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MARCH7 E3 ubiquitin ligase is highly expressed in developing spermatids of rats and its possible involvement in head and tail formation

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Abstract

Spermatogenesis is a highly complicated metamorphosis process of male germ cells. Recent studies have provided evidence that the ubiquitin–proteasome system plays an important role in sperm head shaping, but the underlying mechanism is less understood. In this study, we localized membrane-associated RING-CH (MARCH)7, an E3 ubiquitin ligase, in rat testis. Northern blot analysis showed that March7 mRNA is expressed ubiquitously but highly in the testis and ovary. In situ hybridization of rat testis demonstrated that March7 mRNA is expressed weakly in spermatogonia and its level is gradually increased as they develop. Immunohistochemical analysis detected MARCH7 protein expression in spermiogenic cells from late round spermatids to elongated spermatids and in epididymal spermatozoa. Moreover, MARCH7 was found to be localized to the caudal end of the developing acrosome of late round and elongating spermatids, colocalizing with β-actin, a component of the acroplaxome. In addition, MARCH7 was also detected in the developing flagella and its expression levels were prominent in elongated spermatids. We also showed that MARCH7 catalyzes lysine 48 (K48)-linked ubiquitination. Immunolocalization studies revealed that K48-linked ubiquitin chains were detected in the heads of elongating spermatids and in the acrosome/acroplaxome, neck, midpiece and cytoplasmic lobes of elongated spermatids. These results suggest that MARCH7 is involved in spermiogenesis by regulating the structural and functional integrity of the head and tail of developing spermatids.

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Acknowledgments

We thank Kazue Okubo of Genostaff for in situ hybridization histochemistry, Yoko Yamamoto, Keiko Yamamichi and Ayako Takada for DNA sequencing and electron microscopy, Akira Kato for technical assistance and Yuriko Ishii for secretary assistance. This work was supported by Grant-in-Aid Scientific Research (24570209, 24117707) and by the Global COE Programs of the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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  1. Department of Biological Sciences, Tokyo Institute of Technology, 4259-B13 Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan

    Boqiang Zhao, Kunitoshi Ito, Prasanna Vasudevan Iyengar, Shigehisa Hirose & Nobuhiro Nakamura

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  1. Boqiang Zhao
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  2. Kunitoshi Ito
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Correspondence to Nobuhiro Nakamura.

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418_2012_1043_MOESM1_ESM.tif

Supplementary Fig. S1 a COS7 cells transiently transfected with FLAG-MARCH7 were stained with anti-MARCH7 antiserum (green) and anti-FLAG antibody (red). b COS7 cells transiently transfected with MARCH7 were stained with anti-MARCH7 antiserum (green) and anti-vimentin antibody (red). Nuclei were stained with Hoechst 33342 (blue). Signals were observed with a confocal microscope. Bars, 20 μm. (TIFF 1302 kb)

418_2012_1043_MOESM2_ESM.tif

Supplementary Fig. S2 Sections of rat testis were stained with preimmune serum (green in a–f). Nuclei were stained with Hoechst 33342 (blue). Signals were observed with a confocal microscope. Bar, 20 μm. (TIFF 704 kb)

418_2012_1043_MOESM3_ESM.tif

Supplementary Fig. S3 The stage-dependent expression of March7 mRNA (pink) and MARCH7 protein (green) is summarized on a map of the developmental cycle of rat spermatogenic cells in seminiferous tubules. Roman numbers indicate the stage of seminiferous epithelial cycle. Arabic numbers indicate the step of spermiogenesis. (TIFF 830 kb)

418_2012_1043_MOESM4_ESM.tif

Supplementary Fig. S4 Immunoelectron microscopy localization of MARCH7 in the elongating spermatids. Rat testicular sections were stained with anti-MARCH7 antiserum (a) or preimmune serum (b) by the pre-embedding immunoperoxidase procedure. Arrowheads indicate the acroplaxome. N, nucleus, Ac, acrosome, MT, manchette. Bars, 1 μm. (TIFF 464 kb)

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Zhao, B., Ito, K., Iyengar, P.V. et al. MARCH7 E3 ubiquitin ligase is highly expressed in developing spermatids of rats and its possible involvement in head and tail formation. Histochem Cell Biol 139, 447–460 (2013). https://doi.org/10.1007/s00418-012-1043-z

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