In-situ competition between protamine and fluorochromes for sperm DNA

Abstract

In this study we investigated the relationship between the presence of bound protamine on mouse and human sperm DNA and the level of chromomycin A3 (CMA3) and 4'6-diamidino-2-phenylindole (DAPI) fluorescence. This was accomplished by performing a competition assay between salmon protamine and fluorochromes on decondensed spermatozoa that had their nuclear proteins extracted and were fixed on slides. Various concentrations (0, 0.005, 0.0225, 0.05, 0.225, 0.5 and 5 mg/ml) of salmon protamine were added to either the CMA3 or DAPI staining solutions. Fluorescence emission measurements of stained sperm nuclei were then performed using a microfluorometer. When the treated decondensed sperm heads were stained with either CMA3 or DAPI all spermatozoa were found to fluoresce intensely. The addition of protamines to the spermatozoa led to an elimination of CMA3 fluorescence, while the intensity of DAPI staining was decreased to approximately 50% at the highest concentrations of protamine. The addition of increasing amounts of salmon protamine also induced the sperm nuclei to regain their initial condensed appearance. This study shows that protamine retains a strong affinity for sperm DNA in situ and that CMA3 fluorescence is a strong indicator of the protamination state of spermatozoa.

Changes in spermatozoal chromatin packaging and susceptibility to oxidative challenge during aging

“…The CMA3 assay is becoming more extensively used for evaluating sperm quality as part of male fertility assessment (52,53). It provides an indirect measure of assessing protamines in sperm (54,55), where CMA3 competes with protamines for chromatin binding (47,56).…”

Human Cervical Mucus Can Act in Vitro as a Selective Barrier Against Spermatozoa Carrying Fragmented Dna and Chromatin Structural Abnormalities

“…CMA 3 is a useful tool for rapid screening of subfertility in man, as it seems to allow an indirect visualization of protamine-deficient DNA (14,15,22,23). This molecule has been shown to bind as a Mg 2+ -coordinated dimer at the minor groove of GC-rich DNAs and to induce a conformational perturbation in the double helix resulting in a wider and shallower minor groove at its binding site (24,25).…”

“…For these reasons the conformational arrangement adopted by the DNA-protamine complex can limit the access of CMA 3 to the minor groove. So CMA 3 can also be used as an indicator of underprotaminated spermatozoa (23).…”

MicroRNA-122 Influences the Development of Sperm Abnormalities from Human Induced Pluripotent Stem Cells by Regulating TNP2 Expression

“…Major chromatin restructuring occurs in elongating and condensing spermatids, which are in turn replaced by protamines (PRMs) [12][13][14][15]. TNPs are arginine-and lysine-rich basic proteins that strongly bind to DNA, and are expressed exclusively in postmeiotic haploid spermatids.…”

“…Furthermore, TNPs are phosphorylated by sperm-specific protein kinase A, and this phosphorylation/dephosphorylation cycle has been found to play an important role in the chromatin condensation process [9][10][11]. TNPs are removed from condensing chromatin and replaced by PRMs [13], which constitute the major nuclear proteins of condensed spermatids and mature spermatozoa [12,[14][15][16]. Miyagawa et al demonstrated single-nucleotide polymorphisms (SNPs) and specific variability in the TNP genes of sterile male patients [1].…”