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Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

Tanphaichitr N, Kongmanas K, Kruevaisayawan H, Saewu A, Sugeng C, Fernandes J, Souda P, Angel JB, Faull KF, Aitken RJ, Whitelegge J, Hardy D, Berger T, Baker M - Asian J. Androl. (2015 Jul-Aug)

Bottom Line: The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing.Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes.Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes.

View Article: PubMed Central - PubMed

Affiliation: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa; Department of Obstetrics and Gynaecology, University of Ottawa; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada, .

ABSTRACT
The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm-ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm-ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm-ZP binding step.

No MeSH data available.


Related in: MedlinePlus

(a) Left panel: Presence of HMW complexes in pig APM vesicles as shown by blue native gel electrophoresis/silver staining; Right panel: Far western blotting showing the binding of Complex I (1000-1300 kDa), Complex II (850-1000 kDa) and Complex III (750-850 kDa) to biotinylated pig ZP3 (sperm receptor). (b) Immunoblotting of Cap sperm APM proteins separated by blue native gel electrophoresis, showing zonadhesin bands in the three Complexes with patterns similar to the far western bands of ZP3 binding. (c) Preincubation of APM Complexes with anti-zonadhesin (anti-ZAN) IgG inhibits the Complex binding to ZP3 in a dose dependent manner. (d) Identity and spectral counts of proteins in the three HMW Complexes. For experimental details of results described throughout this article, see Kongmanas et al.93. All figures shown in this review are also adapted from this article.
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Figure 1: (a) Left panel: Presence of HMW complexes in pig APM vesicles as shown by blue native gel electrophoresis/silver staining; Right panel: Far western blotting showing the binding of Complex I (1000-1300 kDa), Complex II (850-1000 kDa) and Complex III (750-850 kDa) to biotinylated pig ZP3 (sperm receptor). (b) Immunoblotting of Cap sperm APM proteins separated by blue native gel electrophoresis, showing zonadhesin bands in the three Complexes with patterns similar to the far western bands of ZP3 binding. (c) Preincubation of APM Complexes with anti-zonadhesin (anti-ZAN) IgG inhibits the Complex binding to ZP3 in a dose dependent manner. (d) Identity and spectral counts of proteins in the three HMW Complexes. For experimental details of results described throughout this article, see Kongmanas et al.93. All figures shown in this review are also adapted from this article.

Mentions: Blue native gel electrophoresis further revealed the presence of HMW protein complexes (>200 kDa) in APM vesicles from Noncap and Cap sperm (Figure 1a, left panel), but HMW complexes sized 1000–1300 kDa (named Complex I), 850–1000 kDa (Complex II) and 750–850 kDa (Complex III) from Cap sperm had a significantly higher capacity to bind to pig ZP3 glycoproteins (hetero-oligomers of pig ZP3α and pig ZP3β; sperm receptor; Figure 1a, right panel). As expected, LC–MS/MS revealed that proteins known for their affinity for the ZP scored the highest for the spectral counts in all three complexes and the amounts of most of these proteins were higher in Cap HMW complexes (Figure 1d). Interestingly, ZAN had the highest spectral counts in the three complexes. This finding was in contrast to the LC–MS/MS results of the whole APM vesicle extracts where SED1 scored the highest in spectral counts and ZAN the lowest in the protein category of sperm–egg interaction (Table 1). The presence of ZAN in Complexes I, II and III was confirmed by immunoblotting (Figure 1b). The anti-ZAN reactive bands in the three complexes corresponded to the pig ZP3 binding patterns (Figure 1a). Significantly, ZAN contributed to the ZP affinity of the three complexes. Preincubation of the complexes with various concentrations of anti-ZAN IgG inhibited their binding to pig ZP3 in a dose-dependent manner (Figure 1c).


Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

Tanphaichitr N, Kongmanas K, Kruevaisayawan H, Saewu A, Sugeng C, Fernandes J, Souda P, Angel JB, Faull KF, Aitken RJ, Whitelegge J, Hardy D, Berger T, Baker M - Asian J. Androl. (2015 Jul-Aug)

(a) Left panel: Presence of HMW complexes in pig APM vesicles as shown by blue native gel electrophoresis/silver staining; Right panel: Far western blotting showing the binding of Complex I (1000-1300 kDa), Complex II (850-1000 kDa) and Complex III (750-850 kDa) to biotinylated pig ZP3 (sperm receptor). (b) Immunoblotting of Cap sperm APM proteins separated by blue native gel electrophoresis, showing zonadhesin bands in the three Complexes with patterns similar to the far western bands of ZP3 binding. (c) Preincubation of APM Complexes with anti-zonadhesin (anti-ZAN) IgG inhibits the Complex binding to ZP3 in a dose dependent manner. (d) Identity and spectral counts of proteins in the three HMW Complexes. For experimental details of results described throughout this article, see Kongmanas et al.93. All figures shown in this review are also adapted from this article.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492047&req=5

Figure 1: (a) Left panel: Presence of HMW complexes in pig APM vesicles as shown by blue native gel electrophoresis/silver staining; Right panel: Far western blotting showing the binding of Complex I (1000-1300 kDa), Complex II (850-1000 kDa) and Complex III (750-850 kDa) to biotinylated pig ZP3 (sperm receptor). (b) Immunoblotting of Cap sperm APM proteins separated by blue native gel electrophoresis, showing zonadhesin bands in the three Complexes with patterns similar to the far western bands of ZP3 binding. (c) Preincubation of APM Complexes with anti-zonadhesin (anti-ZAN) IgG inhibits the Complex binding to ZP3 in a dose dependent manner. (d) Identity and spectral counts of proteins in the three HMW Complexes. For experimental details of results described throughout this article, see Kongmanas et al.93. All figures shown in this review are also adapted from this article.
Mentions: Blue native gel electrophoresis further revealed the presence of HMW protein complexes (>200 kDa) in APM vesicles from Noncap and Cap sperm (Figure 1a, left panel), but HMW complexes sized 1000–1300 kDa (named Complex I), 850–1000 kDa (Complex II) and 750–850 kDa (Complex III) from Cap sperm had a significantly higher capacity to bind to pig ZP3 glycoproteins (hetero-oligomers of pig ZP3α and pig ZP3β; sperm receptor; Figure 1a, right panel). As expected, LC–MS/MS revealed that proteins known for their affinity for the ZP scored the highest for the spectral counts in all three complexes and the amounts of most of these proteins were higher in Cap HMW complexes (Figure 1d). Interestingly, ZAN had the highest spectral counts in the three complexes. This finding was in contrast to the LC–MS/MS results of the whole APM vesicle extracts where SED1 scored the highest in spectral counts and ZAN the lowest in the protein category of sperm–egg interaction (Table 1). The presence of ZAN in Complexes I, II and III was confirmed by immunoblotting (Figure 1b). The anti-ZAN reactive bands in the three complexes corresponded to the pig ZP3 binding patterns (Figure 1a). Significantly, ZAN contributed to the ZP affinity of the three complexes. Preincubation of the complexes with various concentrations of anti-ZAN IgG inhibited their binding to pig ZP3 in a dose-dependent manner (Figure 1c).

Bottom Line: The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing.Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes.Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes.

View Article: PubMed Central - PubMed

Affiliation: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa; Department of Obstetrics and Gynaecology, University of Ottawa; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada, .

ABSTRACT
The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm-ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm-ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm-ZP binding step.

No MeSH data available.


Related in: MedlinePlus