Localization of a syntaxin isoform, syntaxin 2, to the acrosomal region of rodent spermatozoa

Abstract

The acrosome reaction includes a membrane fusion event that is a prerequisite for sperm penetration through the zona pellucida and subsequent fertilization. Since SNARE (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis. RT‐PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B, and 2C) and syntaxin 4A, in rat testes. Immunoblot analysis with anti‐syntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation. Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was colocalized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail. Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro. In view of the putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa. Mol. Reprod. Dev. 57:375–383, 2000. © 2000 Wiley‐Liss, Inc.

A New ENU-Induced Mutant Mouse with Defective Spermatogenesis Caused by a Nonsense Mutation of the Syntaxin 2/Epimorphin (Stx2/Epim) Gene

“…Stx2/Epim has also been reported to be localized in the acrosomal region of sperm [26,27] and to be involved in the acrosome reaction in which calcium-stimulated exocytosis mediated by membrane fusion is required [28]. The present finding concerning the mutant mice did not provide any evidence regarding the involvement of Stx2/Epim in the acrosome reaction, as no mature sperm was obtained from them.…”

A New ENU-Induced Mutant Mouse with Defective Spermatogenesis Caused by a Nonsense Mutation of the Syntaxin 2/Epimorphin (Stx2/Epim) Gene

“…Stx2/Epim has also been reported to be localized in the acrosomal region of sperm [26,27] and to be involved in the acrosome reaction in which calcium-stimulated exocytosis mediated by membrane fusion is required [28]. The present finding concerning the mutant mice did not provide any evidence regarding the involvement of Stx2/Epim in the acrosome reaction, as no mature sperm was obtained from them.…”

Deficiency in the Omega-3 Fatty Acid Pathway Results in Failure of Acrosome Biogenesis in Mice1

“…Although both syntaxin 2 and syntaxin 6 isoforms show gene expression in round spermatids during spermiogenesis, according to the Mammalian Reproductive Genetics Database from University of Washington (http://mrg.genetics.washington.edu), our results demonstrated that syntaxin 2 is the main isoform involved based on its colocalization with acrosin in Fads2þ/þ and its abnormal localization in Fads2À/À. The localization of syntaxin 2 in the acrosome of Fads2þ/þ is consistent with its presence in the acrosome of mature sperm in rodents [42]. Also, our finding of the presence of syntaxin 2 in both spermatocytes and spermatids is consistent with the syntaxin 2À/À mouse phenotype [43,44].…”

Expression and Localization of Caveolin-1, and the Presence of Membrane Rafts, in Mouse and Guinea Pig Spermatozoa

“…In somatic cells, caveolins have been shown to bind specifically to G␣ subunits of heterotrimeric G proteins (Li et al, 1995), and a G protein-mediated signaling cascade is known to regulate acrosomal exocytosis (Endo et al, 1987;Ward et al, 1992;Ward et al, 1994). Further in support of this hypothesis, we demonstrated that syntaxin-2, a SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) protein suggested to be important in vesicle fusion leading to acrosomal exocytosis (Katafuchi et al, 2000), fractionates in part to membrane rafts (Fig. 8).…”

A New ENU-Induced Mutant Mouse with Defective Spermatogenesis Caused by a Nonsense Mutation of the Syntaxin 2/Epimorphin (Stx2/Epim) Gene

“…Stx2/Epim has also been reported to be localized in the acrosomal region of sperm [26,27] and to be involved in the acrosome reaction in which calcium-stimulated exocytosis mediated by membrane fusion is required [28]. The present finding concerning the mutant mice did not provide any evidence regarding the involvement of Stx2/Epim in the acrosome reaction, as no mature sperm was obtained from them.…”

Deficiency in the Omega-3 Fatty Acid Pathway Results in Failure of Acrosome Biogenesis in Mice1

“…Although both syntaxin 2 and syntaxin 6 isoforms show gene expression in round spermatids during spermiogenesis, according to the Mammalian Reproductive Genetics Database from University of Washington (http://mrg.genetics.washington.edu), our results demonstrated that syntaxin 2 is the main isoform involved based on its colocalization with acrosin in Fads2þ/þ and its abnormal localization in Fads2À/À. The localization of syntaxin 2 in the acrosome of Fads2þ/þ is consistent with its presence in the acrosome of mature sperm in rodents [42]. Also, our finding of the presence of syntaxin 2 in both spermatocytes and spermatids is consistent with the syntaxin 2À/À mouse phenotype [43,44].…”

Expression and Localization of Caveolin-1, and the Presence of Membrane Rafts, in Mouse and Guinea Pig Spermatozoa

“…In somatic cells, caveolins have been shown to bind specifically to G␣ subunits of heterotrimeric G proteins (Li et al, 1995), and a G protein-mediated signaling cascade is known to regulate acrosomal exocytosis (Endo et al, 1987;Ward et al, 1992;Ward et al, 1994). Further in support of this hypothesis, we demonstrated that syntaxin-2, a SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) protein suggested to be important in vesicle fusion leading to acrosomal exocytosis (Katafuchi et al, 2000), fractionates in part to membrane rafts (Fig. 8).…”

A New ENU-Induced Mutant Mouse with Defective Spermatogenesis Caused by a Nonsense Mutation of the Syntaxin 2/Epimorphin (Stx2/Epim) Gene

“…Stx2/Epim has also been reported to be localized in the acrosomal region of sperm [26,27] and to be involved in the acrosome reaction in which calcium-stimulated exocytosis mediated by membrane fusion is required [28]. The present finding concerning the mutant mice did not provide any evidence regarding the involvement of Stx2/Epim in the acrosome reaction, as no mature sperm was obtained from them.…”

Deficiency in the Omega-3 Fatty Acid Pathway Results in Failure of Acrosome Biogenesis in Mice1

“…Although both syntaxin 2 and syntaxin 6 isoforms show gene expression in round spermatids during spermiogenesis, according to the Mammalian Reproductive Genetics Database from University of Washington (http://mrg.genetics.washington.edu), our results demonstrated that syntaxin 2 is the main isoform involved based on its colocalization with acrosin in Fads2þ/þ and its abnormal localization in Fads2À/À. The localization of syntaxin 2 in the acrosome of Fads2þ/þ is consistent with its presence in the acrosome of mature sperm in rodents [42]. Also, our finding of the presence of syntaxin 2 in both spermatocytes and spermatids is consistent with the syntaxin 2À/À mouse phenotype [43,44].…”

Expression and Localization of Caveolin-1, and the Presence of Membrane Rafts, in Mouse and Guinea Pig Spermatozoa

“…In somatic cells, caveolins have been shown to bind specifically to G␣ subunits of heterotrimeric G proteins (Li et al, 1995), and a G protein-mediated signaling cascade is known to regulate acrosomal exocytosis (Endo et al, 1987;Ward et al, 1992;Ward et al, 1994). Further in support of this hypothesis, we demonstrated that syntaxin-2, a SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) protein suggested to be important in vesicle fusion leading to acrosomal exocytosis (Katafuchi et al, 2000), fractionates in part to membrane rafts (Fig. 8).…”