Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

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)

References

  • Allan J, Hortman P, Crone-Robinson C, Aviles F (1980) The structure of histone H1 and its location in chromatin. Nature 288:675–679

    Article  PubMed  CAS  Google Scholar 

  • Anzar M, He L, Buhr M, Kroetsch T, Pauls K (2002) Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility. Biol Reprod 66:354–360

    Article  PubMed  CAS  Google Scholar 

  • Aziz N, Said T, Paasch U, Agarwal A (2007) The relationship between human sperm apoptosis, morphology and the sperm deformity index. Hum Reprod 22:1413–1419

    Article  PubMed  Google Scholar 

  • Boaz S, Domonguez K, Shaman J, Ward S (2008) Mouse spermatozoa contain a nuclease that is activated by pretreatment with EGTA and subsequent calcium incubation. J Cell Biochem 103:1636–1645

    Article  PubMed  CAS  Google Scholar 

  • Jin J-O, Shastina VV, Park JI, Han JY, Makarieva TN, Fedorov SN, Rasskazov V, Stonik V, Kwak JY (2009) Differential induction of apoptosis of leukemic cells by rhizochalin, two headed sphingolipids from sponge and its derivatives. Biol Pharm Bull 32:955–962

    Article  PubMed  CAS  Google Scholar 

  • Makarieva TN, Denisenko VA, Stonik VA, Milgrom YM, Rashkes YV (1989) Rhizochalin, a novel secondary metabolite of mixed biosynthesis from the sponge Rhizochalina incrústala. Tetrahedron Lett 30:6581–6584

    Article  CAS  Google Scholar 

  • Marchetti C, Gallego MA, Defossez A, Formstecher P, Marchetti P (2004) Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters. Hum Reprod 19:1127–1134

    Article  PubMed  CAS  Google Scholar 

  • Martin G, Cagnon N, Sabido O, Sion B, Grizard G, Durand P, Levy R (2007) Kinetics of occurrence of some features of apoptosis during the cryopreservation process of bovine spermatozoa. Hum Reprod 22:380–388

    Article  PubMed  CAS  Google Scholar 

  • Oehninger S, Morshedi M, Weng SL, Taylor S, Duran H, Beebe S (2003) Presence and significance of somatic cell apoptosis markers in human ejaculated spermatozoa. Reprod Biomed Online 7:469–476

    Article  PubMed  Google Scholar 

  • Ortega-Ferrusola C, Sotillo-Galan Y, Varela-Fernandez E, Gallardo-Bolanos JM, Muriel A, Gonzalez-Fernandez L, Tapia JA, Pena FJ (2008) Detection of “apoptosis-like” changes during the cryopreservation process in equine sperm. J Androl 29:213–221

    Article  PubMed  CAS  Google Scholar 

  • Paasch U, Sharma RK, Gupta AK, Grunewald S, Mascha EJ, Thomas AJ, Glander HJ, Agarwal A (2004) Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Biol Reprod 71:1828–1837

    Article  PubMed  CAS  Google Scholar 

  • Pandey S, Wolker PR, Sikorska M (1997) Identification of a novel 97 kDa endonuclease capable of internucleosomal DNA cleavage. Biochemistry 36:711–720

    Article  PubMed  CAS  Google Scholar 

  • Peitsch MC, Mannherz HG, Tschopp J (1994) The apoptosis endonucleases: cleaning up after cell death? Trends Cell Biol 4:37–41

    Article  PubMed  CAS  Google Scholar 

  • Print CG, Loveland KL (2000) Germ cell suicide: new insights into apoptosis during spermatogenesis. Bioessays 22:423–430

    Article  PubMed  CAS  Google Scholar 

  • Shaman JA, Prisztoka R, Ward WS (2006) Topoisomerase IIB and an extracellular nuclease interact to digest sperm DNA in an apoptotic-like manner. Biol Reprod 75:741–748

    Article  PubMed  CAS  Google Scholar 

  • Shastina VV, Menzorova NI, Sibirtzev YT, Rasskazov VA (2003) Purification and characteristics of Ca2+, Mg2+- and Ca2+, Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius. Biochemistry (Moscow) 68:712–724

    Article  Google Scholar 

  • Shi Y (2002) Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 9:459–470

    Article  PubMed  CAS  Google Scholar 

  • Shiokawa D, Ohyama H, Yamada T, Tanuma S (1997) Purification and properties of DNase gamma from apoptotic rat thymocytes. Biochem J 326:675–681

    PubMed  CAS  Google Scholar 

  • Sotolongo B, Huang T, Isenberger E, Ward S (2005) An endogenous nuclease in hamster, mouse and human spermatozoa cleaves DNA into loop-sized fragments. J Androl 26:272–280

    PubMed  CAS  Google Scholar 

  • Voronina E, Wessel G (2001) Apoptosis in sea urchin oocytes, eggs, and early embryos. Mol Reprod Dev 60:553–561

    Article  PubMed  CAS  Google Scholar 

  • Widlak P, Garrard WT (2005) Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. J Cell Biochem 94:1078–1087

    Article  PubMed  CAS  Google Scholar 

  • Wündrich K, Paasch U, Leicht M, Glander HJ (2006) Activation of caspases in human spermatozoa during cryopreservation—an immunoblot study. Cell Tissue Bank 7:81–90

    Article  PubMed  Google Scholar 

  • Yakovlev AG, Wang G, Stoika BA, Simbulan-Rosenthal CM, Yoshihara K, Smulson ME (1999) Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments. Nucleic Acids Res 27:1999–2005

    Article  PubMed  CAS  Google Scholar 

  • Yoshihara K, Tonigawa Y, Burzio L, Koide SS (1975) Evidence for adenosine diphosphate ribosylation of Ca2+, Mg2+-dependent endonucleases. Proc Natl Acad Sci U S A 72:289–293

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Laboratory of Marine Biochemistry, Pacific Institute of Bioorganic Chemistry, Far East Division of Russian Academy of Sciences, 690022, pr. 100 let Vladivostoku, 159, Vladivostok, Russian Federation

    Juriy T. Sibirtsev, Valeria V. Shastina, Natalia I. Menzorova, Tatyana N. Makarieva & Valeriy A. Rasskazov

Authors
  1. Juriy T. Sibirtsev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Valeria V. Shastina
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Natalia I. Menzorova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tatyana N. Makarieva
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Valeriy A. Rasskazov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Valeria V. Shastina.

Additional information

Juriy T. Sibirtsev and Valeria V. Shastina have contributed equally.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sibirtsev, J.T., Shastina, V.V., Menzorova, N.I. et al. Ca2+, Mg2+-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin Strongylocentrotus intermedius Induced by Two-Headed Sphingolipid Rhizochalin. Mar Biotechnol 13, 536–543 (2011). https://doi.org/10.1007/s10126-010-9324-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-010-9324-9

Keywords

Search

Navigation