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Significance of plasmalemma disruption in bovine and equine spermatozoa
J V Abraham-Peskir1, E Chantler, E Uggerhøj
1Institute for Storage Ring Facilities, University of Aarhus, Denmark. jabraham@ifa.au.dk
Abstract:
We have investigated fresh and cryopreserved bovine and equine spermatozoa using light and transmission soft X-ray microscopy. Spermatozoa were examined, in the presence or absence of semen, after using Percoll gradient centrifugation and re-suspending in medium. X-ray microscopy provided high resolution (30 nm) transmission images of whole cells in solution with high contrast, while retaining the simple preparation techniques used in light microscopy. We demonstrated translucent, membrane-bound vesicles in the acrosomal and midpiece regions that were similar in size and we noted their incidence in both fresh and frozen-thawed material from both animals. The vesicles were formed by the separation and expansion of the plasmalemma away from the underlying structure but were not caused by the freeze-thaw process. We suggest that these structures form part of the normal ultrastructure of spermatozoa and are damaged during preparation of the samples for transmission electron microscopy, resulting in a structure previously and incorrectly identified as damaged by the freezing and thawing process.
Insights
Soft X-ray microscopy revealed translucent vesicles in bovine and equine sperm, challenging previous beliefs about freeze-thaw damage. These vesicles are part of normal sperm ultrastructure, not artifacts of cryopreservation.
Area of Science:
- Reproductive Biology
- Microscopy Techniques
- Cellular Ultrastructure
Background:
- Cryopreservation of spermatozoa can lead to structural damage, impacting fertility.
- Previous studies suggested freeze-thaw processes damage sperm ultrastructure, particularly the plasmalemma.
- Transmission electron microscopy (TEM) preparation methods may introduce artifacts.
Purpose of the Study:
- To investigate the ultrastructure of fresh and cryopreserved bovine and equine spermatozoa.
- To evaluate the utility of transmission soft X-ray microscopy (TSXM) for sperm analysis.
- To clarify the nature of observed vesicles in sperm and their relationship to cryopreservation.
Main Methods:
- Spermatozoa from bovine and equine sources were examined in fresh and cryopreserved states.
- Percoll gradient centrifugation and resuspension in medium were employed.
- Light microscopy and transmission soft X-ray microscopy (TSXM) were utilized for imaging.
Main Results:
- TSXM provided high-resolution (30 nm) transmission images of whole cells in solution with high contrast.
- Translucent, membrane-bound vesicles were observed in the acrosomal and midpiece regions of both fresh and cryopreserved sperm.
- These vesicles were formed by plasmalemma separation and expansion, independent of the freeze-thaw process.
Conclusions:
- The observed vesicles represent normal sperm ultrastructure, not damage from cryopreservation.
- TSXM offers a valuable method for studying sperm with minimal preparation artifacts.
- Previous interpretations of freeze-thaw damage may have been influenced by preparation-induced artifacts in TEM.