1. Is there a finite number of binding sites in the caudal isthmus region of the oviduct to regulate the numbers of viable spermatozoa stored during the preovulatory period?

The answer to this question is almost certainly ‘yes’, based on evidence coming principally from experimental observations in domestic farm and laboratory animals. Surgical insemination of freshly ejaculated spermatozoa (Hunter, Reference Hunter1973, Reference Hunter1976) or of frozen–thawed spermatozoa (Polge et al., Reference Polge, Salamon and Wilmut1970) directly into the caudal tip of the oviduct via the utero-tubal junction overrides the available binding sites and generates a high incidence of polyspermic penetration in freshly ovulated eggs. Not only can the incidence of polyspermic fertilization reach 60% or more (Polge et al., Reference Polge, Salamon and Wilmut1970; Hunter, Reference Hunter1976), but there may be multiple sperm penetration of each vitellus. Indeed, massive polyspermy with 20–30 or more sperm heads per vitellus has been observed after surgical insemination into the caudal isthmus of pigs (Hunter, Reference Hunter1976). Such remarkable numbers of vitelline spermatozoa lead to fusion of the enlarging sperm heads and the formation of sperm chromatin aggregates.

Further evidence in support of a finite number of endosalpingeal binding sites comes from so-called delayed mating or insemination when semen is introduced into the female tract after the time of ovulation: here the incidence of polyspermy increases with post-ovulatory age of the eggs (Hancock, Reference Hancock1959; Hunter, Reference Hunter1967). Although ageing of the eggs per se would be a contributing explanation for the observed polyspermy, as would a potential post-ovulatory reduction in the number and nature of binding sites (see below), large numbers of competent spermatozoa are overriding epithelial arrest in the caudal isthmus. As a consequence, sperm:egg ratios at the site of fertilization would have increased in step with the post-ovulatory delay.

2. Does the density of binding sites decrease along the isthmus towards the site of fertilization?

Based on experimental observations, there is not yet a direct answer to this question, but it can be approached by straightforward reasoning. After passage through the utero-tubal junction, the concentration of spermatozoa entering the oviduct would be greatest in the caudal-most portion of the isthmus.

Classical histological observations (Hafez & Blandau, Reference Hafez and Blandau1969; Hafez & Thibault, Reference Hafez and Thibault1973; Thibault, Reference Thibault1973) and more detailed scanning electron microscopic images (Fléchon & Hunter, Reference Fléchon and Hunter1981; Hunter et al., Reference Hunter, Fléchon and Fléchon1987, Reference Hunter, Fléchon and Fléchon1991; Mburu et al., Reference Mburu, Einarsson, Lundeheim and Rodriguez-Martinez1996, Reference Mburu, Rodriguez-Martinez and Einarsson1997) portray large numbers of spermatozoa arrested before ovulation in the caudal portion of the isthmus, in marked contrast to the numbers of spermatozoa revealed close to the isthmo-ampullary junction. Although such a demonstrable sperm gradient could have been established by a variety of means such as physical restriction in the size of the duct lumen, differing viscosity of the accumulated glycoprotein secretions and small differences in temperature, a decreasing density of sperm binding sites might have been a contributory factor. Such a putative reduction in binding sites would have implications for the ad-ovarian progression of spermatozoa activated and released close to the time of ovulation (see below), and could reflect subtle changes in the underlying epithelial histology and distribution of cilia and microvilli and their associated surface molecules. This, ofcourse, bears on the major question of precisely what regulates the expression and loss of epithelial sperm binding sites? See Questions 5 and 10, below.

Although viewed with caution in this assay, evidence from in vitro studies reports differences between the isthmus and ampullary epithelium from the same patient in sperm binding ability (Baillie et al., Reference Baillie, Pacey, Warren, Scudamore and Barratt1997).

3. Are there multiple molecular forms of oviduct sperm binding within a given species?

More specifically, are there binding sites with different molecular characteristics distributed along the caudal portion of the isthmus? The notion of sub-populations of spermatozoa with differing cell surface molecules entering the oviduct has been raised elsewhere (Hunter, Reference Hunter2003b, Reference Hunter2008; Rodriguez-Martinez, Reference Rodriguez-Martinez2007; Rath et al., Reference Rath, Schuberth, Coy and Taylor2008; Petrunkina & Harrison, Reference Petrunkina and Harrison2010; Henning et al., Reference Henning, Petrunkina, Harrison and Waberski2012; Schmid et al., Reference Schmid, Henning, Oldenhof, Wolkers, Petrunkina and Waberski2013). As one consequence, there would be an affinity between specific sperm surface molecules and corresponding endosalpingeal molecules. Not only may the precise nature of the epithelial binding reaction differ between sub-populations of spermatozoa but individual spermatozoa may bind by different domains on the plasma membrane overlying the sperm head. Such considerations would be in line with the proposal that each and every viable sperm cell in the oviduct expresses surface differences and thus has an individual identity (Hunter, Reference Hunter2003b). Moreover, the presence of binding sites with differing molecular characteristics would offer a potential means of organising the preovulatory distribution of spermatozoa in different portions of the storage site, possibly related to the physiological maturity of a sperm cell and its potential for fertilizing an egg. Spermatozoa least avidly bound and presumed to be more mature would be released first.

A related consideration would be that binding reactions on the same sperm cell may be modified with time during remodelling of the surface membranes as the process of capacitation proceeds. Although remodelling at this time is, by definition, dynamic, it does not involve actual loss of membranes.

4. Are some binding sites more favourable than others for maintaining sperm fertilizing potential during the preovulatory interval?

Let us suppose that favourable binding sites are located at some distance from the utero-tubal junction. In such a location, they could be privileged because they would be exposed to a lower density of spermatozoa and closer to the site of fertilization at the isthmo-ampullary junction. Furthermore, such binding sites would be situated beyond the most viscous accumulation of secreted glycoproteins and vestiges of seminal plasma and/or uterine fluids (Jansen, Reference Jansen1978; Hunter, Reference Hunter2001, Reference Hunter2002; Rodriguez-Martinez et al., Reference Rodriguez-Martinez, Tienthai, Suzuki, Funahashi, Ekwall and Johannisson2001; Suarez, Reference Suarez, Neill, Plant, Pfaff, Challis, de Kretser, Richards and Wassarman2006). Likewise, they would be beyond the coolest portion of the oviduct during the preovulatory period (Hunter & Nichol, Reference Hunter and Nichol1986; Bahat et al., Reference Bahat, Tur-Kaspa, Gakamsky, Giojalas, Breitbart and Eisenbach2003). In addition, such binding sites might be attractive for temporary storage of viable spermatozoa due to a greater number of exposed chemical bonds or to more avid bonds related to differences in surface charge. And, as a topographical consideration, binding sites might be more suited to maintaining and preparing spermatozoa for fertilization because of their location between the ridges and troughs of the highly-folded isthmus epithelium (see figures in Fléchon & Hunter, Reference Fléchon and Hunter1981; Hunter et al., Reference Hunter, Fléchon and Fléchon1987; Mburu et al., Reference Mburu, Einarsson, Lundeheim and Rodriguez-Martinez1996, Reference Mburu, Rodriguez-Martinez and Einarsson1997; Yaniz et al., Reference Yaniz, Lopez-Gatius, Santolaria and Mullins2000). Individually or in combination, these diverse factors deserve systematic consideration in a context of the nature of favourable binding sites.

5. Are the isthmus binding sites stable throughout the pre- and peri-ovulatory period of sperm storage?

Responses to this question can be drawn from two lines of evidence. First, cyclic changes in oviduct morphology and physiology are regulated by the ratio of circulating ovarian steroid hormones; synthesis and secretion by preovulatory Graafian follicles change progressively from oestradiol to progesterone after the surge of gonadotrophic hormones. One consequence could be a modification in the number and sperm binding affinity of sites on the isthmus epithelium. Second, the numbers of spermatozoa activated and released from the isthmus reservoir increase progressively from impending ovulation and for many hours or even days thereafter, according to species. Although sperm detachment from binding is associated with increasing flagellar movement (Smith & Yanagimachi, Reference Smith and Yanagimachi1991; Suarez et al., Reference Suarez, Katz, Owen, Andrew and Powell1991a, Reference Suarez, Redfern, Raynor, Martin and Phillips1991b; Suarez, Reference Suarez, Neill, Plant, Pfaff, Challis, de Kretser, Richards and Wassarman2006), a gradual reduction in the avidness of binding prompted by progesterone would explain the increasing sperm:egg ratios at the site of fertilization (Hunter, Reference Hunter1995, Reference Hunter, Croxatto, Schürmann, Fuhrmann and Schellschmidt2005, Reference Hunter2008). It would likewise explain the increasing numbers of accessory spermatozoa on and in the zona pellucida with time after fertilization (Thibault, Reference Thibault1959; Hunter & Dziuk, Reference Hunter and Dziuk1968; Hunter, Reference Hunter1972, Reference Hunter1974).

The observation of a difference in human isthmus versus ampullary sperm binding potential in vitro may be relevant here (Baillie et al., Reference Baillie, Pacey, Warren, Scudamore and Barratt1997), but it would not and could not mimic the influence of changing patterns of ovarian steroid secretion in vivo.

Whether LH or hCG receptors in oviduct tissues (Gawronska et al., Reference Gawronska, Paukku, Huhtaniemi, Wasowicz and Ziecik1999, Reference Gawronska, Stepien and Ziecik2000) act to influence preovulatory sperm release remains to be clarified. Steeply increasing concentrations of endogenous LH in the systemic circulation shortly before ovulation might implicate the oviduct LH receptors in the downgrading of macromolecules underlying sperm-epithelial binding.

6. Are sperm binding sites located only on oviduct cilia or are they also expressed on microvillous regions of the oviduct epithelium?

The emphasis in almost all papers so far concerns binding sites expressed on the extensive tracts of cilia (see Suarez & Pacey, Reference Suarez and Pacey2006). However, in the light of published photographs portraying sperm heads intimately engaged with dense tracts of microvilli (figures in Fléchon & Hunter, Reference Fléchon and Hunter1981; Hunter et al., Reference Hunter, Fléchon and Fléchon1987, Reference Hunter, Fléchon and Fléchon1991), it is worth keeping an open mind on this question. Moreover, if development of binding sites in part involves secretions generated de novo in the oviduct, then microvilli could be involved. During the actual process of fertilization, a sperm head clearly interacts with microvilli of the egg surface (oolemma) – albeit after major membranous modification – so a prior interaction with comparable organelles on the epithelium certainly appears possible. A recent in vitro observation reports sperm head binding to non ciliated oviduct epithelial cells from pig (Chen et al., Reference Chen, Einspanier and Schoen2013).

Turning the emphasis of this paper from binding sites to spermatozoa themselves, further questions are posed below.

7. After passage through the utero-tubal junction and entry into the caudal isthmus, do vanguard spermatozoa interact with the first available binding sites or those closer to the site of fertilization?

In the first instance, this may depend on the molecular configuration expressed on the sperm surface, especially around the sperm head, in relation to molecules expressed on the endosalpinx. However, the rate of progressive sperm motility would also be a consideration and likewise the viscosity of accumulated glycoproteins encountered by such vanguard spermatozoa in the tightly constricted isthmus. Small gradients in temperature might also influence the motility of sperm cells at this stage of their maturation and thus their potential for epithelial interactions (Hunter & Nichol, Reference Hunter and Nichol1986; Bahat et al., Reference Bahat, Tur-Kaspa, Gakamsky, Giojalas, Breitbart and Eisenbach2003).

8. After mating early in the period of receptivity (oestrus), are the first sperm entering the oviduct and binding to the endosalpinx those that will be released first when the time of ovulation approaches?

It may be possible to visualise the sequence of sperm release with improvements in the techniques for labelling individual spermatozoa in conjunction with microscopy using powerful trans-illumination or other detection and tracking procedures (see Druart et al., Reference Druart, Cognie, Baril, Clement, Dacheux and Gatti2009; Rickard et al., Reference Rickard, Pini, Soleilhavoup, Cognie, Bathgate, Lynch, Evans, Maxwell, Druart and de Graaf2014). Ultimately, it would be valuable to learn whether the sequence suggested in the question represents a consistent pattern. Even so, the state of a sperm cell's surface membrane maturity may override any priority gained in sperm progression by the sequence of oviduct entry. Of course vanguard spermatozoa, those entering a preovulatory oviduct first, will enjoy a longer period of maturation.

9. After multiple matings before the time of ovulation, do spermatozoa from second or subsequent matings bind closer to the site of fertilization?

If the answer to this question is ‘yes’, then this situation would suggest that the latter ‘leap-frog’ over already established vanguard spermatozoa to occupy binding sites nearer to the isthmo-ampullary region. In addition, depending on the sequence of activation events close to the time of ovulation, sperm that have bound nearer to the site of fertilization may have a potential advantage in the preliminaries to egg penetration. Indeed, such an advantage may be exploited by experienced males living in free-breeding populations (see Jewell et al., Reference Jewell, Hall and Rosenberg1986).

10. Do activated and detaching spermatozoa contribute to the degradation of binding sites on the oviduct epithelium?

If so, then such a process would be involved in the release of further spermatozoa. There are diverse means by which this scenario for regulating the reserves of stored spermatozoa could be achieved (Hunter, Reference Hunter2008), but direct experimental evidence from in vivo studies is still awaited. Nonetheless, it is possible to envisage a role for macromolecules shed from the surface of liberated spermatozoa acting to remodel and downgrade binding sites on the epithelium (Hunter & Gadea, Reference Hunter and Gadea2014). Tentative support in this direction comes from the in vitro observations of (Hung & Suarez, Reference Hung and Suarez2012) concerning a modified composition of certain proteins on the sperm surface. In parallel, spermatozoa in the hyperactive state may release metabolic and molecular cues to prompt detachment of additional viable spermatozoa in a quantitatively controlled manner. An exchange of ions between detaching and bound spermatozoa still in surface contact might underscore yet another aspect of Ca²⁺ involvement in the events of mammalian fertilization. Sophisticated experiments are needed.

As to whether there is a sperm race – a preovulatory sperm competition to enter the oviduct and colonize the most favourable binding sites – this option has been considered in a recent publication and will not be enlarged upon here (Hunter & Gadea, Reference Hunter and Gadea2014). But it needs to be emphasised that the fertilizing spermatozoon may avoid a phase of endosalpingeal binding altogether, at least under experimental circumstances in vivo (Hunter, Reference Hunter2011).

Summarising some of the key points raised in this short paper, do the site, sequence and intensity of sperm binding in the oviduct isthmus confer any specific advantage in the preliminaries to fertilization? Irrespective of the answer to this question and the broader range of questions posed above, the full impact of oviduct function on the physiology and membranous reactions of individual spermatozoa competent to penetrate and activate an oocyte still requires much painstaking investigation.

Experiments performed in vivo should offer a more meaningful interpretation than those employing autopsy tissues subjected to diverse in vitro procedures, not least as all known components of oviduct physiology are subjected to dynamic influences from the ipsilateral ovary.