swati Varshney ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Modern Biology ◽  
Spectrometry Analysis ◽  
Affinity Enrichment ◽  
Unbiased Manner ◽  
Gas Phase Ion

Co-immunoprecipitation of proteins coupled to mass spectrometry has transformed modern biology understanding of protein interaction networks. These approaches exploit the selective isolation of tagged proteins by affinity enrichment / purification to identify protein binding partners at scale and in an unbiased manner. In instances where a suitable antibody is not be available it is common to graft synthetic tags such as FLAG or His Tags onto target protein sequences allowing the use of commercially available and validated antibodies for affinity purification. To allow the selective elution of protein complexes competitive displacement using a large molar excess of the tag peptide is widely used. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of a contaminating peptide. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device can be applied to FLAG-Tag and His-Tag pull down assay to increase the depth of protein coverage in these experiments. By excluding tag peptides based on their ion mobility profiles we demonstrate that single compensation voltage, or stepped compensation voltages strategies can significantly increase the coverage of total proteins by up to 2.5-fold and unique proteins by up to 15-fold versus experiments that do not use FAIMS. Combined these results highlight FAIMS is able to improve proteome depth by excluding interfering peptides without the need for additional sample handling or altering sample preparation protocols.

scholarly journals Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrinβ1

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Cordula Klockenbusch ◽  
Juergen Kast
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Interaction Analysis ◽  
Protein Complexes ◽  
Human Platelets ◽  
Jurkat Cells ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
High Molecular Weight Complex ◽  
Spectrometry Analysis

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrinβ1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrinβ1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrinβ1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrinβ1,α4 andα6 orβ1,α6,α2, andα5, respectively.

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