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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Almog T, Naor Z (2008) Mitogen activated protein kinases (MAPKs) as regulators of spermatogenesis and spermatozoa functions. Mol Cell Endocrinol 282:39–44. doi:10.1016/j.mce.2007.11.011
Amjad F, Zhou MM (2004) Structure and regulation of MAPK phosphatases. Cell Signal 16:769–779. doi:10.1016/j.cellsig.2003.12.008
Ashizawa K, Hashimoto K, Higashio M, Tsuzuki Y (1997) The addition of mitogen-activated protein kinase and p34 kinase substrate peptides inhibits the flagellar motility of demembranated fowl spermatozoa. Biochem Biophys Res Commun 240:116–121. doi:10.1006/bbrc.1997.7626
Austin CR (1951) Observations on the penetration of the sperm into the mammalian egg. Aust J Biol Sci 4:581–596. doi:10.1071/B19510581
Awda BJ, Buhr MM (2010) Extracellular signal-regulated kinases (ERKs) pathway and reactive oxygen species regulate tyrosine phosphorylation in capacitating boar spermatozoa. Biol Reprod 83:750–758. doi:10.1095/biolreprod.109.082008
Brunet A, Roux D, Lenormand P, Dowd S, Keyse S, Pouyssegur J (1999) Nuclear translocation of p42/p44 mitogen activated protein kinase is required for growth factor induced gene expression and cell cycle entry. EMBO J 18:664–674
Du NS (1998) The fertilization of Eriocheir sinensis. Aquat Sci Technol Inf 25:001
Du NS, Lai W, Xue LZ (1987a) Studies on the sperm of Chinese mitten-handed crab, Ericheir sinensis. Oceanol Limnol Sin 2:001
Du NS, Lai W, Xue LZ (1987b) Acrosome reaction of the sperm of Chinese mitten-handed crab. Ericheir sinensis Acta Zool Sin 1:001
Du NS, Xue LZ, Lai W (1988) Histology of male reproductive system in Chinese mitten-handed crab, Ericheir sinensis. Acta Zool Sin 1:001
Elisabetta T (1994) Sperm activation in species with external fertilisation. Cambridge University Press 359:361
Feld M, Dimant B, Delorenzi A, Coso O, Romano A (2005) Phosphorylation of extra-nuclear ERK/MAPK is required for long-term memory consolidation in the crab. Chasmagnathus Behav Brain Res 158:251–261. doi:10.1016/j.bbr.2004.09.005
Go H, Hwang HJ, Nam TJ (2011) Polysaccharides from Capsosiphon fulvescens stimulate the growth of IEC-6 Cells by activating the MAPK signaling pathway. Mar Biotechnol (NY) 13:433–440. doi:10.1007/s10126-010-9314-y
Gutkind JS (1998) The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 273:1839–1842. doi:10.1074/jbc.273.4.1839
He L, Jiang H, Cao DD, Liu LH, Hu SN, Wang Q (2013) Comparative transcriptome analysis of the accessory sex gland and testis from the Chinese mitten crab (Eriocheir sinensis). PLoS ONE 8:e53915. doi:10.1371/journal.pone.0053915
He L, Wang Q, Jin XK, Wang Y, Chen LL, Liu LH, Wang Y (2012) Transcriptome profiling of testis during sexual maturation stages in Eriocheir sinensis using Illumina sequencing. PLoS ONE 7:e33735. doi:10.1371/journal.pone.0033735
Inselman A, Handel MA (2004) Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes. Biol Reprod 71:570–578. doi:10.1095/biolreprod.104.027938
Kang XJ, Li GL, Mu SM, Guo MS, Ge SQ (2009) Acrosome reaction of Chinese mitten-handed crab Eriocheir sinensis (Crustacea: Decapoda) spermatozoa: promoted by long-term cryopreservation. Aquaculture 295:195–199. doi:10.1016/j.aquaculture.2009.06.041
Li MW, Mruk DD, Cheng CY (2009) Mitogen-activated protein kinases in male reproductive function. Trends Mol Med 15:159–168. doi:10.1016/j.molmed.2009.02.002
Li Z, Zhu JQ, Yang WX (2010) Acrosome reaction in Octopus tankahkeei induced by calcium ionophore A23187 and a possible role of the acrosomal screw. Micron 41:39–46. doi:10.1016/j.micron.2009.08.001
Luconi M, Krausz C, Barni T, Vannelli GB, Forti G, Baldi E (1998) Progesterone stimulates p42 extracellular signal-regulated kinase in human spermatozoa. Mol Hum Reprod 4:251–258
Ma AN, Wang YL, Zou ZH, Fu MJ, Lin P, Zhang ZP (2012) Erk2 in ovarian development of green mud crab Scylla paramamosain. DNA Cell Biol 31:1233–1244. doi:10.1089/dna.2011.1458
Ma Q, Ding YT, Qu D (2007) The spermatozoa viability detected by eosin and Trypan blue Chinese. J Zool 42:65–69
Minagawa M, Chiu J-R, Kudo M, Takashima F (1994) Male reproductive biology of the red frog crab, Ranina ranina, off Hachijojima, Izu Islands, Japan. Mar Biol 118:393–401. doi:10.1007/BF00350296
Mizuno R et al (2014) In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines. J Exp Med 211:1123–1136
Naz RK, Rajesh PB (2004) Role of tyrosine phosphorylation in sperm capacitation/acrosome reaction. Reprod Biol Endocrinol : RB&E 2:75. doi:10.1186/1477-7827-2-75
Nixon B, Bielanowicz A, Anderson AL, Walsh A, Hall T, McCloghry A, Aitken RJ (2010) Elucidation of the signaling pathways that underpin capacitation-associated surface phosphotyrosine expression in mouse spermatozoa. J Cell Physiol 224:71–83. doi:10.1002/jcp.22090
Nixon B, MacIntyre DA, Mitchell LA, Gibbs GM, O'Bryan M, Aitken RJ (2006) The identification of mouse sperm-surface-associated proteins and characterization of their ability to act as decapacitation factors. Biol Reprod 74:275–287. doi:10.1095/biolreprod.105.044644
O'Flaherty C, de Lamirande E, Gagnon C (2005) Reactive oxygen species and protein kinases modulate the level of phospho-MEK-like proteins during human sperm capacitation. Biol Reprod 73:94–105. doi:10.1095/biolreprod.104.038794
Slotta KH, Primosigh J (1951) Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168:697–698
Sun X, He Y, Hou L (2010) Myosin VA participates in acrosomal formation and nuclear morphogenesis during spermatogenesis of Chinese mitten crab Eriocheir sinensis. PLoS ONE 5:e12738. doi:10.1371/journal.pone.0012738.g001
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P, Kopf GS (1995) Capacitation of mouse spermatozoa I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 121:1129–1137
Wang J, Fang DA, Wang Y (2013) Cathepsin A protein from the accessory sex gland of the Chinese mitten crab (Eriocheir sinensis) plays a key role in spermatophore digestion. Insect Physiol 59:953–960. doi:10.1016/j.jinsphys.2013.07.003
Wang YL, Li Q, Xie J, Zhu M, Sun WJ, He L, Wang Q (2014) Involvement of the single Cul4 gene of Chinese mitten crab Eriocheir sinensis in spermatogenesis. Gene 536:9–17. doi:10.1016/j.gene.2013.11.099
Widmann C, Gibson S, Jarpe MB, Johnson GL (1999) Mitogen-activated protein kinase conservation of a three-kinase module from yeast to human. Am Physiol Soc 79:143–180
Yoshinaga K, Toshimori K (2003) Organization and modifications of sperm acrosomal molecules during spermatogenesis and epididymal maturation. Microsc Res Tech 61:39–45. doi:10.1002/jemt.10315
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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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)
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)
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)
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)
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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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DOI: https://doi.org/10.1007/s10126-015-9619-y