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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) Structural domains of ZAN (adapted from Bi et al.38 and Herlyn and Zischler.98 (b) Immunoprecipitation of APM proteins with anti-ZAN IgG captured on Protein G paramagnetic beads93. Input = whole APM protein extracts; Elute = APM proteins bound to anti-ZAN beads. Results indicate interaction among ZAN, proacrosin/acrosin (ACRO) and ACRBP but not SED1 (MFGM). ZAN: zonadhesin; APM: anterior head plasma membrane.
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Figure 3: (a) Structural domains of ZAN (adapted from Bi et al.38 and Herlyn and Zischler.98 (b) Immunoprecipitation of APM proteins with anti-ZAN IgG captured on Protein G paramagnetic beads93. Input = whole APM protein extracts; Elute = APM proteins bound to anti-ZAN beads. Results indicate interaction among ZAN, proacrosin/acrosin (ACRO) and ACRBP but not SED1 (MFGM). ZAN: zonadhesin; APM: anterior head plasma membrane.

Mentions: Zonadhesin is a mosaic protein comprising a number of cell adhesion-related domains (MAM, mucin, and von Willebrand factor D [VWF D]) (Figure 3a). It is synthesized in spermatids as a precursor protein and then processed to mature forms p45 and p105, present in mature spermatozoa.37 Both p45 and p105 still contain VWF D domains: VWF D1 + D2 in p45 and VWF D2 + D3 + D4 in p105. These VWF D domains are likely the basis for the ZP affinity of p45 + p105, as well as their multimerization and interactions with other proteins.3738 Even on SDS–PAGE, HMW forms of Zan (300–500 kDa and higher) are present along with p45 and p105 mature forms,37 a result that corroborates the presence of ZAN using blue native gel electrophoresis (Figure 1). With this molecular adhesion property, ZAN might interact with other acrosomal proteins, scaffolding them as HMW complexes for transport to the sperm APM. In fact, our immunoprecipitation results using anti-ZAN IgG captured on paramagnetic beads93 indicated that ZAN interacts with proacrosin/acrosin and ACRBP in the APM extracts (Figure 3). However, it is still unclear whether ZAN interacts directly with ACRBP or through the association of ACRBP with proacrosin. On the other hand, ZAN does not interact with SED1 (Figure 3). This result is not surprising considering that ZAN and SED1 on the sperm surface originate from different sources: ZAN from the acrosome, and SED1 from the epididymal lumen. Regardless, the interaction among the three acrosomal proteins (ZAN, proacrosin/acrosin and ACRBP) would form a basis for their co-existence in the APM complexes, and transportation of a fraction of them to the sperm APM region during capacitation would partially account for the increased amount in isolated APM vesicles in Cap sperm and the higher ZP binding affinity of the Cap gametes. A better understanding of the capacitation process should be gained by unraveling the mechanisms of how ZAN moves to the sperm APM site. ZAN should be the focus in such a protein transport study because it is the main component of the APM HMW complexes with ZP affinity, and transport of ZAN to the sperm head surface is observed in mouse spermatozoa during capacitation.65 Notably, VWF is known to form multimeric complexes, which are stored intracellularly. However, a soluble fraction of these complexes can be secreted upon the rise of [Ca2+]i, intracellular cAMP levels and pHi.95 All of these stimulations take place as part of physiological changes during sperm capacitation,9697 which might possibly trigger the traffic of ZAN to the sperm APM. In addition, the roles of TCP-1 subunits in chaperoning ZAN and other acrosomal proteins to the sperm head surface cannot be ruled out.


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) Structural domains of ZAN (adapted from Bi et al.38 and Herlyn and Zischler.98 (b) Immunoprecipitation of APM proteins with anti-ZAN IgG captured on Protein G paramagnetic beads93. Input = whole APM protein extracts; Elute = APM proteins bound to anti-ZAN beads. Results indicate interaction among ZAN, proacrosin/acrosin (ACRO) and ACRBP but not SED1 (MFGM). ZAN: zonadhesin; APM: anterior head plasma membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: (a) Structural domains of ZAN (adapted from Bi et al.38 and Herlyn and Zischler.98 (b) Immunoprecipitation of APM proteins with anti-ZAN IgG captured on Protein G paramagnetic beads93. Input = whole APM protein extracts; Elute = APM proteins bound to anti-ZAN beads. Results indicate interaction among ZAN, proacrosin/acrosin (ACRO) and ACRBP but not SED1 (MFGM). ZAN: zonadhesin; APM: anterior head plasma membrane.
Mentions: Zonadhesin is a mosaic protein comprising a number of cell adhesion-related domains (MAM, mucin, and von Willebrand factor D [VWF D]) (Figure 3a). It is synthesized in spermatids as a precursor protein and then processed to mature forms p45 and p105, present in mature spermatozoa.37 Both p45 and p105 still contain VWF D domains: VWF D1 + D2 in p45 and VWF D2 + D3 + D4 in p105. These VWF D domains are likely the basis for the ZP affinity of p45 + p105, as well as their multimerization and interactions with other proteins.3738 Even on SDS–PAGE, HMW forms of Zan (300–500 kDa and higher) are present along with p45 and p105 mature forms,37 a result that corroborates the presence of ZAN using blue native gel electrophoresis (Figure 1). With this molecular adhesion property, ZAN might interact with other acrosomal proteins, scaffolding them as HMW complexes for transport to the sperm APM. In fact, our immunoprecipitation results using anti-ZAN IgG captured on paramagnetic beads93 indicated that ZAN interacts with proacrosin/acrosin and ACRBP in the APM extracts (Figure 3). However, it is still unclear whether ZAN interacts directly with ACRBP or through the association of ACRBP with proacrosin. On the other hand, ZAN does not interact with SED1 (Figure 3). This result is not surprising considering that ZAN and SED1 on the sperm surface originate from different sources: ZAN from the acrosome, and SED1 from the epididymal lumen. Regardless, the interaction among the three acrosomal proteins (ZAN, proacrosin/acrosin and ACRBP) would form a basis for their co-existence in the APM complexes, and transportation of a fraction of them to the sperm APM region during capacitation would partially account for the increased amount in isolated APM vesicles in Cap sperm and the higher ZP binding affinity of the Cap gametes. A better understanding of the capacitation process should be gained by unraveling the mechanisms of how ZAN moves to the sperm APM site. ZAN should be the focus in such a protein transport study because it is the main component of the APM HMW complexes with ZP affinity, and transport of ZAN to the sperm head surface is observed in mouse spermatozoa during capacitation.65 Notably, VWF is known to form multimeric complexes, which are stored intracellularly. However, a soluble fraction of these complexes can be secreted upon the rise of [Ca2+]i, intracellular cAMP levels and pHi.95 All of these stimulations take place as part of physiological changes during sperm capacitation,9697 which might possibly trigger the traffic of ZAN to the sperm APM. In addition, the roles of TCP-1 subunits in chaperoning ZAN and other acrosomal proteins to the sperm head surface cannot be ruled out.

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