title: Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy.
abstract: Spermatids must precisely integrate specific molecules into structurally supported domains that develop during spermatogenesis. Once established, the architecture of the acrosome contributes to the acrosome reaction, which occurs prior to gamete interaction in mammals. The present study aims to clarify the morphology associated with the integration of the mouse fertilization-related acrosomal protein equatorin (mEQT) into the developing acrosome. EQT mRNA was first detected by in situ hybridization in round spermatids but disappeared in early elongating spermatids. The molecular size of mEQT was approximately 65 kDa in the testis. Developmentally, EQT protein was first detected on the nascent acrosomal membrane in round spermatids at approximately step 3, was actively integrated into the acrosomal membranes of round spermatids in the following step and then participated in acrosome remodeling in elongating spermatids. This process was clearly visualized by high-resolution fluorescence microscopy and super-resolution stimulated emission depletion nanoscopy by using newly generated C-terminally green-fluorescent-protein-tagged mEQT transgenic mice. Immunogold electron microscopy revealed that mEQT was anchored to the acrosomal membrane, with the epitope region observed as lying 5-70 nm away from the membrane and was associated with the electron-dense acrosomal matrix. This new information about the process of mEQT integration into the acrosome during spermatogenesis should provide a better understanding of the mechanisms underlying not only acrosome biogenesis but also fertilization and male infertility.
authors.affiliation: Department of Reproductive Biology and Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. chizuru@faculty.chiba-u.jp
authors.forename: Chizuru
authors.initials: C
authors.lastname: Ito
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamatoya
authors.forename: Keiichi
authors.initials: K
authors.lastname: Yoshida
authors.forename: Lisa
authors.initials: L
authors.lastname: Fujimura
authors.forename: Masahiko
authors.initials: M
authors.lastname: Hatano
authors.forename: Kenji
authors.initials: K
authors.lastname: Miyado
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Toshimori
mesh_terms: D000177:Acrosome / Q000378:metabolism* / Q000648:ultrastructure*; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D005260:Female; D005306:Fertilization / Q000235:genetics; D005452:Fluoresceins / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017403:In Situ Hybridization; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016253:Microscopy, Immunoelectron / Q000379:methods*; D019887:Peanut Agglutinin / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013091:Spermatogenesis / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism; D013737:Testis / Q000166:cytology
chemical_list: D000906:Antibodies; C512717:Eqtn protein, mouse; D005452:Fluoresceins; D008565:Membrane Proteins; D019887:Peanut Agglutinin; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; C468515:fluorescein isothiocyanate-peanut agglutinin; D049452:Green Fluorescent Proteins