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ERK Is Involved in the Process of Acrosome Reaction In Vitro of the Chinese Mitten Crab, Eriocheir sinensis

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Abstract

Mitogen-activated protein kinases (MAPKs), also termed extracellular signal-regulated kinases (ERKs), are cytoplasmic and nuclear serine/threonine kinases involved in signal transduction of several extracellular effectors. In mammals, ERKs participate in the regulation of spermatogenesis, mature spermatozoa motility, hyperactivation, and the acrosome reaction. To investigate ERK functions in Eriocheir sinensis reproduction, we successfully cloned the full-length ERK from the testis of E. sinensis (ES-ERK). The 1098-nucleotide open reading frame encodes a 365-amino-acid protein with a predicted molecular weight of 42 kDa. Expressions of ES-ERK in different tissues and testis development stages were detected by the quantitative RT-PCR and Western blotting. ES-ERK is expressed relatively highly in the testis. The expression of ES-ERK protein gradually increased in the spermatid stage, reaching a peak in sperm stage. Western blotting showed a similar expression pattern for the total ES-ERK protein, but phospho-ERK (p-ERK) showed the higher expression in spermatid than sperm stage. We also used trypan blue and hematoxylin and eosin staining to identify structural changes in E. sinensis spermatozoa during the process of acrosome reaction (AR). After stimulating the process of AR, the ES-ERK has translocated from the nucleus to the acrosomal tubule. This result suggested that the ERK MAPK might be involved in the regulation of the E. sinensis acrosome reaction.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 41376157 and No. 31172393), the Shanghai Natural Science Fund Committee (No. 12ZR1408900). We thank the Instruments Sharing Platform of School of Life Sciences, East China Normal University for the equipment support.

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    Authors and Affiliations

    1. School of Life Science, East China Normal University, Shanghai, China

      Wen-Juan Sun, Ming Zhu, Yuan-Li Wang, Qing Li, Hong-Dan Yang, Ze-Lin Duan, Lin He & Qun Wang

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    1. Wen-Juan Sun
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    2. Ming Zhu
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    3. Yuan-Li Wang
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    4. Qing Li
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    Correspondence to Lin He or Qun Wang.

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    Wen-Juan Sun, Ming Zhu and Yuan-Li Wang contributed equally to this work.

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    Fig. S1

    The cDNA of E.s-ERK gene and its deduced amino acid sequences. Red font indicates the T-E-Y motif (TIFF 2016 kb)

    High resolution image (GIF 153 kb)

    Fig. S2

    Multiple sequence alignment of ERK proteins. “ * ” indicates identical, “ · ” indicates very similar, “ : ” indicates similar; red triangles mark ATP binding sites; yellow triangles mark substrate binding sites; blue triangles mark KIM docking sites; a black rectangle surrounds the A-loop ; and a red rectangle surrounds the T-E-Y motif (TIFF 3704 kb)

    High resolution image (GIF 498 kb)

    Fig. S3

    Neighbor-joining phylogenetic tree of the ES-ERK protein and similar proteins. Numbers indicate the percentage frequencies at which the phylogram topology was representative for 1000 bootstrap replicates. A red triangle indicates the E.s-ERK protein (TIFF 445 kb)

    High resolution image (GIF 73 kb)

    Fig. S4

    Sperm of Eriocheir sinensis stained with trypan blue. Live sperm (LS) have radium arms (RA) and acrosome (A) is transparent (arrows in a). The RA of dead sperm (DS) has disappeared and the acrosome is stained dark blue (arrow in a) (TIFF 519 kb)

    High resolution image (GIF 92 kb)

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    Sun, WJ., Zhu, M., Wang, YL. et al. ERK Is Involved in the Process of Acrosome Reaction In Vitro of the Chinese Mitten Crab, Eriocheir sinensis . Mar Biotechnol 17, 305–316 (2015). https://doi.org/10.1007/s10126-015-9619-y

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