Patricio Morales1 and Miguel Llanos2

1 P. Catholic University of Chile, Faculty of Biological Sciences. P. O. Box 114-D. Santiago, Chile.

2INTA, University of Chile, P. O. Box 138-11. Santiago, Chile.


7.1. Gi proteins

A major shortcoming in the study of spermatozoa-oocyte interaction in humans is the scarcity of ZP material. Since the electrophoretic pattern of the ZP isolated from unfertilized eggs is identical to that of non-inseminated eggs, this has been overcome by the use of inseminated but unfertilized eggs (16). Using this biological material, it was demonstrated that the human ZP-induced AR is mediated by a sperm Gi protein (68, 69). This is also true for the murine and bovine ZP-induced AR (70, 71). Thus, after the spermatozoa bind to the ZP, a Gi protein may be a crucial transducing signal. Activation of a Gi protein mediates the production of intracellular signal (s) involved in the molecular and cellular events of the AR. In cells other than spermatozoa, Gi proteins are involved in receptor-mediated regulation of: inward rectifying K+ channels, Ca2+ mobilization, phospholipase C activity, and adenyl cyclase activity (72). Several of these processes have already been implicated in the mechanism that triggers the AR (73).

7.2. Intracellular free calcium

An extracellular Ca2+-dependent increase in the concentration of free intracellular Ca2+ is usually required for the initiation of the AR. This seems to be true at least for the mouse and bovine ZP-induced AR (71, 74). Pertussis toxin inhibits the ZP/ZP3 induced elevations of intracellular Ca2+ (71, 75), suggesting the involvement of a Gi protein in these events. Working with bovine spermatozoa, Florman et al. proposed a model which explains the Gi-dependent Ca2+ influx promoted by ZP/ZP3 after interaction with its sperm receptor (76). Briefly, this model states that ZP/ZP3 stimulates: i) a poorly selective, Gi protein-insensitive cation channel responsible for membrane depolarization and ii) a Gi protein-dependent alkalinization of the sperm cytoplasm. These changes activate an L-type voltage-sensitive Ca2+ channel which, in turn, causes the massive Ca2+ influx necessary for the AR (76).

The relative contribution of intracellular sources of Ca2+, however, has not been properly evaluated. There is increasing evidence for the presence of Ca2+ stores within spermatozoa which may have a role in the AR. In several somatic cells, Ca2+ found in the endoplasmic reticulum can be released by thapsigargin, a plant-derived sesquiterpene lactone that is a highly specific inhibitor of the Ca2+-ATPase responsible for Ca2+ accumulation by that organelle (77, 78). Thapsigargin stimulates the AR of capacitated human (79), mouse (80), and hamster spermatozoa (81) and it requires external Ca2+ and influx of this ion (79, 81). Thus, the Ca2+ release from intracellular stores by thapsigargin may lead to an influx of extracellular Ca2+ and subsequently to the AR. Putative sites for thapsigargin-sensitive intracellular Ca2+ stores in the spermatozoa may include the cytoplasmic droplet, the sperm nucleus and the acrosome (79, 80).

Recently, the selective localization of inositol 1,4,5-trisphosphate receptors (IP3R) in the acrosomes of rat, mouse, hamster and dog spermatozoa was reported (80). The authors also described the presence of Galphaq/11 and phospholipase Cß1, and suggested a role for inositol 4,5-trisphosphate- (IP3) gated Ca2+ release in the mammalian sperm AR. Walensky and Snyder presented a model where a multivalent interaction between ZP/ZP3 and sperm plasma membrane binding proteins/receptors leads to the production of multiple intracellular signals (80). Receptor activation of Gq (a pertussis toxin-insensitive process) leads to activation of phospholipase Cß1 with the subsequent hydrolysis of phosphatydilinositol 4,5-trisphosphate and the generation of IP3 and diacylglycerol (DAG). Then, the binding of IP3 to IP3R localized in the outer acrosomal membrane would induce the release of acrosomal Ca2+. A subsequent capacitative Ca2+ entry, through focal voltage-insensitive channels, would produce a further elevation of intracytoplasmic Ca2+, triggering membrane depolarization and activation of voltage-sensitive L-type Ca2+ channels (80). The high intracellular free Ca2+ concentration together with DAG production would be required for molecular events leading to membrane fusion and finally for the acrosomal exocytosis (82-84).

7.3. Polyphosphoinositides

Hydrolysis of polyphosphoinositides could be involved in the ZP-induced AR. Hydrolysis of polyphosphoinositides produces IP3 and DAG, both of which are involved in the AR as stated above. Polyphosphoinositides are constituted by phosphatydilinositol, phosphatydilinositol 4-phosphate and phosphatydilinositol 4,5-bisphosphate. In mammalian spermatozoa, polyphosphoinositides seem to be located in the inner and outer leaflets of the plasma and outer acrosomal membranes, respectively (85). In the classic model, IP3 and DAG produced after hydrolysis of phosphatydilinositol 4,5-bisphosphate by a phospholipase C are involved in modulation of intracellular Ca2+ concentration and activation of a PKC (86). In several mammalian spermatozoa, including human, the activation of PKC (a molecule able to phosphorylate proteins) is involved in the AR (87, 88). Besides the activation of PKC, DAG produced in the spermatozoa activates a phospholipase C, able to hydrolyze phosphatydilcholine and to further increase the level of DAG (84). This high level of DAG would modulate a phospholipase A2 (PLA2) (82, 83). This key enzyme, which produces fusogenic compounds such as lysophospholipids and free fatty acids, is required for the development of AR (41, 42, 89).

The role of ZP in the hydrolysis of polyphosphoinositides in human spermatozoa is not known. However, in mouse spermatozoa, stimulation with ZP or P results in hydrolysis of phosphatydilinositol 4-phosphate and phosphatydilinositol 4,5-bisphosphate (84). Recently, it was reported that mouse ZP/ZP3 activates a phosphatydilinositol 4,5-bisphosphate-PLCgamma1 through a mechanism involving tyrosine phosphorylation (90). In human spermatozoa, a fraction of hFF induces hydrolysis of polyphosphoinositides that rely upon a primary Ca2+ entry (91). This may contradict the model proposed by Walensky and Snyder (80), in which Ca2+ influx occurs subsequent to and is stimulated by the mobilization of internal Ca2+. Early events of the AR require micromolar Ca2+ levels whereas later events require millimolar levels of Ca2+ (82, 92). Those findings suggest that the small, rapid, and IP3-dependent Ca2+ increase is due to intracellular Ca2+ mobilization, and that this is followed by extracellular Ca2+ influx.

7.4. Adenylyl cyclase activity and cAMP

Phosphodiesterase inhibitors and analogs of cAMP stimulate the AR (73). Activators of the protein kinase A pathway (PKA), such as forskolin and dibutiryl cAMP, also induce the AR of capacitated human spermatozoa (93). In addition, the hFF-induced and solubilized human ZP-induced AR are blocked by KT5720, which is an inhibitor of PKA (93). PKA inhibitors prevent the stimulation of the AR by a PKC stimulator and vice-versa (94). De Jonge suggested that a cross talk between the PKA and PKC pathways may lead to phosphorylation of proteins followed by AR (94).

An elevation of cAMP was observed during the ZP-induced mouse sperm AR (95), indicating that ZP modulates the activity of adenylyl cyclase (AC). Lecler and Kopf reported that ZP and forskolin, in a concentration dependent manner and in the presence of Mg2+, but not Mn2+, can stimulate a membrane associated AC in capacitated mouse spermatozoa (96). Forskolin (a stimulator of the AC) has been also reported to induce the human sperm AR, an effect that can be blocked by adenosine analogs known to inhibit AC. The latter blockage is inhibited by cAMP analogs, suggesting that forskolin acts through AC (93). Though several G proteins are present in mammalian spermatozoa, the presence of a Gs protein in these cells has not yet been documented (97). Therefore, it is not clear as how the sperm AC is activated. Further investigation is needed to elucidate the mechanism (s) by which ZP stimulates the membrane-associated AC and to reveal its role in the AR. A link between AC activation, increased cAMP concentration, Ca2+ channel modulation, and the development of the AR were recently suggested (96).

7.5. Membrane tyrosine kinases

Solubilized ZP stimulates a protein tyrosine kinase activity involved in the mouse sperm AR. A 95 kDa protein, found on the sperm surface, was suggested to bind ZP3, possesses tyrosine kinase activity and participates in the AR (98, 99). The role of this protein as a ZP3 receptor in the mouse, however, should be re-examined, since it was demonstrated that this protein is indeed a unique, phosphotyrosine-containing form of hexokinase, only found in the testis and spermatozoa (30). It was shown that a 95 kDa protein in the human spermatozoa interacts with human ZP (ZRK; distinct from hexokinase) and that this protein contains phosphotyrosine and tyrosine kinase activity. Moreover, recombinant human ZP3, obtained from human ZP3 cDNA transfected COS cells, stimulates kinase activity (28). In addition, in capacitated human spermatozoa, tyrphostin prevents the human ZP3-induced AR (29). Taken together, these results indicate that ZP3-induced tyrosine phosphorylation of ZRK coincides with the ZP3-induced AR, strongly suggesting a direct role for the tyrosine kinase activity in the AR of human spermatozoa. The function of tyrosine phosphorylation in the AR is still obscure. A recent report showed modulation of mouse sperm phospholipase C activity by ZP-stimulated tyrosine phosphorylation, linking the protein tyrosine kinase signaling pathway and phosphatydilinositol 4,5-bisphosphate hydrolysis (90). As discussed above, this pathway seems to play a crucial role in the AR.

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