P-630: Similarity in tyrosine phosphorylation patterns in human sperm bound to hyaluronic acid and to zona pellucida of oocytes

Abstract

During spermiogenesis there is a sperm plasma membrane remodeling that facilitates the formation of zona pellucida and hyaluronic acid (HA) binding sites. Sperm with arrested maturation can fail to bind to the zona pellucida and HA. Also, the sperm selection attributes of HA and zona pellucida are similar: HA-bound sperm show no cytoplasmic retention, persistent histones or DNA fragmentation. Further, in HA-selected sperm the frequencies of chromosomal aneuploidies are normal, regardless of their frequencies in semen (Fert.Ster., 2005). A number of previous studies have shown specific protein tyrosine phosphorylation (TP) changes in spermatozoa related to events associated with capacitation and zona-binding. In the present study, we have studied TP patterns in spermatozoa isolated from media or bound to HA. We have also explored whether sperm with arrested maturation and surplus cytoplasm would undergo TP changes. Time related study of TP patterns in sperm suspended in media or bound to HA. Immunofluorescence was performed with antisera for TP and CK. We prepared sperm smears and HA-bound mature sperm from each semen sample suspended in HTF medium (Irvine Scientific) and using HA-coated slides (MidAtlantic Diagnostics). The sperm were fixed with 3.7% formaldehyde after 0 time and 4hr incubations. The slides were treated with either a monoclonal anti-phosphotyrosine antibody (Sigma) or with polyclonal anti-creatine kinase(CK) antiserum (Chemicon Co., USA), or with both. These steps were followed with FITC or Rhodamine labeling for sperm assessment with fluorescence microscopy. Statistical evaluation by paired T-test and Wilcoxon ranked sum test were carried out with SigmaStat (Jandel, CA). All values are mean±SEM. TP occurred in the equatorial segment, acrosomal area, neck, and principal piece of sperm. To investigate whether TP may occur in sperm of diminished maturity we explored the potential relationship between sperm maturity and TP by double-labeling sperm for TP and CK (a marker of surplus cytoplasm). Double staining revealed that the TP changes occur exclusively in mature spermatozoa, and there were no TP changes in sperm with cytoplasmic retention. When we assessed TP in sperm at 0 time and 4h in HTF medium, and after HA-binding, the proportion of sperm with TP at 0 time and 4h in HTF were 41.7±2.6% and 54.7±4.2% (NS, N=7873 sperm). The respective TP values in the HA-bound sperm fractions were 54.7±4.2% and 67.0±3.8% (NS, N=7508 sperm). We observed major TP changes, with time and HA-binding, only in the neck region of mature sperm. The % sperm with neck phosphorylation in HTF at 0 time and 4 hours were: 14.4±5.3 and 41.5±8.8 (p=0.02, N=3777 sperm) compared to 21.9%±4.9% and 51.5±5.5% (p=0,01, N=3555 sperm) in the HA-bound sperm fraction, respectively. The extent of TP in the sperm neck, a marker of sperm activation, increases with time and contact with HA. We have demonstrated for the first time that TP does not occur in diminished maturity sperm with cytoplasmic retention. Moreover, in line with other sperm attributes, the sperm TP pattern changes are also similar in sperm that are bound to HA or to zona pellucida of oocytes.