Male Pronuclei Formation Release of Phosphorylation of Histone H-3 During Decondensation of Human Sperm Nuclei Activated in Vitro by Heparin

Abstract

The release and phosphorylation/dephosphorylation mechanisms of human spermatozoa histone during nuclei in vitro decondensation by heparin was studied. Washed sperm cells were incubated in the presence of ³²P and in the absence or presence of heparin. The results showed an increase in the incorporation of ³²P of 20 times greater in the presence of heparin than in the absence of heparin (the control sample). In some cases the incorporation of ³²P into histones was confirmed by its isolation. To validate these results a phosphorylation kinetic of isolated sperm histone, used as a substrate, was performed. The amount of ³²P was not a linear function of time, and maximal phosphorylation was reached in 60 min. A measurement of ³²P incorporated as a function of the amount of histone, shows a linear relationship of up to 50 μg of protein, with a rapid saturation thereafter with the incorporation of 220 nm and with a KD - 442 × 10⁻⁶ mol/L. ³²P incorporation, independent of exogenous cAMP, was related to alkaline pH but was totally dependent on temperature—with a maximum of 37 °C. The only histone released was histone H-3. Phosphorylation/dephosphorylation is involved during male pronuclei formation.

Key Words:

• Histone
• Protamine
• Decondensation
• Phosphorylation
• Glycosamineglycans.