Introduction
The sperm chromatin stability sets the degree of access to the DNA. The stability appears to depend on bridges between protamine molecules; bridges that could be salt bridges with zinc (S-Zn-S) or disulfide bridges (S-S).
Aim: To evaluate how zinc depletion affects sperm chromatin stability and the DNA stainability
Material and methods
Samples were processed within 1 hour after ejaculation: washed in a buffer salt solution with zinc (BSS-Zn) and resuspended in BSS-Zn or BSS-EDTA (BSS-Zn with zinc-chelating EDTA), kept 2 hours at +4°C. Test aliquots of spermatozoa were exposed to dithiothreitol (DTT) for different durations. Then exposed to acid and stained with acridine orange. Red and green fluorescence were assessed with flow-cytometry in duplicates. Three samples were exposed to DTT for 0, 1, 3, 5, 7 and 10 minutes, four samples for 0, 10, 20 and 60 minutes. A novel gating technique was developed, as DFI% was unable to reflect changes in scattergram. MP% is an oval gate comprising the Main Population. SP% is a rhombic gate of population above MP as seen in green/red scattergram.
Results
Short-term incubation: All three samples kept in BSS-Zn showed linear decrease in MP% over 10 minutes (average slope of -2.0). From BSS-EDTA only one sample showed similar decrease (slope of -1.9). Two were not affected by DTT.
Long-term incubation: Two samples in BSS-EDTA showed linear decrease in MP% (average slope of -0.2). Other two samples were not affected by DTT.
Conclusions
In 4 of 7 spermatozoa, EDTA made the chromatin inaccessible to the effect of DTT in both short-term and long-term incubation. Therefore, buffers containing EDTA might interfere with normal sperm function in oocyte. DFI% and HGS% were both unable to identify a subpopulation of spermatozoa reacting more readily to DTT, indicating that they cannot detect changes in chromatin packaging.