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Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy

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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.

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Acknowledgment

This study was partly performed by using a nanoscopy TCS STED CW (Leica Microsystems, Tokyo). The authors are grateful to Professor Ikawa and Professor Okabe at the Research Institute for Microbial Diseases, Osaka University for kindly providing the pCM vector for the mouse Calmegin promotor and to Ms. K Kamimura and Mr. T Mutoh for their excellent technical assistance.

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Authors and Affiliations

  1. Department of Reproductive Biology and Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan

    Chizuru Ito, Kenji Yamatoya & Kiyotaka Toshimori

  2. Biomedical Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan

    Kenji Yamatoya, Lisa Fujimura & Masahiko Hatano

  3. Ministry of Education, Culture, Sports, Science and Technology (MEXT), 3-2-2 Kasumigaseki, Chiyoda-ku, Tokyo, 100-8959, Japan

    Keiichi Yoshida

  4. Department of Biomedical Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan

    Masahiko Hatano

  5. Department of Reproductive Biology, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo, 157-8535, Japan

    Kenji Miyado

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  1. Chizuru Ito
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  2. Kenji Yamatoya
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  4. Lisa Fujimura
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  5. Masahiko Hatano
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  6. Kenji Miyado
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  7. Kiyotaka Toshimori
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Corresponding author

Correspondence to Chizuru Ito.

Additional information

This work was supported by the Japan Society for the Promotion of Science partially via grants to K.T. (22112504, 22390033, 24112706) and in part by grants to C.I. (24592441) and to K.Y. (23791809).

The authors declare no conflicts of interest.

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Ito, C., Yamatoya, K., Yoshida, K. et al. Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy. Cell Tissue Res 352, 739–750 (2013). https://doi.org/10.1007/s00441-013-1605-y

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  • DOI: https://doi.org/10.1007/s00441-013-1605-y

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