A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures

Abstract

Protamine crosslinking by disulphide (-SS-) bonds in the main factor responsible for the stability of chromatin structure in mammalian spermatozoa. Sperm chromatin containing arginine/cysteine-rich protamines shows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatic. After methanol or ethanol-acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smear from human fertile donors exhibited a pale blue (orthochromatic) colour, while a few sperm heads exhibited violet-blue or violet (metachromatic) staining. Smears from infertile patients showed an increased amount of metachromatic sperm nuclei. After reduction of -SS- bonds by dithiothreitol, sperm heads from all smears were metachromatic, suggesting that DNA phosphates then become available for TB stacking. Micro- and macro-spectrophotometric studies confirmed the microscopic colour reaction of sperm nuclei. The ortho-/metachromatic ration seems a useful parameter for evaluation of altered chromatin structure in spermatozoa cells. Taking into account the current interest in complementary staining tests for evaluation of semen quality, the metachromatic TB reaction represents a simple cytochemical approach for detecting sperm chromatin abnormalities based on differences in -SS- crosslinking.

SUBLETHAL DOSES OF MALATHION ALTER MALE REPRODUCTIVE PARAMETERS OF Eisenia foetida

“…Increases in the number of spermatozoa with abnormalities could be related to the alkylating properties of these insecticides, acting directly on the complex DNA-proteins (Sorour & Larink;Green et al, 2001). The test of fluorescence with acridine orange, based on the determination of the relation between green orthocromatic fluorescence (normal) and red metachromatic (altered) chromatin, is an adequate test to measure effects of chemical agents on male gametes (Andreetta et al, 1995;Evenson et al, 2002). Therefore, malathion could affect the sperm count, but in addition, its metabolites could affect sperm quality.…”

“…Smears were stained with acridine orange at a final concentration of 0.19mg/ml at pH 2.5 for 5 min in a cold (4ºC) and dark chamber and was observed in a epifluorescence microscope with a 436 nm filter (Zeiss III RS, Germany) (Tejada et al, 1984). Orthochromatic or metachromatic reactions in sperm head are originated when chromatin is highly condensed or decondensed, respectively (Andreetta et al, 1995).…”

Assessment of Chromatin Maturity in Human Spermatozoa: Useful Aniline Blue Assay for Routine Diagnosis of Male Infertility

“…Many analytical techniques have been proposed to assess nuclear compaction in ejaculated sperm. Some cytochemical or fluorescent dyes were used: acidic aniline blue to detect excessive presence of histones [11–15], chromomycin A3 applied for the evaluation of protamine deficiency [16, 17], and acridine orange [17] and toluidine blue staining used for assessing sperm chromatin structure and packaging [7, 18]. These different dyes have the advantage of providing suitable slides for use on a light or fluorescent microscope.…”

Extra‐epididymal spermatozoa express nuclear abnormalities

“…This produces a highly stable and condensed chromatin structure. Toluidine blue staining of spermatozoa produces mainly orthochromatic (pale blue) nuclei, whilst a small percentage of cells stain metachromatically (violet or violetblue) due to de®cient protamine disulphide crosslinking and chromatin condensation (Andreetta et al, 1995). Toluidine blue staining of spermatozoa produces mainly orthochromatic (pale blue) nuclei, whilst a small percentage of cells stain metachromatically (violet or violetblue) due to de®cient protamine disulphide crosslinking and chromatin condensation (Andreetta et al, 1995).…”

“…This was performed by staining cells with toluidine blue and recording their spectra, allowing the detection of de®cient nuclear protamine disulphide crosslinking and reduced chromatin condensation (Andreetta et al, 1995). This was performed by staining cells with toluidine blue and recording their spectra, allowing the detection of de®cient nuclear protamine disulphide crosslinking and reduced chromatin condensation (Andreetta et al, 1995).…”