Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes

The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matur ed in vitro was assessed. For this purpose, protamine was purified from boa r sperm nuclei and an antiserum against protamine was developed. After affi nity purification, the antiserum reacted exclusively with boar protamine du ring western blotting, showing no crossreactivity with core histones. Immun ohistochemical evaluation revealed that only fully developed spermatid nucl ei in boar testes stained strongly with the antiserum. When pig oocytes mat ured in vitro were fertilized in vit ro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermi c penetration Paraffin wax sections of the inseminated oocytes were examine d by immunohistochemical analysis with the antiserum. The proportion of con densed sperm nuclei that reacted with the antiserum was 87% of the sperm nu clei that penetrated by 2 h after insemination, and this decreased to 20 an d 13% at 3 and 5 h after insemination, respectively. However, none of the d econdensing sperm nuclei or male pronuclei reacted with the antiserum durin g the entire insemination period. These results indicate that a specific an tiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei befo re decondensation during in vitro fertilization.