Protein tyrosine phosphorylation is associated with capacitation of human sperm in vitro but is not sufficient for its completion.
Capacitation is a prerequisite step that mammalian sperm must take before being able to undergo the acrosome reaction (AR). The molecular mechanisms of this process remain incompletely understood and can be studied only in vitro. Human sperm preparation for insemination of oocytes requires the removal of seminal plasma and incubation in defined media that usually contain albumin and calcium. In various conditions of capacitation, we have studied the relationship between protein tyrosine phosphorylation as measured by Western blotting and the capacitation state as evaluated by ionomycin-induced AR in the presence of calcium. Several proteins with molecular masses from 60 to 200 kDa became increasingly phosphorylated with time. The kinetics of phosphorylation showed an inter-individual variability, with a maximal level reached between 1 and 4 h, and was associated with the capacitation state. Albumin increased phosphorylation in a concentration-dependent manner. Lack of albumin prevented both phosphorylation and capacitation. Conversely, lack of CaCl2 in the capacitating medium enhanced phosphorylation and did not impair the induced AR. Surprisingly, sperm incubation in seminal plasma, which is supposed to contain "decapacitation factors," did not affect the time-dependent increase in tyrosine phosphorylation. However, in seminal plasma-free medium, it inhibited induction of the AR. Protein tyrosine phosphorylation is associated with in vitro capacitation. Although it cannot by itself complete capacitation, it could represent an important step in this process by priming the transductive pathways that are activated during exocytosis.
Emiliozzi
C
C
Groupe de Recherche sur l'Interaction Gamétique, CJF INSERM 95-04, Faculté de Médecine, Nice, France.
Fenichel
P
P
D000177:Acrosome / Q000502:physiology; D000418:Albumins / Q000502:physiology; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012661:Semen / Q000502:physiology; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology*
D000418:Albumins; D011506:Proteins; D017027:Protein Tyrosine Phosphatases; D002118:Calcium