Priming of signal transducing mechanisms during capacitation

The following model of the ionic events in ZP signal transduction is largely based on Ž . the information reviewed by Florman et al. 1998 . According to this model the activation of the sperm surface receptor by the association with mouse ZP3 initiates two separate pathways. One signaling sequence leads to the activation of a cation channel C producing inward currents depolarizing sperm membrane potential and opening low voltage-activated T-type Ca 2q channel. The other pathway initiates internal alkaliniza- tion by mechanisms likely reflecting the mediation of G proteins. This pH increase and transient Ca 2q current in response to ZP or membrane depolarization promote a sustained Ca 2q increase. The elevation of Ca 2q level, a rise of intracellular pH and the enhancement of membrane fusogenity are postulated to be the driving forces triggering Ž the cascade of acrosomal expcytosis Harrison and Roldan, 1990; Roldan and Harrison, . 1990; Kopf and Gerton, 1991; Aitken, 1997 .

6. Priming of signal transducing mechanisms during capacitation

Ejaculated mammalian sperm need a period of incubation in the female reproductive Ž . tract in order to acquire the capacity to fertilize an egg Yanagimachi, 1994 . This period Ž . of attaining functional competence, referred to as capacitation Austin, 1951 , is required for undergoing the acrosome reaction induced by physiological stimuli such as ZP Ž . Florman and First, 1988 . Since the response to the oocyte signals can be initiated only in capacitated sperm, the signal-transducing mechanisms appear to be primed during capacitation. Capacitation is accompanied by an increase of membrane fluidity and remodeling of the sperm surface, protein phosphorylation, an increase of internal Ca 2q and pH and Ž . membrane hyperpolarization Storey, 1995 . Generally accepted is the concept of Ž . capacitation as series of positive destabilizing events Harrison, 1996 . The initial event is the BSArLDH-mediated cholesterol efflux resulting in an increase of plasma Ž membrane fluidity thus supporting membrane remodeling Davis et al., 1979; Visconti et . al., 1999 . Kinase-mediated tyrosine posphorylations are tightly connected with this Ž . event Visconti et al., 1994a,b . Tyrosine phosphorylation appears to be an essential process of capacitation: if tyrosine kinase inhibitors block it the spermatozoa lose the ability to respond to Ž . physiological agonists Aitken et al., 1996 . Since cross talk with cAMP is involved in the regulation of protein tyrosine phosphorylation, it is tempting to speculate that the loss of cholesterol may be involved in the regulation of cAMP pathway. The monotonic elevating of Ca 2q with the rate about 0.5 nmrmin seems to be insufficient to initiate acrosome reaction and might occur due to the modulation of Ca 2q -ATPase activity. Ž The mechanism related to voltage-dependent T-channel described above Florman et . al., 1998 and references therein appears to be responsible for the explosive increase of Ca 2q in response to physiological agonists leading to the acrosomal exocytosis. The sperm T-channel may be primed and held in steady state during capacitating by Ž q . hyperpolarization probably due to K -permeability contribution at the level of an upstream action channel and by tyrosine phosphorylation. The tyrosine phosphorylation of T-channel or channel regulator decreases Ca 2q current through the channel prevent- ing the acrosome reaction, whereas, the dephosphorylation may stimulate the activation Ž . of the channel Arnoult et al., 1997 . However, it appears that further signal transduction mechanisms may be involved in the activation of Ca 2q pathways. A mild hypoosmotic shock has been shown to be a Ž . potent stimulus of the acrosome reaction. Rossato et al. 1996 demonstrated that human spermatozoa possess Ca 2q influx pathways activated by plasma membrane stretching. A 2q Ž q . blocker of mechano-sensitive Ca channels Gd diminished osmotically sensitive Ca 2q -rises in a dose-dependent manner and completely blocked the osmotically sensitive acrosome reaction. The formal evidence is missing that Ca 2q rise occurs via Ca 2q channels, but this hypothesis seems likely due to fast kinetics, sensitivity to membrane depolarization and Gd q treatment. Osmosensitive mechanisms may also play a role during preparation steps of the Ž . acrosome reaction capacitation . The regulation mediated by osmotic events may be reflected in cell volume behavior during capacitation. Boar sperm cell volume tends to show cyclical variations during incubation under capacitating conditions with the frequency varying dependent on the incubation conditions. The expression of different levels of swelling at fixed osmotic conditions is coupled to the change in cell osmole content. The osmosensing regulatory performance, such as swelling and subsequent regulatory volume decrease, could be modulated during capacitation and may reflect the ongoing destabilization of the membrane on earlier stages of capacitation treatment Ž . Ž . Petrounkina et al., 2000 . Preliminary studies on boar sperm let suggest that i changes in cell volume are closely related to the expression of tyrosine phosphorylation as shown by indirect immunfluorescence on the earlier stages of incubation under capacitation Ž . conditions, and ii cell volume regulation is mediated in the first line by quinine-sensi- Ž . tive channels Petrounkina et al., 2000 . The participation of quinine-sensitive channels Ž . on cell volume regulation of bull sperm was reported by Kulkarni et al. 1997 . For Ž . other cell types e.g. in erythrocytes protein tyrosine phosphorylation has already been Ž . found to be osmo- and volume-sensitive Mush et al., 1994, 1998 . Hypoosmotic shock triggered a rapid increase in tyrosine phosphorylation; furthermore, the volume regula- tion of swollen cells was sensitive to tyrosine phosphatase inhibition. Phosphorylation of a band 3 protein in human erythrocytes, observed by ionophore treatment or shrinking- induced volume regulation, was suppressed by quinine, an inhibitor of Ca 2q -activated q Ž . K channel Minetti et al., 1996 . Moving into field of speculation, the similar modulation of osmosensing signal transduction by tyrosine phosphorylation may be expected also in sperm cells while priming for activation in response to ZP and for zona penetration. Further studies are in progress to investigate this hypothetical regulatory mechanism of signal transduction during capacitation.

7. Conclusion