The fine structure of the glycocalyx of equine spermatozoa: a high-resolution cytochemical study.

The research article presents a study using high-resolution cytochemistry to examine the fine structure of glycocalyx, a protective coating, on equine spermatozoa, whereby a series of chemical testing and electron microscopy uncovered a distinct electron-dense layer on mature sperm cells.

Sample Preparation and Process

• The researchers obtained the equine spermatozoa from the ejaculates of young stallions.
• The semen was then processed to isolate the spermatozoa. The seminal plasma, which is the fluid supporting the sperm, was removed. The remaining sperm pellets were washed three times with a salt solution (M-NaCl) and then subjected to a high-speed centrifuge, ensuring only the sperm cells were left.

Chemical Treatment and Microscopy

• The pellets were then fixed in a mixture of glutaraldehyde and cacodylate buffer. Glutaraldehyde is a strong disinfectant and fixative, preserving the structure of the sperm cells, while the cacodylate buffer maintained the pH at a level conducive for the process.
• The treated samples were then colored with Alcian blue, a stain known for its high affinity for certain types of glycans (which make up the glycocalyx).
• Following primary fixation, the samples were post-fixed by treating them with osmium tetroxide, which reacts with lipids (fatty acids) and proteins helping to further preserve the structure of the samples, and lanthanum nitrate, used as a contrast agent.
• Some of the initial sperm samples were handled separately and treated with a different compound, ruthenium red, which is known to remarkably contrast and delineate the glycocalyx.
• All samples were then dehydrated using increasing concentrations of ethanol, providing a medium that allows for the samples to be readily embedded in a plastic resin called araldite, a step necessary for proper sectioning and examination under an electron microscope.

Results and Observations

• The high-resolution images from electron microscopy demonstrated electron-dense deposits present on the cell surface, specifically the head, mid-piece, and tail of 70% of mature spermatozoa, suggesting the presence of a glycocalyx.
• The images also indicated comparable deposits on samples that were treated with ruthenium red, further supporting the initial findings and confirming the presence of a surface glycocalyx.
• No such glycocalyx was observed in the samples that were not subjected to any special treatment, implying that special staining techniques must be used to visualize the glycocalyx.

This methodology and the observations made provide a pathway for further exploration of the structural properties of equine spermatozoa, potentially uncovering more about the nature and function of the glycocalyx in this context.