Skip to main content
Download PDF

Flow cytometry based techniques to study testicular acidophilic granulocytes from the protandrous fish gilthead seabream (Sparus aurata L.)

Biological Procedures Online volume 6, pages 129–136 (2004)Cite this article

Abstract

The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation, and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular interleukin-1β. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead seabream testicular acidophilic granulocytes and permits their quantification.

Abbreviations

BW:

body weight

BSA:

bovine serum albumin

SR-2:

steroid-free synthetic serum

PBS:

phosphate-buffered saline

FCS:

fetal calf serum

FITC:

fluorescein isothiocyanate

FSC:

forward scatter

SSC:

side scatter

TSB:

trypticase soy broth

FC:

flow cytometry

ANOVA:

one-way analysis of variance

mAb:

monoclonal antibody

IL-1β:

interleukin-1β

References

  1. Hess RA. Spermatogenesis, overview. In: Knobil E, Neil JD, editors. Encyclopedia of Reproduction volumen 4. New York: Academic Press; 1999. p.539–545.

    Google Scholar 

  2. Miura T. Spermatogenetic cycle in fish. In: Knobil E, Neil JD, editors. Encyclopedia of Reproduction volumen 4. New York: Academic Press; 1999. p. 571–578.

    Google Scholar 

  3. Hales DB. Testicular macrophage modulation of Leydig cell steroidogenesis. J Reprod Immunol 2002; 57:3–18.

    Article  PubMed  CAS  Google Scholar 

  4. Loir M. In vitro approach to the control of spermatogonia proliferation in the trout. Mol Cell Endocrinol 1994; 102:141–150.

    Article  PubMed  CAS  Google Scholar 

  5. Miura T, Yamauchi K, Takahashi H, Nagahama Y. Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica). Proc Natl Acad Sci USA 1991; 88:5774–5778.

    Article  PubMed  CAS  Google Scholar 

  6. Loir M, Sourdaine P. Testes cells: isolation and culture. In: Hochachka, Mommsen editors. Biochemistry and Molecular Biology of Fishes volumen 3. Elsevier Science Press; 1994. p. 249–272.

  7. Sepulcre MP, PelegrÍn P, Mulero V, Meseguer J. Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response. Cell Tissue Res 2002; 308:97–102.

    Article  PubMed  CAS  Google Scholar 

  8. Esteban MA, Mulero V, Muñoz J, Meseguer J. Methodological aspects of assesing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy. Cell Tissue Res 1998; 293:133–141.

    Article  PubMed  CAS  Google Scholar 

  9. Stanley H, Chieffi G, Botte V. Histological and histochemical observations on the testis of Gobius paganellus. Z Zellforsch 1965; 65:350–362.

    Article  PubMed  CAS  Google Scholar 

  10. Shrestha TK, Khanna SS. Histology and seasonal changes in the testes of a hill-stream fish Schizothorax plagiostomus. Z Mikrosk Anat Forsch 1976; 90:749–761.

    PubMed  CAS  Google Scholar 

  11. Shrestha TK, Khanna SS. Seasonal changes in the testes of a hill stream teleost, Garra gotyla (Gray). Acta Anat 1978; 100:210–220.

    Article  PubMed  CAS  Google Scholar 

  12. Micale V, Perdichizzi F, Santangel G. The gonadal cycle of captive white bream, Diplodus sargus (L.). J Fish Biol 1986; 31:435–440.

    Article  Google Scholar 

  13. Besseau L, Faliex E. Resorption of unemitted gametes in Lithognathus mormyrus (Sparidae, Teleostei): a possible synergic action of somatic and immune cells. Cell Tissue Res 1994; 276:123–132

    Article  PubMed  CAS  Google Scholar 

  14. Bruslé-Sicard S, Fourcault B. Recognition of sex-inverting protandric Sparus aurata: ultrastructural aspect. J Fish Biol 1997; 50:1094–1103.

    Google Scholar 

  15. Chaves-Pozo E, Pelegrín P, Mulero V, Meseguer J, García Ayala A. A role for acidophilic granulocytes in the testis of the gilthead seabream (Sparus aurata L., Teleostei). J Endocrinol 2003; 179:165–174.

    Article  PubMed  CAS  Google Scholar 

  16. Van den Hurk R, Peute J, Vermeij JA. Morphological and enzyme cytochemical aspects of the testis and vas deferens of the rainbow trout, Salmo gairdneri. Cell Tissue Res 1978; 186:309–325.

    Article  PubMed  Google Scholar 

  17. Billard R, Takashima F. Resorption of spermatozoa in the sperm duct of rainbow trout during the post-spawning period. Bull Japan Soc Sci Fish 1983; 49:387–392.

    Google Scholar 

  18. Scott AP, Sumpter JP. Seasonal variations in testicular germ cell stages and in plasma concentrations of sex steroids in male rainbow trout (Salmo gairdneri) maturing at 2 years old. Gen Comp Endocrinol 1989; 73:46–58.

    Article  PubMed  CAS  Google Scholar 

  19. Meseguer J, Esteban MA, Muñoz J, López-Ruiz A. Ultrastructure of the peritoneal exudate cells of seawater teleosts, seabream (Sparus aurata) and sea bass (Dicentrarchus labrax). Cell Tissue Res 1993; 273:301–307.

    Article  Google Scholar 

  20. López-Ruiz A, Esteban MA, Meseguer J. Blood cells of the gilthead seabream (Sparus aurata L.). Light and electron microscopic studies. Anat Rec 1992; 234:161–171.

    Article  PubMed  Google Scholar 

  21. Miura T, Ando N, Miura C, Yamauchi K. Comparative studies between in vivo and in vitro spermatogenesis of Japanese eel (Anguilla japonica). Zoolog Sci 2002; 19:321–329.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cell Biology, Faculty of Biology, University of Murcia, Campus Universitario de Espinardo, 30100, Murcia, Spain

    Elena Chaves-Pozo, Victoriano Mulero, José Meseguer & Alfonsa García Ayala

Authors
  1. Elena Chaves-Pozo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Victoriano Mulero
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José Meseguer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alfonsa García Ayala
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alfonsa García Ayala.

Additional information

Published: June 29, 2004.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chaves-Pozo, E., Mulero, V., Meseguer, J. et al. Flow cytometry based techniques to study testicular acidophilic granulocytes from the protandrous fish gilthead seabream (Sparus aurata L.). Biol. Proced. Online 6, 129–136 (2004). https://doi.org/10.1251/bpo81

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo81

Indexing terms

Biological Procedures Online

ISSN: 1480-9222

Contact us