Testis/sperm-specific histone 2B in the sperm of donors and subfertile patients: variability and relation to chromatin packaging

Abstract

BACKGROUND: The compaction of human sperm chromatin is the result of replacement of ~85% of histones with protamines. Germ-line testis/sperm-specific histone 2B (TSH2B) has been detected in only ~30% of mature spermatozoa. Its level in the semen of subfertile patients varies; its function is unknown. We evaluated TSH2B in the sperm samples of 23 donors and 49 subfertile patients and assessed its association with chromatin compaction status. METHODS: TSH2B level was measured using immunoblotting. Chromatin packaging quality was evaluated by staining with chromomycin A3 (CMA3) which marked spermatozoa with defective packaging. To assess both TSH2B and chromatin status in the same spermatozoon, CMA3 staining and TSH2B immunolocalization were performed sequentially. RESULTS: A significant correlation (r = 0.55, P = 0.0027) was found between TSH2B level and percentage of CMA3-positive sperm in patient and donor semen samples. When individual spermatozoa were assessed for these parameters, 92% of TSH2B-containing cells were also CMA3 positive. Variation in the total sperm TSH2B level was less in donors than in patients. CONCLUSIONS: CMA3 positive staining of TSH2B-containing individual spermatozoa and a significant correlation between the total TSH2B level and CMA3 percentage in semen samples suggest a structural role for TSH2B in sperm chromatin organization. Low variability of TSH2B level in donors implies a mechanism (however unknown) regulating this parameter.

DNA Damage in Human Spermatozoa Is Highly Correlated with the Efficiency of Chromatin Remodeling and the Formation of 8-Hydroxy-2′-Deoxyguanosine, a Marker of Oxidative Stress1

“…The latter competes with nucleoproteins for binding sites in the minor groove of GC-rich DNA and serves as a marker for the efficiency of DNA protamination during spermiogenesis. Accordingly, staining with this probe has been shown to be positively correlated with the presence of nuclear histones [28] and ultrastructural evidence of poor chromatin compaction [29] but negatively correlated with the presence of protamines [30]. Our observation that the binding of CMA3 correlates with the presence of DNA strand breaks (Fig.…”

Developmental sperm contributions: fertilization and beyond

“…Human testis/sperm-specific histone H2B (hTSH2B) is a variant of the histone H2B expressed exclusively in spermatogenic germline cells and present in some mature sperm cells. Thus, this protein marks a subpopulation of sperm cells in the ejaculate (165,166). Using indirect immunofluorescence, Singleton et al (165) examined the influence of hTSH2B on ZP binding and sperm head decondensation in amphibian egg cell-free extract.…”

Kinetics of human male pronuclear development in a heterologous ICSI model

“…Significant correlation between TSH2B level and CMA3 staining of individual spermatozoa resulting from loosened packaging of DNA has been demonstrated [33]. In studies using the Xenopus egg extract model, sperm cells containing TSH2B decondensed more rapidly and to a greater extent than did spermatozoa without this histone [34]. Other possible reasons for size heterogeneity of mPN may include sperm DNA damage, cell to cell variability in sperm DNA methylation, or heterogeneity of oocytes.…”