scholarly journals Ligands and Receptors Involved in the Sperm-Zona Pellucida Interactions in Mammals

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 133
Author(s):  
Lucie Tumova ◽  
Michal Zigo ◽  
Peter Sutovsky ◽  
Marketa Sedmikova ◽  
Pavla Postlerova
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Present Article ◽  
Zona Pellucida ◽  
Protein Complexes ◽  
Model Organisms ◽  
Carbohydrate Composition ◽  
Comprehensive Understanding ◽  
Sperm Surface ◽  
Complex Process

Sperm-zona pellucida (ZP) interaction, involving the binding of sperm surface ligands to complementary carbohydrates of ZP, is the first direct gamete contact event crucial for subsequent gamete fusion and successful fertilization in mammals. It is a complex process mediated by the coordinated engagement of multiple ZP receptors forming high-molecular-weight (HMW) protein complexes at the acrosomal region of the sperm surface. The present article aims to review the current understanding of sperm-ZP binding in the four most studied mammalian models, i.e., murine, porcine, bovine, and human, and summarizes the candidate ZP receptors with established ZP affinity, including their origins and the mechanisms of ZP binding. Further, it compares and contrasts the ZP structure and carbohydrate composition in the aforementioned model organisms. The comprehensive understanding of sperm-ZP interaction mechanisms is critical for the diagnosis of infertility and thus becomes an integral part of assisted reproductive therapies/technologies.

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.

scholarly journals Optimisation of DNA extraction from the crustaceanDaphnia

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2004 ◽  
Author(s):  
Camila Gonçalves Athanasio ◽  
James K. Chipman ◽  
Mark R. Viant ◽  
Leda Mirbahai
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dna Extraction ◽  
Organic Contaminants ◽  
High Throughput Sequencing ◽  
Model Organisms ◽  
High Yield ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Mate Pair

Daphniaare key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA fromDaphniaspp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that ifDaphnia magnaare homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin inDaphnia’scarapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses.

Spermatozoa from a marsupial, the brushtail possum, contain β1,4-galactosyltransferase

2008 ◽  
Vol 20 (3) ◽  
pp. 402 ◽  
Author(s):  
A. G. Braundmeier ◽  
William G. Breed ◽  
D. J. Miller
Keyword(s):  
Molecular Weight ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Brushtail Possum ◽  
Laboratory Mice ◽  
Sperm Surface ◽  
Protein Functions ◽  
Sperm Acrosome Reaction ◽  
Porcine Spermatozoa ◽  
Egg Coat

β1,4-Galactosyltransferase-I (GalTase-I) is one of the key molecules on the sperm surface of eutherian mammals that is likely to be involved in binding to the egg coat, the zona pellucida, to mediate sperm–egg interaction. In laboratory mice, the species for which most data are available, this protein functions as a receptor for the zona pellucida protein ZP3 of the oocyte and, upon binding, triggers the sperm acrosome reaction. In the present study, we investigated the presence and abundance of GalTase-I in epididymal sperm extracts of a marsupial, the brushtail possum, Trichosurus vulpecula. For this, spermatozoa were collected from cauda epididymides and the amount of β1,4-galactosyltransferase activity in washed sperm extracts was compared with that of porcine spermatozoa. Overall β1,4-galactosyltransferase enzyme activity was found to be more abundant in possum sperm extracts than those from porcine spermatozoa (P < 0.05). Immunoblots with an antibody to mouse GalTase-I revealed that the molecular weight of possum spermatozoa GalTase-I was 66 kDa, which is similar to the molecular weight of GalTase-I in spermatozoa from eutherian mammals. The molecular weight of GalTase-I was the same in sperm extracts collected from the caput and cauda epididymides. These results demonstrate that GalTase-I is indeed present in possum spermatozoa and thus it may be a gamete receptor molecule on the sperm surface of marsupials as well as those of eutherian mammals.

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.

scholarly journals Sperm surface galactosyltransferase activities during in vitro capacitation

1982 ◽  
Vol 95 (2) ◽  
pp. 567-573 ◽  
Author(s):  
BD Shur ◽  
NG Hall
Keyword(s):  
Molecular Weight ◽  
Zona Pellucida ◽  
Low Molecular Weight ◽  
Sperm Capacitation ◽  
Vitro Fertilization ◽  
F9 Cells ◽  
Sperm Surface ◽  
Sperm Binding ◽  
Fertilizing Ability

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as "decapacitation factors" when added back to in vitro fertilization assays. These glycoside "decapacitation factors" inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces "decapacitation factio" competition. On the other hand "conventional" low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of "decapacitation factos" is a necessary prerequisite for sperm binding to the zona pellucida.

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