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.


ZP is involved in several essential events in fertilization of mammalian eggs. This includes species-specific sperm recognition and binding, stimulation of the sperm AR, and the egg-induced block to polyspermy. All these events are specially well documented in the mouse (1, 3). Since only acrosome-reacted spermatozoa are capable of penetrating through the ZP and fusing with the egg plasma membrane, the AR is crucial for successful fertilization of eggs in mammals (37).

The morphological characteristics of the AR in human spermatozoa are similar to that of other mammalian species investigated thus far (38). AR implies a sequential process of fusion and fenestration of the outer acrosomal membrane and its overlying plasma membrane, which permits release of soluble contents of acrosome and apparently facilitates the passage of spermatozoa through the ZP. A subsequent formation of mixed vesicles, composed of outer acrosomal membrane and plasma membrane, occurs. These vesicles remain close to the sperm head for a short time and then disperse. Since only acrosome-reacted spermatozoa penetrate the zona, the inner acrosomal membrane and the associated hydrolytic enzymes (11, 39) together with the hyperactivated motility (37, 40) should play important functions in the penetration process.

6.1. Physiological inducers of the acrosome reaction of human spermatozoa

AR of human spermatozoa may be initiated by different molecules that can be classified as physiological or pharmacological inducers of this event. Within the pharmacological inducers, the most extensively used agents are the calcium ionophores A23187 and ionomycin. Fusogenic compounds such as lysophospholipids have also been reported to stimulate the AR in mammalian, including human spermatozoa (41-43). The preovulatory human follicular fluid (hFF; (44)) and the human ZP (45) are considered to be among the physiological stimuli for the AR in human spermatozoa. Furthermore, the human cumulus oophorus, the human mural granulosa cells and the progestins progesterone (P) and 17-alpha-P also stimulate AR (44, 46, 47). The AR-inducing activity of hFF and cumulus oophorus is, in part, related to their P content. Indeed, individual hFF samples exhibited great differences in their ability to induce the AR and this ability was significantly correlated with the P content of each fluid (48). Moreover, charcoal-dextran treated hFF lost the AR-initiating ability, which could be only restored after re-addition of P (48). The cumulus cells may be an additional source of P. P level in the cumulus has been estimated at 1-2 µg/ml (49), which is sufficient for induction of AR.

Extensive work has been performed to elucidate the molecules and mechanisms involved in the P-induced human sperm AR (for reviews see (50, 51)). There are some similarities between the way P and the ZP induce the AR. Effects of P on the human spermatozoa are mediated by the presence of binding proteins/receptors located on the acrosomal area of a sperm subpopulation (52, 53). P acts on the human sperm surface to induce at least three different events: i) opening of a plasma membrane Ca2+ channel to induce Ca2+ influx (54, 55). This, in turns, provokes a transient, protein kinase C (PKC)-dependent elevation of intracellular free Ca2+ concentration (56); ii) opening of a plasma membrane Cl- channel to induce Cl- efflux (57). This channel appears to be part of a receptor resembling, but no identical to, the neuronal g-aminobutyric acid receptor type A (GABAA) (58); and iii) activation of a protein tyrosine kinase (59, 60). All these events seem to be due to independent actions of the steroid and are perhaps mediated by three different binding receptors (61). Thus, P may interact with a multireceptor system rather than a single receptor on the plasma membrane of spermatozoa. This hypothesis can explain the variety of responses due to nongenomic action of steroids, and reconcile experimental results that seem contradictory at the first glance (61).

The site where the fertilizing spermatozoon undergoes the AR in vivo is still a matter of debate. In the mouse, only acrosome-intact spermatozoa are able to bind to the ZP and, therefore, the fertilizing spermatozoon is presumed to undergo the AR on the zona surface (62). However, in several species, both acrosome-intact and acrosome-reacted spermatozoa are equally able to bind to the surface of the ZP and penetrate through it (63, 64). Acrosome-reacted human spermatozoa are also able to bind to the human ZP (65). However, it is yet not clear whether they are able to penetrate the ZP and fertilize the oocyte.

Studies on the effect of the ZP on sperm function have been carried out with intact and solubilized ZP and the purified glycoproteins, especially ZP3. More recently, some studies on human spermatozoa have demonstrated that a recombinant human ZP3 is able to promote the AR (29, 66). Also, acrosome-intact mouse spermatozoa are able to bind to ZP3 covalently linked to silica beads; such bound spermatozoa, subsequently, undergo AR and are released from the beads (67). Spermatozoa do not bind to silica beads to which a variety of other glycoproteins, including other zona glycoproteins, are covalently linked. The properties and mode of action of solubilized and purified ZP3 or recombinant ZP3, however, may not be necessarily identical to those of ZP3 immobilized on a three dimensional extracellular matrix. The ordered cross-linking of ZP2/ZP3 heterodimers by ZP1 generates the presence of stereospecific domains. These domains could be playing a role in mediating the effects of ZP3 on the sperm function. Their effects may not be apparent in studies using solubilized or purified ZP components.

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