In-vitro human spermatozoa nuclear decondensation assessed by flow cytometry

Abstract

The process of sperm chromatin decondensation occurs when a spermatozoon enters an ovum. Protamine disulphide bonds are reduced to SH and the polycationic protamines combine with the polyanionic egg protein, nucleoplasmin, thus being stripped from DNA which then combines with histones. Defective chromatin decondensation will thus prevent further development of the male pronucleus. In this study human sperm samples were incubated in vitro at 28 degrees C (using a medium in which the polyanion, heparin, substitutes for nucleoplasmin and beta-mercaptoethanol for egg glutathione) for 10, 20 and 30 min before stopping the reaction with formalin (to 3.6%). The DNA of the fixed cells was stained with Acridine Orange by a one-step method and subjected to flow cytometry and data analysis, in which a zone characteristic of condensed chromatin is outlined on red-green fluorescence contour plots. After 20 min of incubation 97% of the control spermatozoa that were in the mature window (WIN M) had decondensed and moved out of this region. Defects in sperm decondensation were seen in four semen samples of the 20 that were tested. In cases where spermatozoa fail to produce a fertilized egg the cause may lie with defective chromatin quality, including failure of the sperm chromatin to decondense. The method described here is a simple procedure for detecting sperm samples containing such defective cells.

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

“…Zucker et al 137 detected head decondensation of rat and hamster spermatozoa prior to assessment by increases in light scattering. Samocha-Bone et al 138 evaluated decondensation with the help of acridine orange dye in human spermatozoa. Use of the dye PI with ethylenediaminetetraacetic acid and sodium dodecylsulphate is able to determine decondensation in boar spermatozoa.…”

Nucleons, I: A Model for Studying the Mechanism of Sperm Nucleus Swelling in Vitro

“…Studies may shed light upon the following chromatin decondensation mechanisms: (1) role of the perinuclear theca, lamin, and calicin [33,40,53], (2) sperm structure integrity, membrane influence, and the requirement of being alive or dead [1,13,20,23,26], and (3) clinical semen analysis to predict the sperm fertilizing ability [12,19,27,47].…”

Relationship between nuclear chromatin decondensation (NCD) in vitro and other sperm parameters and their predictive value on fertilization rate in IVF program

“…These methods tend to be somewhat subjective because it is difficult to decide what degree of sperm head enlargement is to be the criterion of decondensation. Samocha-Bone et al (33) measured sperm chromatin decondensation in vitro by acridine orange staining followed by flow cytometry.…”