Limits...
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 and b) Zan targets to the pig sperm head surface during incubation in capacitating medium. (a) Merged immunofluorescence (panels a–d) and phase contrast (panels g–j) images are shown in the left column, whereas the corresponding flow cytometry histograms are shown on the right. Spermatozoa were incubated in capacitating medium for 30 and 60 min. (b) Kinetics of the numbers of sperm with ZAN on the head surface is shown as the function of capacitation time, together with the population of acrosome reacted sperm (negatively stained with PSA). (c) Immunofluorescence and flow cytometry indicating that only a fraction of ZAN was targeted to the sperm surface and that most of the protein remained in the acrosome of nitrogen cavitated sperm. (d) Immunofluorescence of SED1 (MFGM) on spermatozoa before and after nitrogen cavitation. Results indicate the same fluorescence pattern/intensity of SED1 in Noncap and Cap sperm and the absence of the protein in the acrosome of nitrogen-cavitated gametes. Zan: zonadhesin; Noncap: noncapacitated; Cap: capacitated; PSA: pisum sativum agglutinin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492047&req=5

Figure 2: (a and b) Zan targets to the pig sperm head surface during incubation in capacitating medium. (a) Merged immunofluorescence (panels a–d) and phase contrast (panels g–j) images are shown in the left column, whereas the corresponding flow cytometry histograms are shown on the right. Spermatozoa were incubated in capacitating medium for 30 and 60 min. (b) Kinetics of the numbers of sperm with ZAN on the head surface is shown as the function of capacitation time, together with the population of acrosome reacted sperm (negatively stained with PSA). (c) Immunofluorescence and flow cytometry indicating that only a fraction of ZAN was targeted to the sperm surface and that most of the protein remained in the acrosome of nitrogen cavitated sperm. (d) Immunofluorescence of SED1 (MFGM) on spermatozoa before and after nitrogen cavitation. Results indicate the same fluorescence pattern/intensity of SED1 in Noncap and Cap sperm and the absence of the protein in the acrosome of nitrogen-cavitated gametes. Zan: zonadhesin; Noncap: noncapacitated; Cap: capacitated; PSA: pisum sativum agglutinin.

Mentions: Besides ZAN, proacrosin, ACRBP, SP10 and ZPBP1 are ZP binding proteins present in the three HMW complexes (Figure 1d). All of these proteins are known to localize in the acrosome. Previous studies indicated that vesicles of hybrid membranes (APM and outer acrosomal membrane) existed in the vesicle preparation from pig sperm subjected to nitrogen cavitation at 650 psi (called APM vesicles in this review).94 ZAN was localized to the outer acrosomal membrane and acrosomal matrix.3857 Therefore, its existence as revealed by LC–MS/MS in the HMW complexes might reflect these previous findings. The question relevant to the physiology of sperm–ZP interaction, however, remains: are acrosomal proteins present in the APM HMW complexes exposed on the sperm head surface, so that they can bind to the ZP? Our immunofluorescence and flow cytometry of ZAN on intact pig sperm indeed revealed that ZAN was not present on the head surface in the majority of spermatozoa resuspended in medium that did not support capacitation. However, the percentage of sperm that were positively labeled with anti-ZAN increased when sperm were incubated in capacitating medium (containing albumin, bicarbonate, and CaCl2) for 30 min (Figure 2a). In addition, the immunofluorescence intensity of ZAN increased in these spermatozoa. Both the percentage of anti-ZAN labeled sperm and the immunofluorescence intensity peaked at 60 min incubation in capacitating medium (Figure 2b). However, most spermatozoa (≥80%) were still acrosome-intact as shown by the binding of FITC-labeled Pisum sativum agglutinin to their acrosomal matrix (Figure 2b). Corroborating this result is the observation that ZAN remained in the acrosome of nitrogen-cavitated spermatozoa with a much higher level of immunofluorescence intensity than that present on the head surface of the corresponding Cap acrosome-intact sperm (Figure 2c). All of these results indicate that a fraction of ZAN is transported from the acrosome to the sperm head surface during capacitation. Immunofluorescence and flow cytometry of proacrosin/acrosin show the same trend as ZAN in terms of their transport to the sperm head surface.93 However, the transportation of ACRBP (sp32) to the sperm head surface appeared to be much earlier than that of ZAN and proacrosin/acrosin. ACRBP was present on the head surface of almost all Noncap spermatozoa and this distribution remained the same when sperm were incubated in capacitating medium, although the intensity of the immunofluorescence increased slightly. ACRBP possesses a specific affinity to proacrosin (53 kDa) but not to the intermediate and mature forms of acrosin (43 and 35 kDa, respectively). All of these proacrosin/acrosin forms are present in Noncap and Cap spermatozoa. Therefore, the overall results suggest that ACRBP targets to the head surface of Noncap sperm independently of proacrosin. While the transport of ZAN and proacrosin (both with known ZP affinity) to the APM region of Cap sperm is likely beneficial for interaction with the ZP, the benefit of having ACRBP is still a matter of investigation. To date, direct affinity of ACRBP for the ZP has not been demonstrated.


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 and b) Zan targets to the pig sperm head surface during incubation in capacitating medium. (a) Merged immunofluorescence (panels a–d) and phase contrast (panels g–j) images are shown in the left column, whereas the corresponding flow cytometry histograms are shown on the right. Spermatozoa were incubated in capacitating medium for 30 and 60 min. (b) Kinetics of the numbers of sperm with ZAN on the head surface is shown as the function of capacitation time, together with the population of acrosome reacted sperm (negatively stained with PSA). (c) Immunofluorescence and flow cytometry indicating that only a fraction of ZAN was targeted to the sperm surface and that most of the protein remained in the acrosome of nitrogen cavitated sperm. (d) Immunofluorescence of SED1 (MFGM) on spermatozoa before and after nitrogen cavitation. Results indicate the same fluorescence pattern/intensity of SED1 in Noncap and Cap sperm and the absence of the protein in the acrosome of nitrogen-cavitated gametes. Zan: zonadhesin; Noncap: noncapacitated; Cap: capacitated; PSA: pisum sativum agglutinin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: (a and b) Zan targets to the pig sperm head surface during incubation in capacitating medium. (a) Merged immunofluorescence (panels a–d) and phase contrast (panels g–j) images are shown in the left column, whereas the corresponding flow cytometry histograms are shown on the right. Spermatozoa were incubated in capacitating medium for 30 and 60 min. (b) Kinetics of the numbers of sperm with ZAN on the head surface is shown as the function of capacitation time, together with the population of acrosome reacted sperm (negatively stained with PSA). (c) Immunofluorescence and flow cytometry indicating that only a fraction of ZAN was targeted to the sperm surface and that most of the protein remained in the acrosome of nitrogen cavitated sperm. (d) Immunofluorescence of SED1 (MFGM) on spermatozoa before and after nitrogen cavitation. Results indicate the same fluorescence pattern/intensity of SED1 in Noncap and Cap sperm and the absence of the protein in the acrosome of nitrogen-cavitated gametes. Zan: zonadhesin; Noncap: noncapacitated; Cap: capacitated; PSA: pisum sativum agglutinin.
Mentions: Besides ZAN, proacrosin, ACRBP, SP10 and ZPBP1 are ZP binding proteins present in the three HMW complexes (Figure 1d). All of these proteins are known to localize in the acrosome. Previous studies indicated that vesicles of hybrid membranes (APM and outer acrosomal membrane) existed in the vesicle preparation from pig sperm subjected to nitrogen cavitation at 650 psi (called APM vesicles in this review).94 ZAN was localized to the outer acrosomal membrane and acrosomal matrix.3857 Therefore, its existence as revealed by LC–MS/MS in the HMW complexes might reflect these previous findings. The question relevant to the physiology of sperm–ZP interaction, however, remains: are acrosomal proteins present in the APM HMW complexes exposed on the sperm head surface, so that they can bind to the ZP? Our immunofluorescence and flow cytometry of ZAN on intact pig sperm indeed revealed that ZAN was not present on the head surface in the majority of spermatozoa resuspended in medium that did not support capacitation. However, the percentage of sperm that were positively labeled with anti-ZAN increased when sperm were incubated in capacitating medium (containing albumin, bicarbonate, and CaCl2) for 30 min (Figure 2a). In addition, the immunofluorescence intensity of ZAN increased in these spermatozoa. Both the percentage of anti-ZAN labeled sperm and the immunofluorescence intensity peaked at 60 min incubation in capacitating medium (Figure 2b). However, most spermatozoa (≥80%) were still acrosome-intact as shown by the binding of FITC-labeled Pisum sativum agglutinin to their acrosomal matrix (Figure 2b). Corroborating this result is the observation that ZAN remained in the acrosome of nitrogen-cavitated spermatozoa with a much higher level of immunofluorescence intensity than that present on the head surface of the corresponding Cap acrosome-intact sperm (Figure 2c). All of these results indicate that a fraction of ZAN is transported from the acrosome to the sperm head surface during capacitation. Immunofluorescence and flow cytometry of proacrosin/acrosin show the same trend as ZAN in terms of their transport to the sperm head surface.93 However, the transportation of ACRBP (sp32) to the sperm head surface appeared to be much earlier than that of ZAN and proacrosin/acrosin. ACRBP was present on the head surface of almost all Noncap spermatozoa and this distribution remained the same when sperm were incubated in capacitating medium, although the intensity of the immunofluorescence increased slightly. ACRBP possesses a specific affinity to proacrosin (53 kDa) but not to the intermediate and mature forms of acrosin (43 and 35 kDa, respectively). All of these proacrosin/acrosin forms are present in Noncap and Cap spermatozoa. Therefore, the overall results suggest that ACRBP targets to the head surface of Noncap sperm independently of proacrosin. While the transport of ZAN and proacrosin (both with known ZP affinity) to the APM region of Cap sperm is likely beneficial for interaction with the ZP, the benefit of having ACRBP is still a matter of investigation. To date, direct affinity of ACRBP for the ZP has not been demonstrated.

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