Protein Tyrosine Phosphorylation Is Associated with Capacitation of Human Sperm In Vitro but Is Not Sufficient for Its Completion1

Abstract

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.

Evidence for the involvement of PECAM-1 in a receptor mediated signal-transduction pathway regulating capacitation-associated tyrosine phosphorylation in human spermatozoa

“…2). For instance, initial studies conducted with the mild zwitterionic detergent, CHAPS, revealed a complex pattern of phosphotyrosine proteins whereas those performed with a more stringent SDS-based solubilization, were biased by the presence of two predominant proteins of approximately 85 and 105 kDa, as previously reported (Emiliozzi and Fenichel, 1997). Overexposure of the latter blots revealed a more complex pattern of tyrosine phosphorylation, approximating that seen in the CHAPS extract (Fig.…”

Signal transduction mechanisms involved in in vitro ram sperm capacitation

“…These phosphorylations should not be considered strictly indicative of full capacitation in ram sperm, as described in human (Emiliozzi & Fenichel 1997) and boar (Tardif et al 2003), where protein tyrosine phosphorylation alone was not sufficient for the completion of capacitation. These data indicate that the signal transduction mechanisms of capacitation in ram sperm differ from those in other mammals, which suggests that species specificities might exist with respect to this process.…”

Effect of Progesterone on Acrosome Reaction, Hypoosmotic Swelling Test, and Dna Stability in Human Spermatozoa

“…In one spermatozoon the progesterone interacts with a cell surface steroid receptor, causing an immediate increase in [Ca], but in other sperm their action was on the membrane phospholipids not inducing AR [2]. Several reports have shown the capacitating effect of progesterone on ejaculated spermatozoa obtained with two layers of Percoll, but those effects cannot be addressed to other sperm parameters as DNA stability or plasma membrane integrity [4,5].…”