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Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas

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Abstract

Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P<0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally isotonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum volume of flagella obtained after hypotonic fixation was approximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Although the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.

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Acknowledgements

We thank B. Eudeline and Taylor Shellfish, for supplying the oysters, Y. Xiao and B. Liu for laboratory assistance, and R. Downer of the LSU Department of Experimental Statistics for statistical consultation. This manuscript was approved for publication by the Director of the Louisiana Agricultural Experiment Station (no. 05-11-0578).

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Authors and Affiliations

  1. Aquaculture Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA, 70803, USA

    Qiaoxiang Dong, Changjiang Huang & Terrence R. Tiersch

  2. Institute of Marine Sciences, Wenzhou Medical College, Wenzhou, 325035, People’s Republic of China

    Qiaoxiang Dong & Changjiang Huang

  3. Socolofsky Microscopy Center, Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 70803, USA

    Margaret C. Henk

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  1. Qiaoxiang Dong
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  2. Changjiang Huang
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  4. Terrence R. Tiersch
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Correspondence to Terrence R. Tiersch.

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This work was supported in part by funding from the USDA-SBIR program, 4Cs Breeding Technologies, and the Louisiana Sea Grant College Program.

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Dong, Q., Huang, C., Henk, M.C. et al. Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas . Cell Tissue Res 324, 335–345 (2006). https://doi.org/10.1007/s00441-005-0141-9

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  • DOI: https://doi.org/10.1007/s00441-005-0141-9

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