Presence and Release of Bovine Sperm Histone H1 During Chromatin Decondensation by Heparin-Glutathione

Abstract

During spermatogenesis, changes in sperm nuclear morphology are associated with the replacement of core somatic histones by protamines. Although protamines are the major nucleoproteins of mature sperm, not all species totally replace the histones. Histone H1, along with protamines, mediates chromatin condensation into an insoluble complex that is transcriptionally inactive. In vitro, heparin-reduced glutathione causes sperm decondensation, and the structures formed are morphologically similar to the in vivo male pronucleus. To study the participation of histone H1 in bovine sperm chromatin remodelling, we measured the presence and release of histone H1 by immunofluorescence, acetic acid-urea-triton-polyacrylamide gel electrophoresis, and immunoblotting. Nuclear decondensation was induced by 80 microM heparin and 15.0 mM reduced glutathione (GSH) for 7, 14, and 21 h at 37 degrees C. Additionally, nucleons, composed of nuclei isolated from the sperm, were decondensed with 20.0 microM heparin and 5.0 mM GSH for 4.0 h at 37 degrees C. Controls were incubated in buffer for similar periods of time. Immunofluorescent localization of histone H1 was carried out with mouse monoclonal antibody, and DNA localization was visualized by 0.001% quinacrine staining. Chromatin decondensation was accompanied by increased sperm nuclei and nucleon surface area. We observed that histone H1 was localized exclusively in the nuclei of intact sperm and nucleons. Histone H1 immunofluorescent intensity did not change in control samples but decreased over time in samples treated with heparin-GSH. There was a negative correlation between the surface area of sperm nuclei and immunohistochemical intensity of histone H1 (P < 0.05). Nucleon decondensation showed a similar relationship. By electrophoresis and immunoblotting, we verified the loss of histone H1 from the sperm and nucleons and its release into the incubation media. Based on these results, we propose that histone H1 is present in the bovine sperm nuclei. H1 depletion may participate in chromatin decondensation and nuclear swelling induced by heparin-GSH.

Chromatin condensation and morphometry of the bovine sperm head afterin vitrosperm selection and capacitation

“…This occurs as a result by the action of sulfated glycosaminoglycans (GAGs), such as heparin, both in vivo (Romanato et al 2003;Romanato et al 2005;Sa´nchez-Va´zquez et al 2008) and in vitro (Reckova et al 2008;Lymberopoulos and Khalifa 2010), during fertilization. The GAGs bind to specific receptors on the plasma membrane (SanchezPrieto et al 1996) of spermatozoa inducing capacitation (Miller et al 1990), acrosome reaction (Reyes et al 1984) and release of basic nucleoproteins (Reyes et al 1991), such as histones and protamines (Romanato et al 2003;Sa´nchez-Va´zquez et al 2008), and transform strongly condensed and inert chromatin to decondensed and competent chromatin for transcription. In human sperm, it has been proposed that efficient and necessary dissolution of chromatin after fertilization is facilitated by zinc binding in regions where disulfide bridges would normally form, thus preventing bridge formation.…”

What is the role of histone H1 heterogeneity? A functional model emerges from a 50 year mystery

“…H1oo’s chief characteristic is its ability to rapidly, within 5 min., occupy exogenous chromatin introduced into the egg whether it arrives on the head of a sperm or by injection of a somatic nucleus [93]. Even in the protamine packaged sperm nucleus, ~ 15% of the chromatin is still organized by histones [157], including H1s [158], in stretches of DNA that can be up 50,000 bp [157]. This exogenous chromatin is stripped of any residual H1s possibly by the citrullination of a single arginine site (R-54) near the NTD–GD boundary [105] (Figures 8A).…”

Improved Bovine ICSI Outcomes by Sperm Selected after Combined Heparin-Glutathione Treatment

“…Sperm were also treated with 80 lM heparin and 15 mM L-GSH alone, as described by Delgado et al (2001). Treated sperm were incubated for 7 h at 39°C in 20% O 2 and 5% CO 2 according to Sanchez-Vazquez et al (2008). Sperm pretreatment with DTT was carried out according to Rho et al (1998a).…”