We used atomic force microscopy (AFM), which utilizes a novel 3D image-contrast mechanism, to obtain nanometer-resolved, topographic data images of the natural surface structures of untreated bovine sperm cells. Freshly ejaculated, thawed, sonicated, and demembranated bovine sperm were adsorbed passively or by motility from suspension onto a coverglass substrate and directly imaged in normal air and saline environments without damaging the cells. Our AFM images of the surface structures of unfixed sperm imaged in normal air were consistent with previous electron microscope results on frozen or fixed sperm, demonstrating that the accurate preservation of small cellular structures is achievable using greatly simplified AFM sample preparation and imaging environments. Our AFM results also indicate that imaging sperm in physiologic buffer provides more native views of sperm due to the retention of cytoplasmic structures easily disrupted by drying forces. In addition, the AFM images show that numerous nanometersized subcellular structures of the sperm head and tail regions could be clearly visualized on rapidly prepared, unfixed, intact cells. Consequently, AFM should be considered a new tool for studying sperm structure abnormalities and monitoring the specific effects of, or damage caused by, various chemical reactants or other treatments on the structures of metabolically active or partially demembranated sperm. AFM is now emerging as an important new structural technique for imaging hydrated cells and organelles and, in addition, has the capabilities to physically "interrogate" them with the local probe.

Acrosome↗

PubMedDate not supplied

Mapping and measuring DNA to protein ratios in mammalian sperm head by XANES imaging.

We have used XANES imaging, which combines X-ray absorption near edge spectral features (XANES) with 50-nm-resolution X-ray microscopy, to examine the content and distribution of DNA and protein in mature sperm cells. Sperm nuclei from five different species of mammals were examined; these species were chosen for analysis because their sperm contain marked differences in their protamine 1 and protamine 2 contents. The data we've obtained for bull, stallion, hamster, and mouse sperm suggest that the total nuclear protein to DNA ratio is similar in the sperm of many eutherian mammals. Since protamine constitutes the majority of the sperm nuclear protein, these results indicate that the total protamine content of sperm chromatin must be constant among mammalian species, independent of the extent of expression of the protamine 2 gene.

Absorptiometry, Photon↗

PubMedDate not supplied

Characteristics of human sperm chromatin structure following an episode of influenza and high fever: a case study.

Semen samples from a fertile patient presenting with influenza and a 1-day fever of 39.9 degrees C were obtained and analyzed at 18-66 days postfever (dpf) for sperm nuclear proteins, DNA stainability, free thiols (-SH), and susceptibility to DNA denaturation in situ. At 18 dpf, 36% of sperm demonstrated denatured DNA as measured by the sperm chromatin structure assay (SCSA), and decreased to 23% by 39 dpf. Samples at 33 and 39 dpf contained 49% and 30%, respectively, of cells with increased DNA stainability (HIGRN). A unique sperm nuclear protein band migrating between histones and protamines on acid-urea gels appeared at 33 and 39 dpf and nearly disappeared by 52 dpf. Amino acid sequencing of the first 8 N-terminal residues identified this protein as the precursor to protamine 2. The protamine P1 and P2 ratio remained normal, whereas the histone to protamine ratio increased slightly at 33 to 39 dpf. Flow cytometric measurements of nuclear -SH groups revealed the greatest reduction in free nuclear thiols at 33 dpf, and returned to normal by 45 dpf. The time of appearance of the unprocessed protamine 2 precursor and the relative increase in histone suggest a fever-related disruption of the synthesis of mRNA that codes for a P2 processing enzyme or enzymes. Increased DNA staining is likely due to the increased histone/protamine ratio. This case study demonstrates that fever/influenza can have latent effects on sperm chromatin structure and may result in transient release of abnormal sperm.

Cell Nucleus↗