Review

To those analysing the nature of gamete interactions within the oviduct environment of mammals, the statement repeated in recent publications that “sperm adhesion to the oviductal epithelium is essential for successful fertilization” offers an unnecessary diversion. The statement has invariably been made (e. g. Gervasi et al., Reference Gervasi, Rapanelli, Ribeiro, Farina, Billi, Franchi and Martinez2009) or inferred (Gualtieri & Talevi, Reference Gualtieri and Talevi2003; Gualtieri et al., Reference Gualtieri, Boni, Tosti, Zagami and Talevi2005, Reference Gualtieri, Mollo, Duma and Talevi2009) by people working with biochemical or molecular techniques and is not strictly correct. It overlooks a large body of evidence to the contrary.

The background to this erroneous view has been one of in vitro studies employing oviduct tissues obtained post mortem. Epithelial cells harvested from such tissues have been used to examine the binding and release of spermatozoa under different conditions of culture. By focusing on preparations of isolated cells maintained in vitro, colleagues have lost sight of the physiological situation. The latter should always constitute a point of reference for any meaningful interpretation of in vitro results (Hunter & Rodriguez-Martinez, Reference Hunter and Rodriguez-Martinez2002).

Evidence that sperm head binding to the endosalpinx of the caudal (distal) portion of the isthmus is not necessary for subsequent fertilization comes from: (a) observations made after spontaneous mating or artificial insemination; and (b) observations made under conditions of surgical insemination. Relevant studies have been published for the pig, a species with a relatively long period of estrus (2–3 days) that extends beyond the time of ovulation:

1. (a) After post-ovulatory mating or artificial insemination in domestic pigs, large numbers of viable spermatozoa pass rapidly to the ampullary-isthmic junction, resulting in an incidence of polyspermic fertilization that increases with post-ovulatory age of the eggs at sperm penetration (Hunter, Reference Hunter1967). In similar vein, intra-cervical insemination of pigs at the time of ovulation can result in sperm penetration of the zona pellucida within a period of 2 h (Hunter & Dziuk, Reference Hunter and Dziuk1968), once again negating the requirement for a preliminary phase of sperm binding in the caudal portion of the isthmus.

   As reasoned elsewhere from a large body of experimental evidence, ovarian secretion of progesterone regulates the phenomenon of sperm binding to the isthmus epithelium: peri- or post-ovulatory insemination under the influence of increasing concentrations of plasma progesterone increases the patency of the isthmus and reduces the incidence of sperm binding therein (Hunter, Reference Hunter2008). This steroid hormone effect can be demonstrated most dramatically by injection of microdroplets of a solution of progesterone in oil under the serosal layer of the caudal isthmus in preovulatory animals. Such treatment prompts a massive release of bound spermatozoa in previously mated pigs and leads to elevated levels of polyspermic fertilization when compared with results in the contralateral control oviduct (Hunter, Reference Hunter1972).

2. (b) Both intra-peritoneal insemination and surgical insemination directly into the ampulla via the fimbriated extremity lead to fertilization in rabbits (Chang, Reference Chang1951; Hadek, Reference Hadek1958; Rowlands, Reference Rowlands1958; Adams, Reference Adams1969), sheep (Mattner, Reference Mattner1963), pigs (Hunter, Reference Hunter1978; Hunter et al., Reference Hunter, Huang and Holtz1998) and various other mammals (Yaniz et al., Reference Yaniz, Lopez-Bejar, Santolaria, Rutlant and Lopez-Gatius2002), providing further evidence that a phase of sperm binding in the isthmus is not essential. And, in the present context, the observation that surgical resection of the oviduct isthmus or at least removing >90% of this portion of the duct results in polyspermic fertilization in pigs and rabbits (Hunter & Léglise, Reference Hunter and Léglise1971a, Reference Hunter and Légliseb) strongly suggests that a phase of sperm binding in the isthmus is not mandatory for fertilization.

The phenomenon of preovulatory binding of viable spermatozoa to the epithelium of the caudal isthmus seemingly evolved to provide a sophisticated storage mechanism and to regulate the number of viable spermatozoa progressing to the site of fertilization close to the time of ovulation. Maintenance of a reservoir of male gametes beyond the reach of uterine polymorphonuclear leukocytes is an essential function of the oviduct isthmus during the preovulatory interval. Such epithelial binding and a controlled peri-ovulatory release of competent spermatozoa together act to minimise the incidence of polyspermic fertilization. Bearing in mind the substantial numbers of spermatozoa that are established in the caudal isthmus after mating (e. g. ≥4000–5000 per isthmus in pigs; Hunter, Reference Hunter and Motta1997), then an incisive means of regulation is clearly necessary. Polyspermic penetration of the egg cytoplasm is pathological in mammals (Beatty, Reference Beatty1957; Bomsel-Helmreich, Reference Bomsel-Helmreich, Wolstenholme and O'Connor1965; Thibault, Reference Thibault1967).

What is seldom appreciated by specialists in this field of sperm binding is that the oviduct is not a passive or immotile portion of the genital tract during the preovulatory interval. On the contrary, under the overall influence of ovarian steroid hormones and more immediately of the autonomic nervous system, the myosalpinx shows powerful waves of contractile activity (Corner, Reference Corner1923; Seckinger, Reference Seckinger1923; Wislocki & Guttmacher, Reference Wislocki and Guttmacher1924), increasing to a peak around the time of ovulation. Such contractions are enhanced in the proximity of a mature male, and stimulated still further by mounting, intromission and semen deposition. Moreover, multiple mating may occur under natural conditions. Were it not for the phase of avid sperm binding in the caudal isthmus, newly ovulated eggs would risk being overwhelmed by large numbers of spermatozoa displaced to the isthmo-ampullary junction by ad-ovarian waves of myosalpingeal activity. However, initial sperm:egg ratios at the site of fertilization are close to unity (reviewed in Hunter, Reference Hunter1993, Reference Hunter1995, Reference Hunter1996; Yanagimachi, Reference Yanagimachi, Knobil and Neill1994), even after multiple mating of sheep and pigs early in estrus (Hunter & Nichol, Reference Hunter and Nichol1983, Reference Hunter and Nichol1986; Hunter, Reference Hunter1984).

More subtle interactions between bound sperm cells and the endosalpinx, such as the preovulatory suppression of completion of capacitation and then peri-ovulatory influx of Ca²⁺ ions into the male gamete, presumably evolved after the regulatory and storage events were first established. Indeed, some form of sperm-endosalpingeal adhesion may have been required for suppressing sperm cell activity and membrane remodelling around the sperm head in species with a relatively long period of preovulatory storage. Even so, account needs to be taken of the fact that ejaculated boar spermatozoa will adhere to cells of the tracheal epithelium in smears taken during endotracheal intubation of estrous animals (Hunter, Reference Hunter2003). This observation therefore raises questions as to the molecular nature – as distinct from strategic role – of preovulatory sperm binding to the endosalpinx.

The present discussion could be usefully extended to closely related events. Just as sperm binding to the oviduct epithelium is not an essential preliminary to fertilization, so sperm binding to the surface of the zona pellucida may not be an essential preliminary to penetration of this egg coat. When orientated appropriately, a highly motile spermatozoon under the influence of powerful flagellar thrust may drive its way into the substance of the zona pellucida (see Bedford, Reference Bedford, Austin and Short1982, Reference Bedford1983) without the need for specific protein–carbohydrate linkage interactions at the zona surface. Whereas sperm–zona binding may be essential prior to penetration of the mouse egg (Wassarman, Reference Wassarman1987, Reference Wassarman1988), the cumulus-corona cell investment together with the highly uneven and pitted surface of the zona pellucida in domestic farm animals may serve to guide a highly motile spermatozoon into its matrix without an initial phase of molecular binding (Hunter, Reference Hunter2003). Species differences in the events of fertilization underline these remarks.

As suggested above, the ability of living spermatozoa to bind to an epithelial surface appears to be non-specific, in the sense that mammalian sperm may bind to different parts of the oviduct, to the tracheal epithelium, and doubtless to other epithelial surfaces bearing cilia. Non-specificity in the binding phenomenon is also suggested by a different line of observation. The epithelium of the female genital tract is under the cyclic influence of the ovarian steroid hormones, estradiol and progesterone, and yet spermatozoa will apparently bind in vitro to endosalpingeal cells harvested from any portion of the duct at any stage of the cycle (see Fazeli et al., Reference Fazeli, Duncan, Watson and Holt1999; Green et al., Reference Green, Bredl, Holt, Watson and Fazeli2001). These considerations therefore lead to the major question of exactly how non-specific binding is able to regulate sperm cell membrane activity and capacitation status with precision, if such precision is actually found. A complete answer to the question will not be forthcoming until the domains involved in membranous contact and the communicating ion channels have been defined.

Writing as a physiologist, one presumes that very large numbers of different molecules are exposed on the plasmalemma around the sperm head, and that such molecules are expressed in a dynamic state. As time elapses in the female tract, some molecules may be internalized in the sperm head membranes whilst others may be leached from its surface. Large numbers of different molecules would also be anticipated on the apical surface of oviduct epithelial cells. Again, expression of such molecules would be in a dynamic state, not least as prompted by changes in ovarian steroid hormone secretion. Thus, the nature of the binding process could also be dynamic, with the interacting molecules contributing to the cell adhesion process changing with time. Such an interpretation makes it possible to understand sperm head binding to diverse preparations of oviduct epithelial cells and indeed to other types of epithelial cell.

However, the manner in which such diversity of cell contact achieves the specific objectives of preovulatory suppression of sperm activity for a phase of storage and then peri-ovulatory influx of Ca²⁺ ions for completion of capacitation and full activation remains unclear. Interpretations need to take into account the fact that there will be many sub-populations of spermatozoa established in the oviduct isthmus of an animal mated before ovulation. And, as reasoned elsewhere, every viable sperm cell in the oviduct may be different when viewed at the molecular level (Hunter, Reference Hunter2003), inferring that each has a specific individual identity

Perhaps binding, that is molecular adhesion of the sperm head to: (a) the oviduct epithelium; and subsequently to (b) the surface of the zona pellucida; can be viewed as forms of fine tuning that have developed relatively recently during the evolution of eutherian mammals. A phase of sperm binding may contribute to the selection of competent cells for peri-ovulatory release and progression to the site of fertilization at the isthmo-ampullary junction. Coincidentally, binding may be critically involved in the programming of sequential membrane changes in viable spermatozoa. Such hypothetical fine tuning could help to coordinate cellular events leading to successful monospermic fertilization. Nonetheless, the fact remains that sperm head binding to the epithelium of the oviduct isthmus is not essential for fertilization in diverse mammals, even in those with a relatively long period of estrus (e. g. sheep, cow, pig, horse).

Finally, serious concerns have previously been expressed when using in vitro models as an approach to understanding physiological events (Hunter & Rodriguez-Martinez, Reference Hunter and Rodriguez-Martinez2002). These concerns have now reached a new level as ever more sophisticated biochemical interpretations of cellular events are drawn from in vitro preparations. As but one example, the recent study of Gualtieri et al. (Reference Gualtieri, Mollo, Duma and Talevi2009) used bovine oviduct material obtained post mortem with no reference to animal history, health status, stage of the estrous cycle, or even to whether the animals were cyclic. Yet, it has been known for three-quarters of a century that the gonadal steroid hormones act to programme the genital tract and that the relative plasma concentrations of estradiol and progesterone have a potent influence on female reproductive tissues. Furthermore, when viewed at a molecular level, the apical surface of endosalpingeal cells will undergo additional modifications, as influenced by the local oviduct fluid milieu and by adhering sperm cells. The critical question must therefore be posed as to whether oviduct tissues collected randomly at slaughter are appropriate and sufficient for modern analytical experiments?

In the present author's experience, access to the reproductive tissues in a cattle abattoir is seldom within 5 min of actual slaughter, more commonly some 15–20 min or longer. Slaughter itself provokes massive wayward contractions of the genital tract, and the myosalpingeal autonomic nervous system is rendered completely aberrant with a flood of catecholamine discharge. Removal of reproductive tissues from the carcase, transport to the laboratory, preparation of epithelial cells and their establishment in culture exposes such cells to a variety of temperature gradients, fluid environments and other significant perturbations, all of which will impact on the cell surface. Can one seriously believe that the use of such preparations in sperm binding studies will yield results closely representative of the physiological situation? Extrapolation from such work may provide intellectual excitement, but the utmost caution is advised if one is seeking a realistic understanding of events in the living animal.