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Marine Biotechnology

, Volume 13, Issue 3, pp 536–543 | Cite as

Ca2+, Mg2+-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin Strongylocentrotus intermedius Induced by Two-Headed Sphingolipid Rhizochalin

  • Juriy T. Sibirtsev
  • Valeria V. ShastinaEmail author
  • Natalia I. Menzorova
  • Tatyana N. Makarieva
  • Valeriy A. Rasskazov
Original Article

Abstract

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca2+, Mg2+-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn2+ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.

Keywords

Sea urchin Spermatozoa Ca2+ Mg2+-dependent DNase Two-headed sphingolipids Apoptosis Caspase 

Abbreviations

EGTA

Ethyleneglycoltetraacetic acid

EDTA

Ethylenediaminetetraacetic acid

DTT

Dethiothreithol

PMSF

Phenylmethylsulfonyl fluoride

SDS

Sodium dodecyl sulfate

PI

Propidium iodide

FDA

Fluorescein diacetate

FAM-LEHD-fmk

Carboxyfluorescein analog of benzyloxycarbonyl-Leu-Glu-His-Asp(OMe) fluoromethyl ketone (z-LEHD-fmk)

FAM-IETD-fmk

Carboxyfluorescein analog of benzyloxycarbonyl-Ile-Glu-Thr-Asp(OMe)fluoromethyl ketone (z-IETD-fmk)

FAM-DEVD-fmk

Carboxyfluorescein analog of benzyloxycarbonyl-Asp(OMe)-Glu(O-Me)-Val-Asp(O-Me) fluoromethyl ketone (z-DEVD-fmk)

Notes

Acknowledgments

This work was supported by RFBR Grant No. 08-08-00975 and the grant of the Russian Academy of Sciences on Program of Fundamental Research “Physicochemical biology FEB RAS” (No. 09-I-П22-05) and RFBR Grant 09-04-00015-a.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Juriy T. Sibirtsev
    • 1
  • Valeria V. Shastina
    • 1
    Email author
  • Natalia I. Menzorova
    • 1
  • Tatyana N. Makarieva
    • 1
  • Valeriy A. Rasskazov
    • 1
  1. 1.Laboratory of Marine Biochemistry, Pacific Institute of Bioorganic ChemistryFar East Division of Russian Academy of SciencesVladivostokRussian Federation

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